Publications

Gestational diabetes mellitus (GDM), a transient form of diabetes that resolves postpartum, is a major risk factor for type 2 diabetes (T2D) in women. While the progression from GDM to T2D is not fully understood, it involves both genetic and environmental components. By integrating clinical, metabolomic, and genome-wide association study (GWAS) data, we identified associations between decreased sphingolipid biosynthesis and future T2D, in part through the rs267738 allele of the CERS2 gene in Hispanic women shortly after a GDM pregnancy. To understand the impact of the CERS2 gene and risk allele on glucose regulation, we examined whole-body Cers2 knockout and rs267738 knock-in mice. Both models exhibited glucose intolerance and impaired insulin secretion in vivo. Islets isolated from these models also demonstrated reduced β cell function, as shown by decreased insulin secretion ex vivo. Overall, reduced circulating sphingolipids may indicate a high risk of GDM-to-T2D progression and reflect deficits in CERS2 activity that negatively affect glucose homeostasis and β cell function.

The relationship between membrane proteins and the lipid constituents of the membrane bilayer depends on finely-tuned atomic interactions, which itself depends on the precise distribution of amino acids within the 3D structure of the protein. In this regard, tryptophan (Trp), one of the least abundant amino acids, is found at higher levels in transmembrane proteins where it likely plays a role in helping anchor them to the membrane. We now re-evaluate Trp distribution in membrane proteins using all known proteins in the Swiss-Prot database and confirm that it is somewhat higher (∼1.7%) than in soluble proteins (∼1.0%), but not as high as in a well-quoted study (∼3.1%). However, the resident endoplasmic reticulum membrane protein, ceramide synthase (CerS), contains a higher abundance of Trp (3.4%). In the case of CerSs which contain a Hox-like domain, the Trp residues are asymmetrically distributed throughout the protein with a bias towards the lumenal side of the endoplasmic reticulum membrane. Mutation of these residues, even to other hydrophobic amino acids, leads to loss of activity, expression, and/or N-glycosylation. Moreover, five of the ten most conserved amino acids in the CerSs are Trp, and site-directed mutagenesis of numerous conserved Trp residues to alanine had distinct effects. Our data is consistent with other studies suggesting that Trp plays critical roles not only in membrane anchoring of transmembrane proteins but also in their activity and function.

Research on sphingolipids has proliferated exponentially over the past couple of decades, as exemplified in the findings reported at the International Leopoldina Symposium on Lipid Signaling held in Frankfurt in late 2023. Most researchers in the field study how sphingolipids function in regulating a variety of cellular processes and, in particular, how they are dysregulated in numerous human diseases; however, I now propose that we implement a more holistic research program in our study of sphingolipids, which embraces a sense of awe and wonder at the complexities and beauty of sphingolipids and of sphingolipid metabolism. I will outline the chemical complexity of sphingolipids, their modes of interaction within the lipid bilayer, and their biosynthetic pathways. I will then briefly touch upon the ability of current neo-Darwinian mechanisms to explain the emergence of both sphingolipids and of the complex pathways that generate them. Although such discussion is normally considered taboo in biological circles, I nevertheless submit that in-depth analysis of the minutiae of metabolic pathways, such as those of the sphingolipid biosynthetic pathway, raises challenges to current neo-Darwinian mechanisms that should not be shunned or ignored.

Almost all attempts to date at gene therapy approaches for monogenetic disease have used the amino acid sequences of the natural protein. In the current study, we use a designed, thermostable form of glucocerebrosidase (GCase), the enzyme defective in Gaucher disease (GD), to attempt to alleviate neurological symptoms in a GD mouse that models type 3 disease, i.e. the chronic neuronopathic juvenile subtype. Upon injection of an AAVrh10 (adeno-associated virus, serotype rh10) vector containing the designed GCase (dGCase) into the left lateral ventricle of Gba^−/−;Gba^tg mice, a significant improvement in body weight and life-span was observed, compared to injection of the same mouse with the wild type enzyme (wtGCase). Moreover, a reduction in levels of glucosylceramide (GlcCer), and an increase in levels of GCase activity were seen in the right hemisphere of Gba^−/−;Gba^tg mice, concomitantly with a significant improvement in motor function, reduction of neuroinflammation and a reduction in mRNA levels of various genes shown previously to be elevated in the brain of mouse models of neurological forms of GD. Together, these data pave the way for the possible use of modified proteins in gene therapy for lysosomal storage diseases and other monogenetic disorders.

Biological membranes consist of a lipid bilayer in which integral membrane proteins are embedded. Based on the compositional complexity of the lipid species found in membranes, and on their specific and selective interactions with membrane proteins, we recently suggested that membrane bilayers can be best described as \u201cfinely-tuned molecular machines.\u201d We now discuss one such set of lipid-protein interactions by describing a negative feedback mechanism operating in the de novo sphingolipid biosynthetic pathway, which occurs in the membrane of the endoplasmic reticulum, and describe the atomic interactions between the first enzyme in the pathway, namely serine palmitoyl transferase, and the product of the fourth enzyme in the pathway, ceramide. We explore how hydrogen-bonding and hydrophobic interactions formed between Asn13 and Phe63 in the serine palmitoyl transferase complex and ceramide can influence the ceramide content of the endoplasmic reticulum. This example of finely-tuned biochemical interactions raises intriguing mechanistic questions about how sphingolipids and their biosynthetic enzymes could have evolved, particularly in light of their metabolic co-dependence.

Sphingolipids are unique among cellular lipids inasmuch as their biosynthesis is compartmentalized between the endoplasmic reticulum (ER) and the Golgi apparatus. This compartmentalization was first recognized about thirty years ago, and the current review not only updates studies on the compartmentalization of sphingolipid biosynthesis, but also discusses the ramifications of this feature for our understanding of how the pathway could have evolved. Thus, we augment some of our recent studies by inclusion of two further molecular pathways that need to be considered when analyzing the evolutionary requirements for generation of sphingolipids, namely contact sites between the ER and the Golgi apparatus, and the mechanism(s) of vesicular transport between these two organelles. Along with evolution of the individual enzymes of the pathway, their subcellular localization, and the supply of essential metabolites via the anteome, it becomes apparent that current models to describe evolution of the sphingolipid biosynthetic pathway may need substantial refinement.

In eukaryotes, the de novo synthesis of sphingolipids (SLs) consists of multiple sequential steps which are compartmentalized between the endoplasmic reticulum and the Golgi apparatus. Studies over many decades have identified the enzymes in the pathway, their localization, topology and an array of regulatory mechanisms. However, little is known about the evolutionary forces that underly the generation of this complex pathway or of its anteome, i.e., the metabolic pathways that converge on the SL biosynthetic pathway and are essential for its activity. After briefly describing the pathway, we discuss the mechanisms by which the enzymes of the SL biosynthetic pathway are targeted to their different subcellular locations, how the pathway per se may have evolved, including its compartmentalization, and the relationship of the pathway to eukaryogenesis. We discuss the circular interdependence of the evolution of the SL pathway, and comment on whether current Darwinian evolutionary models are able to provide genuine mechanistic insight into how the pathway came into being.

Despite being the second most common neurodegenerative disorder, little is known about Parkinson's disease (PD) pathogenesis. A number of genetic factors predispose towards PD, among them mutations in GBA1, which encodes the lysosomal enzyme acid-beta-glucosidase. We now perform non-targeted, mass spectrometry based quantitative proteomics on five brain regions from PD patients with a GBA1 mutation (PD-GBA) and compare to age- and sex-matched idiopathic PD patients (IPD) and controls. Two proteins were differentially-expressed in all five brain regions whereas significant differences were detected between the brain regions, with changes consistent with loss of dopaminergic signaling in the substantia nigra, and activation of a number of pathways in the cingulate gyrus, including ceramide synthesis. Mitochondrial oxidative phosphorylation was inactivated in PD samples in most brain regions and to a larger extent in PD-GBA. This study provides a comprehensive large-scale proteomics dataset for the study of PD-GBA.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid β-glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra-peritoneal injection of conduritol B-epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non-targeted, mass spectrometry-based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE-injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up-regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE-injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology.

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases.

Untargeted lipidomics has been increasingly adopted for hypothesis generation in a biological context or discovery of disease biomarkers. Most of the current liquid chromatography mass spectrometry (LCMS) based untargeted methodologies utilize a data dependent acquisition (DDA) approach in pooled samples for identification and MS-only acquisition for semi-quantification in individual samples. In this study, we present for the first time an untargeted lipidomic workflow that makes use of the newly implemented Quadrupole Resolved All-Ions (Q-RAI) acquisition function on the Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer to acquire MS2 spectra in data independent acquisition (DIA) mode. This is followed by data processing and analysis on MetaboKit, a software enabling DDA-based spectral library construction and extraction of MS1 and MS2 peak areas, for reproducible identification and quantification of lipids in DIA analysis. This workflow was tested on lipid extracts from human plasma and showed quantification at MS1 and MS2 levels comparable to multiple reaction monitoring (MRM) targeted analysis of the same samples. Analysis of serum from Ceramide Synthase 2 (CerS2) null mice using the Q-RAI DIA workflow identified 88 lipid species significantly different between CerS2 null and wild type mice, including well-characterized changes previously associated with this phenotype. Our results show the Q-RAI DIA as a reliable option to perform simultaneous identification and reproducible relative quantification of lipids in exploratory biological studies.

The unique biophysical and biochemical properties of lipids render them crucial in most models of the origin of life (OoL). Many studies have attempted to delineate the prebiotic pathways by which lipids were formed, how micelles and vesicles were generated, and how these micelles and vesicles became selectively permeable towards the chemical precursors required to initiate and support biochemistry and inheritance. Our analysis of a number of such studies highlights the extremely narrow and limited range of conditions by which an experiment is considered to have successfully modeled a role for lipids in an OoL experiment. This is in line with a recent proposal that bias is introduced into OoL studies by the extent and the kind of human intervention. It is self-evident that OoL studies can only be performed by human intervention, and we now discuss the possibility that some assumptions and simplifications inherent in such experimental approaches do not permit determination of mechanistic insight into the roles of lipids in the OoL. With these limitations in mind, we suggest that more nuanced experimental approaches than those currently pursued may be required to elucidate the generation and function of lipids, micelles and vesicles in the OoL.

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.

Acid-β-glucosidase (GCase, EC 3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme®) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step towards developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine-learning based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.

Modern cell membranes contain a bewildering complexity of lipids, among them sphingolipids (SLs). Advances in mass spectrometry have led to the realization that the number and combinatorial complexity of lipids, including SLs, is much greater than previously appreciated. SLs are generated de novo by four enzymes, namely serine palmitoyltransferase, 3-ketodihydrosphingosine reductase, ceramide synthase and dihydroceramide Δ4-desaturase 1. Some of these enzymes depend on the availability of specific substrates and cofactors, which are themselves supplied by other complex metabolic pathways. The evolution of these four enzymes is poorly understood and likely depends on the co-evolution of the metabolic pathways that supply the other essential reaction components. Here, we introduce the concept of the anteome, from the Latin ante (before) to describe the network of metabolic (omic) pathways that must have converged in order for these pathways to co-evolve and permit SL synthesis. We also suggest that the current origin of life and evolutionary models lack appropriate experimental support to explain the appearance of this complex metabolic pathway and its anteome.

A number of genetic risk factors have been identified over the past decade for Parkinsons Disease (PD), with variants in GBA prominent among them. GBA encodes the lysosomal enzyme that degrades the glycosphingolipid, glucosylceramide (GlcCer), with the activity of this enzyme defective in Gaucher disease. Based on the ill-defined relationship between glycosphingolipid metabolism and PD, we now analyze levels of various lipids by liquid chromatography/electrospray ionization-tandem mass spectrometry in four brain regions from age- and sex-matched patient samples, including idiopathic PD, PD patients with a GBA mutation and compare both to control brains (n=21 for each group) obtained from individuals who died from a cause unrelated to PD. Of all the glycerolipids, sterols, and (glyco)sphingolipids (251 lipids in total), the only lipid class which showed significant differences were the gangliosides (sialic acid-containing complex glycosphingolipids), which were elevated in 3 of the 4 PD-GBA brain regions. There was no clear correlation between levels of individual gangliosides and the genetic variant in Gaucher disease [9 samples of severe (neuronopathic), 4 samples of mild (non-neuronopathic) GBA variants, and 8 samples with low pathogenicity variants which have a higher risk for development of PD]. Most brain regions, i.e. occipital cortex, cingulate gyrus, and striatum, did not show a statistically significant elevation of GlcCer in PD-GBA. Only one region, the middle temporal gyrus, showed a small, but significant elevation in GlcCer concentration in PD-GBA. We conclude that changes in ganglioside, but not in GlcCer levels, may contribute to the association between PD and GBA mutations.

The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide in vitro. In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6.sup.ADAAAIA expression were unaffected in the CerS6.sup.ADAAAIA mouse, and CerS6.sup.ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6.sup.ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6.sup.ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths.

Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders.

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

Ceramide is a lipid moiety synthesized via the enzymatic activity of ceramide synthases (CerSs), six of which have been identified in mammalian cells, and each of which uses a unique subset of acyl-CoAs for ceramide synthesis. The CerSs are part of a larger gene family, the Tram-Lag-CLN8 domain family. Here, we identify a unique, C-terminal motif, the DxRSDxE motif, which is only found in CerSs and not in other Tram-LagCLN8 family members. Deletion of this motif in either CerS2 or in CerS6 did not affect the ability of either enzyme to generate ceramide using both an in vitro assay and metabolic labeling, but deletion of this motif did affect the activity of CerS2 when coexpressed with CerS6. Surprisingly, transfection of cells with either CerS2 or CerS6 lacking the motif did not result in changes in cellular ceramide levels. We found that CerS2 and CerS6 interact with each other, as shown by immunoprecipitation, but deletion of the DxRSDxE motif impeded this interaction. Moreover, proteomics analysis of cells transfected with CerS6^Δ338-³⁴⁴ indicated that deletion of the C-terminal motif impacted cellular protein expression, and in particular, the levels of ORMDL1, a negative regulator of sphingolipid synthesis. We suggest that this novel C-terminal motif regulates CerS dimer formation and thereby impacts ceramide synthesis.

Mutations in GBA, which encodes the lysosomal enzyme glucocerebrosidase, are the highest genetic risk factor for Parkinson's disease (PD), although the mechanistic link between GBA mutations and PD is unknown. An attractive hypothesis is that the lipid substrate of glucocerebrosidase, glucosylceramide, accumulates in patients with PD with a GBA mutation (PD-GBA). Despite the availability of new and accurate methods to quantitatively measure brain glucosylceramide levels, there is little evidence that glucosylceramide, or its deacetylated derivative, glucosylsphingosine, accumulates in human PD or PD-GBA brain or cerebrospinal fluid. Thus, a straightforward association between glucosylceramide levels and the development of PD does not appear valid, necessitating the involvement of other cellular pathways to explain this association, which could involve defects in lysosomal function.

Oncogenic multidrug resistance (MDR) is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin-rich domains. As ceramides are the sole source for sphingomyelins, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-seq databases, we found patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (∼2-fold) in chemoresistant ABCB1high (A498, Caki-1) compared to chemosensitive ABCB1low (ACHN, HPCT) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias towards CerS2-associated C20:0/C20:1-ceramides compared to CerS5/6-associated C14:0/C16:0-ceramides (2:1). Sphingomyelins were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (L-cycloserine) and CerS (fumonisin B1) in cell viability assays. Additionally, downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically-inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and sphingomyelin composition to ultra long-chain species (C22-C26). Inhibitors of ER-associated degradation (ERAD) (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/sphingomyelin species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse MDR in renal cancers.

The complex 16p11.2 deletion syndrome (16pdel) is accompanied by neurological disorders, including epilepsy, autism spectrum disorder, and intellectual disability. We demonstrated that 16pdel iPSC differentiated neurons from affected people show augmented local field potential activity and altered ceramide-related lipid species relative to unaffected. FAM57B, a poorly characterized gene in the 16p11.2 interval, has emerged as a candidate tied to symptomatology. We found that FAM57B modulates ceramide synthase (CerS) activity, but is not a CerS per se. In FAM57B mutant human neuronal cells and zebrafish brain, composition and levels of sphingolipids and glycerolipids associated with cellular membranes are disrupted. Consistently, we observed aberrant plasma membrane architecture and synaptic protein mislocalization, which were accompanied by depressed brain and behavioral activity. Together, these results suggest that haploinsufficiency of FAM57B contributes to changes in neuronal activity and function in 16pdel syndrome through a crucial role for the gene in lipid metabolism.

As editors, we are delighted to announce the launch of BioCosmos: New perspectives on the origin and evolution of life, which, as the name implies, aims to present a wide variety of novel perspectives on the origins and nature of life that go beyond the standard neo-Darwinian paradigm of biological evolution.

Sphingolipids can be differentiated from other membrane lipids by the distinctive chemistry of the sphingoid long chain base (LCB), which is generated by the condensation of an amino acid (normally but not always serine) and a fatty acyl CoA (normally palmitoyl CoA) by the pyridoxal phosphate-dependent enzyme, serine palmitoyl transferase (SPT). The first five carbon atoms of the sphingoid LCB, herein defined as the sphingoid motif, are largely responsible for the unique chemical and biophysical properties of sphingolipids since they can undergo a relatively large number (compared to other lipid species) of molecular interactions with other membrane lipids, via hydrogen-bonding, charge-pairing, hydrophobic and van der Waals interactions. These interactions are responsible, for instance, for the association of sphingolipids with cholesterol in the membrane lipid bilayer. Here, we discuss some of the unique properties of this sphingoid motif, and in addition to outlining how this structural motif drives intra-bilayer interactions, discuss the atomic details of the interactions with two critical players in the biosynthetic pathway, namely SPT, and the ceramide transport protein, CERT. In the former, the selectivity of sphingolipid synthesis relies on a hydrogen bond interaction between Lys379 of SPTLC2 and the l-serine sidechain hydroxyl moiety. In the latter, the entire sphingoid motif is stereoselectively recognized by a hydrogen-bonding network involving all three sphingoid motif heteroatoms. The remarkable selectivity of these interactions, and the subtle means by which these interactions are modified and regulated in eukaryotic cells raises a number of challenging questions about the generation of these proteins, and of their interactions with the sphingoid motif in evolutionary history.

Ceramides are a family of sphingophospholipids synthesized in the endoplasmic reticulum, which mediate cell stress responses, including apoptosis, autophagy and senescence, Serine palmitoyltransferase generates 3-ketosphinganine, which is reduced to dihydrosphingosine. N-Acylation allows the formation of dihydroceramides, which are subsequently reduced to form ceramides. Once synthesized, ceramides are trafficked from the ER to the Golgi bound to the ceramide transfer protein, CERT (COL4A3BP, Q9Y5P4). Ceramide can be metabolized via multiple routes, ensuring tight regulation of its cellular levels. Addition of phosphocholine generates sphingomyelin while carbohydrate is added to form glucosyl- or galactosylceramides. Ceramidase re-forms sphingosine or sphinganine from ceramide or dihydroceramide. Phosphorylation of ceramide generates ceramide phosphate. The determination of accurate kinetic parameters for many of the enzymes in the sphingolipid metabolic pathway is complicated by the lipophilic nature of the substrates.

Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.

Sphingolipids are a family of lipids that are critical to cell function and survival. Much of the recent work done on sphingolipids has been performed by a closely-knit family of sphingolipid researchers, which including our colleague, Dr. Lina Obeid, who recently passed away. We now briefly review where the sphingolipid field stands today, focusing in particular on areas of sphingolipid research to which Dr. Obeid made valued contributions. These include the 'many-worlds' view of ceramides and the role of a key enzyme in the sphingolipid biosynthetic pathway, namely the ceramide synthases (CerS). The CerS contain a number of functional domains and also interact with a number of other proteins in lipid metabolic pathways, fulfilling Dr. Obeid's prophecy that ceramides, and the enzymes that generate ceramides, form the critical hub of the sphingolipid metabolic pathway.

Gaucher disease (GD) is caused by homozygous mutations in the GBA1 gene, which encodes the lysosomal β-glucosidase (GBA) enzyme. GD affects several organs and tissues, including the brain in certain variants of the disease. Heterozygous GBA1 variants are a major genetic risk factor for developing Parkinson's disease. The RIPK3 kinase is relevant in GD and its deficiency improves the neurological and visceral symptoms in a murine GD model. RIPK3 mediates necroptotic-like cell death: it is unknown whether the role of RIPK3 in GD is the direct induction of necroptosis or if it has a more indirect function by mediating necrosis-independent. Also, the mechanisms that activate RIPK3 in GD are currently unknown. In this study, we show that c-Abl tyrosine kinase participates upstream of RIPK3 in GD. We found that the active, phosphorylated form of c-Abl is increased in several GD models, including patient's fibroblasts and GBA null mice. Furthermore, its pharmacological inhibition with the FDA-approved drug Imatinib decreased RIPK3 signaling. We found that c-Abl interacts with RIPK3, that RIPK3 is phosphorylated at a tyrosine site, and that this phosphorylation is reduced when c-Abl is inhibited. Genetic ablation of c-Abl in neuronal GD and GD mice models significantly reduced RIPK3 activation and MLKL downstream signaling. These results showed that c-Abl signaling is a new upstream pathway that activates RIPK3 and that its inhibition is an attractive therapeutic approach for the treatment of GD.

Cell membranes are now emerging as finely tuned molecular systems, signifying that reevaluation of our understanding of their structure is essential. Although the idea that cell membrane lipid bilayers do little more than give shape and form to cells and limit diffusion between cells and their environment is totally passé, the structural, compositional, and functional complexity of lipid bilayers often catches cell and molecular biologists by surprise. Models of lipid bilayer structure have developed considerably since the heyday of the fluid mosaic model, principally by the discovery of the restricted diffusion of membrane proteins and lipids within the plane of the bilayer. In reviewing this field, we now suggest that further refinement of current models is necessary and propose that describing lipid bilayers as \u201cfinelytuned molecular assemblies\u201d best portrays their complexity and function.

Most lysosomal storage diseases (LSDs) have a significant neurological component, including types 2 and 3 Gaucher disease (neuronal forms of Gaucher disease; nGD). No therapies are currently available for nGD since the recombinant enzymes used in the systemic form of Gaucher disease do not cross the bloodbrain barrier (BBB). However, a number of promising approaches are currently being tested, including substrate reduction therapy (SRT), in which partial inhibition of the synthesis of the glycosphingolipids (GSLs) that accumulate in nGD lowers their accumulation. We now induce nGD in mice by injection with conduritol B-epoxide (CBE), an irreversible inhibitor of acid beta-glucosidase (GCase), the enzyme defective in nGD, with or without co-injection with Genz-667161, a prototype for SRT which crosses the BBB. Significant neuropathology, and a reduction in lifespan, was observed upon CBE injection, and this was largely reversed by co-injection with Genz-667161, along with a reduction in glucosylceramide and glucosylsphingosine levels. Analysis of gene expression by RNAseq revealed that Genz-667161 largely reversed the changes in genes and pathways that were differentially expressed upon CBE injection, specifically pathways of GSL metabolism, lipoproteins and other lipid metabolic pathways, lipid droplets, astrocyte activation, neuronal function, and to some extent, neuroinflammation. Together, this demonstrates the efficacy of SRT to reverse the effects of substrate accumulation on pathological components and pathways in nGD brain.

(1) Background: six mammalian ceramide synthases (CerS1-6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22-C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba^-/-;Gba^tg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba^-/-;Gba^tg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba^-/-;Gba^tg mice being a genuine type 3 GD model. Together, the Gba^-/-;Gba^tg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

OBJECTIVE A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins in lysosomes. This event culminates with the loss of cell homeostasis, inflammation and cell death. In this study we propose to identify the chemical etiological factors and understanding the molecular and cellular mechanisms responsible for the impairment of lysosome function in macrophages.APPROACH AND RESULTS Using shotgun lipidomics we have discovered that a family of oxidized lipids (cholesteryl hemiesters, ChE), end products of oxidation of polyunsaturated cholesteryl esters, occurs at higher concentrations in the plasma of two cohorts of cardiovascular disease patients than in the plasma of a control cohort. Macrophages exposed to the most prevalent ChE, cholesteryl hemiazelate (ChA) exhibit lysosome enlargement, peripheral lysosomal positioning, lysosome dysfunction and lipidosis which are irreversible. The transcriptomic profile of macrophages exposed to ChA indicates that the lysosome pathway is deeply affected and is well correlated with lysosome phenotypic and functional changes. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences to the pathology.CONCLUSION We identify ChA not only as one of the molecules involved in the etiology of irreversible lysosome dysfunction culminating with lipidosis but also as a promoter of exocytosis of the dysfunctional lysosomes. The latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.Competing Interest StatementThe authors have declared no competing interest.ACSAcute coronary syndromeChACholesteryl hemiazelateChECholesteryl hemiestersChSCholesteryl hemisuccinateSAPStable angina pectoris

The lipid composition of human immunodefiency virus 1 (HIV-1) virions is enriched in sphingomyelin, but the roles that sphingomyelin or other sphingolipids might play in the HIV-1 replication pathway have not been elucidated. In human cells, sphingolipid levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to sphingomyelins, hexosylceramides, and other sphingolipids. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and sphingomyelin were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the Vesicular Stomatitis Virus (VSV) glycoprotein (G) did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E (Itgae, CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection.

Background: Mucolipidosis type IV (MLIV), an ultra-rare neurodevelopmental and neurodegenerative disorder, is caused by mutations in the MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1). The precise pathophysiogical pathways that cause neurological disease in MLIV are poorly understood. Recently, the first post-mortem brain sample became available from a single MLIV patient, and in the current study we performed mass spectrometry (MS)-based proteomics on this tissue with a view to delineating pathological pathways, and to compare with previously-published data on MLIV, including studies using the Mcoln1^−/− mouse. Results: A number of pathways were altered in two brain regions from the MLIV patient, including those related to the lysosome, lipid metabolism, myelination, cellular trafficking and autophagy, mTOR and calmodulin, the complement system and interferon signaling. Of these, levels of some proteins not known previously to be associated with MLIV were altered, including APOD, PLIN4, ATG and proteins related to interferon signaling. Moreover, when proteins detected by proteomics in the human brain were compared with their orthologs detected in the Mcoln1^−/− mouse by RNAseq, the results were remarkably similar. Finally, analysis of proteins in human and mouse CSF suggest that calbindin 1 and calbindin 2 might be useful as biomarkers to help chart the course of disease development. Conclusions: Despite the sample size limitations, our findings are consistent with the relatively general changes in lysosomal function previously reported in MLIV, and shed light on new pathways of disease pathophysiology, which is required in order to understand the course of disease development and to determine the efficacy of therapies when they become available for this devastating disease.

Sphingolipids are ubiquitous components of eukaryotic cell membranes and have been widely studied over the past couple of decades due to the discovery of their involvement in a wide range of biological processes. This chapter focuses on the structure, biosynthesis and degradation of sphingolipids, with particular emphasis on recent studies documenting the huge numbers of individual sphingolipid structures which are based on variation in both the sphingolipid backbone and in the headgroup. Attention is also paid to the roles of sphingolipids in signalling pathways with particular emphasis on sphingosine 1-phosphate and on ceramide. The significance of sphingolipids in human diseases is highlighted, as sphingolipids are now known to play a role in conditions as diverse as inflammation and infection, cancer, psychiatric disorders and neurological and immunological diseases. Thus, sphingolipids have been repositioned as fundamental players in the regulation of human health and disease.

Homozygous mutations in GBA1 cause the most common lysosomal storage disease (LSD), namely, Gaucher disease (GD). Mutations (homozygous or heterozygous) in GBA1 are also the highest genetic risk factor for Parkinson's disease (PD),1 although the mechanistic basis for this relationship is not known. We now suggest, based on welldocumented data in some cases and on more anecdotal data in others, that there is a resemblance in some nonmotor symptoms (NMSs) between PD and LSDs. This may indicate that brain regions associated with NMSs are more vulnerable to changes in lysosomal activity and αsynuclein deposition as a result of lysosomal dysfunction.

BACKGROUND: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis.METHODS: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels.RESULTS: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER.CONCLUSION: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.

Ceramides are an important group of sphingolipids that modulate several cellular events. The mechanisms underlying biological actions of ceramides are not fully known, but evidence suggests that ceramides can act through regulation of the biophysical properties of the membrane. However, ceramide-induced changes on membrane properties are complex and depend on several factors. To gain further insight into this subject, we characterized the biophysical impact of very-long acyl chain C24-ceramide in a fluid model membrane under thermodynamic equilibrium and non-equilibrium conditions. Our results show that C24-ceramide readily forms two types of gel domains with distinct properties, likely corresponding to different interdigitated metastable gel phases. Upon reaching thermodynamic equilibrium, only partially interdigitated gel phase coexists with the fluid phase. In addition, C24-ceramide promotes strong changes in the shape of the vesicles, including domains with sharp edges and tubule-like structures. The results suggest that the formation of very long acyl chain ceramides in response to stress stimuli will initially induce a multitude of changes in the organization and fluidity of biological membranes that might be responsible for the activation of different cellular processes.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

BACKGROUND: The type 1 interferon (IFN) response is part of the innate immune response and best known for its role in viral and bacterial infection. However, this pathway is also induced in sterile inflammation such as that which occurs in a number of neurodegenerative diseases, including neuronopathic Gaucher disease (nGD), a lysosomal storage disorder (LSD) caused by mutations in GBA.METHODS: Mice were injected with conduritol B-epoxide, an irreversible inhibitor of acid beta-glucosidase, the enzyme defective in nGD. MyTrMaSt null mice, where four adaptors of pathogen recognition receptors (PRRs) are deficient, were used to determine the role of the IFN pathway in nGD pathology. Activation of inflammatory and other pathways was analyzed by a variety of methods including RNAseq.RESULTS: Elevation in the expression of PRRs associated with the IFN response was observed in CBE-injected mice. Ablation of upstream pathways leading to IFN production had no therapeutic benefit on the lifespan of nGD mice but attenuated neuroinflammation. Primary and secondary pathological pathways (i.e., those associated or not with mouse survival) were distinguished, and a set of ~210 genes including those related to sphingolipid, cholesterol, and lipoprotein metabolism, along with a number of inflammatory pathways related to chemokines, TNF, TGF, complement, IL6, and damage-associated microglia were classified as primary pathological pathways, along with some lysosomal and neuronal genes.CONCLUSIONS: Although IFN signaling is the top elevated pathway in nGD, we demonstrate that this pathway is not related to mouse viability and is consequently defined as a secondary pathology pathway. By elimination, we defined a number of critical pathways that are directly related to brain pathology in nGD, which in addition to its usefulness in understanding pathophysiological mechanisms, may also pave the way for the development of novel therapeutic paradigms by targeting such pathways.

Both monogenic diseases and viral infections can manifest in a broad spectrum of clinical phenotypes that range from asymptomatic to lethal, suggesting that other factors modulate disease severity. Here, we examine the interplay between the genetic neuronopathic Gaucher's disease (nGD), and neuroinvasive Sindbis virus (SVNI) infection. Infection of nGD mice with SVNI had no influence on nGD severity. However, nGD mice were more resistant to SVNI infection. Significantly different inflammatory responses were seen in nGD brains when compared with SVNI brains: the inflammatory response in the nGD brains consisted of reactive astrocytes and microglia with no infiltrating macrophages, but the inflammatory response in the brains of SVNI-infected mice was characterized by infiltration of macrophages and altered activation of microglia and astrocytes. We suggest that the innate immune response activated in nGD confers resistance against viral infection of the CNS.

The lysosome is a central player in the cell, acting as a clearing house for macromolecular degradation, but also plays a critical role in a variety of additional metabolic and regulatory processes. The lysosome has recently attracted the attention of neurobiologists and neurologists since a number of neurological diseases involve a lysosomal component. Among these is Parkinson's disease (PD). While heterozygous and homozygous mutations in GBA1 are the highest genetic risk factor for PD, studies performed over the past decade have suggested that lysosomal loss of function is likely involved in PD pathology, since a significant percent of PD patients have a mutation in one or more genes that cause a lysosomal storage disease (LSD). Although the mechanistic connection between the lysosome and PD remains somewhat enigmatic, significant evidence is accumulating that lysosomal dysfunction plays a central role in PD pathophysiology. Thus, lysosomal dysfunction, resulting from mutations in lysosomal genes, may enhance the accumulation of α-synuclein in the brain, which may result in the earlier development of PD.

Sphingolipids (SLs) are an important class of membrane lipids containing a long chain sphingoid base backbone. SL synthesis is compartmentalized between two major cell organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. The initial steps of sphingolipid synthesis take place in the ER, where the simplest SL, ceramide, is synthesized. Although ceramide is a critical membrane component, an imbalance of ceramide levels can have significant deleterious effects on cell properties leading to events such as apoptosis. For this reason and others, ER ceramide levels must be tightly regulated. Here, we describe the biological and biophysical properties of ceramide and discuss how this might impact the ER membrane. This article is part of a special issue entitled: ER Platforms for Membrane Lipid Dynamics.

The endoplasmic reticulum (ER) is not only important for protein synthesis and folding but is also crucial for lipid synthesis and metabolism. In the current study, we demonstrate an important role of ceramide synthases (CerS) in ER stress and NAFLD progression. Ceramide is important in sphingolipid metabolism, and its acyl chain length is determined by a family of six CerS in mammals. CerS2 generates C22-C24 ceramides, and CerS5 or CerS6 produces C16 ceramide. To gain insight into the role of CerS in NAFLD, we used a high-fat diet (HFD)-induced NAFLD mouse model. Decreased levels of CerS2 and increased levels of CerS6 were observed in the steatotic livers of mice fed a HFD. In vitro experiments with Hep3B cells indicated the protective role of CerS2 and the detrimental role of CerS6 in the ER stress response induced by palmitate treatment. In particular, CerS6 overexpression increased sterol regulatory element-binding protein-1 (SREBP-1) cleavage with decreased levels of INSIG-1, leading to increased lipogenesis. Blocking ER stress abrogated the detrimental effects of CerS6 on palmitate-induced SREBP-1 cleavage. In accordance with the protective role of CerS2 in the palmitate-induced ER stress response, CerS2 knockdown enhanced ER stress and SREBP1 cleavage, and CerS2 heterozygote livers exhibited a stronger ER stress response and higher triglyceride levels following HFD. Finally, treatment with a low dose of bortezomib increased hepatic CerS2 expression and protected the development of NAFLD following HFD. These results indicate that CerS and its derivatives impact hepatic ER stress and lipogenesis differently and might be therapeutic targets for NAFLD.

Bi-allelic mutations in the glucocerebrosidase gene (GBA1) cause Gaucher's disease, the most common human lysosomal storage disease. We previously reported a marked increase in miR-155 transcript levels and early microglial activation in a zebrafish model of Gaucher's disease (gba1(-/-)). miR-155 is a master regulator of inflammation and has been implicated in a wide range of different neurodegenerative disorders. The observed miR-155 upregulation preceded the subsequent development of widespread pathology with marked neuroinflammation, closely resembling human Gaucher's disease pathology. We now report similar increases of miR-155 expression in mammalian models of GD, confirming that miR-155 upregulation is a shared feature in glucocerebrosidase (GCase) deficiency across different species. Using CRISPR/Cas9 mutagenesis we then generated a miR-155 mutant zebrafish line (miR-155(-/-)) with completely abolished miR-155 expression. Unexpectedly, loss of miR-155 did not mitigate either the reduced lifespan or the robust inflammatory phenotypes of gba1(-/-) mutant zebrafish. Our data demonstrate that neither neuroinflammation nor disease progression in GCase deficiency are dependent on miR-155 and suggest that miR-155 inhibition would not be a promising therapeutic target in Gaucher's disease.

LAG1 was the first longevity assurance gene discovered in Saccharomyces cerevisiae. The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and in vitro assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Delta lag1 cells have an increased lifespan while Delta lac1 cells do not.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/ db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)-dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosidesnamely monosialoganglioside GM3 and disialoganglioside GD3as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.Author summary Invariant natural killer T (iNKT) cells are a population of unconventional T lymphocytes that activate rapidly during inflammation due to their innate-like features. They are unconventional since they do not react to peptidic antigens (Ags) presented by classical major histocompatibility complex (MHC) molecules; instead, they recognize lipid-based Ags in the context of the MHC class I-like molecule CD1d. While numerous Ags of microbial origins have been described, their endogenous Ags are far less understood and remain a matter of strong debate. Here, we report that engagement of an innate receptor on the Ag-presenting cells leads to modulation of their lipid metabolism. This results in an enrichment of particular glycosphingolipid species that differ in both the nonpolar tail and polar head structures. Among those, two species have the potential to activate iNKT cells in a CD1d-dependent manner after further intracellular modifications. Based on these data, we propose a concept that iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced changes in CD1d-expressing cells. Given the presence of closely related molecules in some pathological conditions such as cancer, it will be interesting to evaluate the biological relevance of these Ags in disease states.

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of β-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages (Gaucher cells) have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. (Figure presented.). This article is part of the Special Issue \u201cLysosomal Storage Disorders\u201d.

Glucocerebrosidase (GBA) is a lysosomal β-glucosidase-degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism-based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity-based probes to visualize catalytic pocket occupancy of candidate off-target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α-glucosidase were identified as major off-targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD-like models. Enzymes: Glucocerebrosidase (EC 3.2.1.45), nonlysosomal β-glucocerebrosidase (EC 3.2.1.45); cytosolic β-glucosidase (EC 3.2.1.21); α-glucosidases (EC 3.2.1.20); β-glucuronidase (EC 3.2.1.31).

The majority of enzymes in the sphingolipid (SL) biosynthetic pathway have been identified over the past couple of decades. Despite significant work, and despite their crucial and central roles in SL synthesis, significant information is still lacking concerning the enzymes that catalyze the N-acylation of sphingoid long chain bases, namely the ceramide synthases (CerS), a family of six mammalian genes originally named longevity assurance (Lass) genes. Each of these six endoplasmic reticulum (ER) membrane-bound enzymes utilizes a relatively restricted sub-set of fatty acyl-CoAs for N-acylation, but are far more promiscuous about the use of long chain bases. The reason that mammals and other species have multiple CerS, generating a specific subset of ceramides, is not yet known, but implies an important role for ceramides containing specific fatty acids in cell physiology. In this brief chapter, we will stroll down the CerS lane and discuss what is known, and what is not known, about this important enzyme family.

Cholesterol, sphingolipids and glycerophospholipids are critical constituents of the brain, subserving neuronal membrane architecture and providing a platform for biochemical processes essential for proper neurodevelopment and function. When lysosomal defects arise in a lipid metabolic pathway, it is therefore easy to imagine that neurological decline will transpire, however for deficits in non-lipid pathways, this becomes harder to envisage. Here we suggest the working hypothesis that neurodegenerative lysosomal storage disorders might manifest as primary and/or secondary disorders of lipid metabolism. Evidence suggests that the mere process of lysosomal substrate accumulation, ubiquitous in all lysosomal storage disorders, impairs lysosome integrity resulting in secondary lipid accumulation. Impaired lysosomal degradation of a specific lipid defines a primary disorder of lipid metabolism and as these lysosomal storage disorders additionally show secondary lipid alterations, all primary disorders can also be considered secondary disorders of lipid metabolism. The outcome is a generalized cellular lipid dyshomeostasis and consequently, the physiological architecture of the lipid-enriched plasma membrane is perturbed, including the lipid composition of specialized membrane microdomains, often termed lipid rafts. Neurotoxicity results from the complex interplay of malfunctioning signaling and vesicular trafficking important for neuronal communication and synaptic plasticity-induced by the imbalance in physiological membrane lipid composition - together with compensatory mechanisms aimed at restoring lipid homeostasis.

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5 (299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4 (291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1/ astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1/ mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1/ astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1/ astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

The mechanisms by which lung structural cells survive toxic exposures to cigarette smoke (CS) are not well defined but may involve proper disposal of damaged mitochondria by macroautophagy (mitophagy), processes that may be influenced by proapoptotic ceramide (Cer) or its precursor dihydroceramide (DHC). Human lung epithelial and endothelial cells exposed to CS exhibited mitochondrial damage, signaled by phosphatase and tensin homologinduced putative kinase 1 (PINK1) phosphorylation, autophagy, and necroptosis. Although cells responded to CS by rapid inhibition of DHC desaturase, which elevated DHC levels, palmitoyl (C16)Cer also increased in CSexposed cells. Whereas DHC augmentation triggered autophagy without cell death, the exogenous administration of C16Cer was sufficient to trigger necroptosis. Inhibition of Cergenerating acid sphingomyelinase reduced both CSinduced PINK1 phosphorylation and necroptosis. When exposed to CS, Pinkldeficient (Pink1−/−) mice, which are protected from airspace enlargement compared with wildtype littermates, had blunted C16Cer elevations and less lung necroptosis. CSexposed Pink1−/− mice also exhibited significantly increased levels of lignoceroyl (C24)DHC, along with increased expression of Cer synthase 2 (CerS2), the enzyme responsible for its production. This suggested that a combination of high C24DHC and low C16Cer levels might protect against CSinduced necroptosis. Indeed, CerS2−/− mice, which lack C24DHC at the expense of increased C16Cer, were more susceptible to CS, developing airspace enlargement following only 1 month of exposure. These results implicate DHCs, in particular, C24DHC, as protective against CS toxicity by enhancing autophagy, whereas C16Cer accumulation contributes to mitochondrial damage and PINKlmediated necroptosis, which may be amplified by the inhibition of C24DHCproducing CerS2. Mizumura, K., Justice, M.J., Schweitzer, K.S., Krishnan, S., Bronova, I., Berdyshev, E. V., Hubbard, W.C., PewznerJung, Y., Futerman, A. H., Choi, A. M. K., Petrache, I. Sphingolipid regulation of lung epithelial cell mitophagy and necroptosis during cigarette smoke exposure.

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ~6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

The role of sphingolipids (SLs) in the immune system has come under increasing scrutiny recently due to the emerging contributions that these important membrane components play in regulating a variety of immunological processes. The acyl chain length of SLs appears particularly critical in determining SL function. Here, we show a role for very-long acyl chain SLs (VLC-SLs) in invariant natural killer T (iNKT) cell maturation in the thymus and homeostasis in the liver. Ceramide synthase 2-null mice, which lack VLC-SLs, were susceptible to a hepatotropic strain of lymphocytic choriomeningitis virus, which is due to a reduction in the number of iNKT cells. Bone marrow chimera experiments indicated that hematopoietic-derived VLC-SLs are essential for maturation of iNKT cells in the thymus, whereas parenchymal-derived VLC-SLs are crucial for iNKT cell survival and maintenance in the liver. Our findings suggest a critical role for VLC-SL in iNKT cell physiology.

The Concise Guide to PHARMACOLOGY 2017/18 is the third in this series of biennial publications. This version provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13882/full. In addition to this overview, in which are identified Other protein targets which fall outside of the subsequent categorisation, there are eight areas of focus: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism.

Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant decrease in cytoplasmic area occupied by autophagic vacuoles. Together, these findings highlight the critical role of GBA1 in mediating enhanced self-consumption of intracellular components and endomembranes, leading to autophagic cell death.

Ceramide and more complex sphingolipids constitute a diverse group of lipids that serve important roles as structural entities of biological membranes and as regulators of cellular growth, differentiation, and development. Thus, ceramides are vital players in numerous diseases including metabolic and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes of ACBP−/− mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial importance in understanding the regulation of ceramide metabolism in pathogenesis.

Sphingolipids modulate clathrin-mediated endocytosis (CME) by altering the biophysical properties of membranes. We now examine CME in astrocytes cultured from ceramide synthase 2 (CerS2) null mice, which have an altered sphingolipid acyl chain composition. The rate of endocytosis of low-density lipoprotein and transferrin, which are internalized via CME, was reduced in CerS2 null astrocytes, although the rate of caveolin-mediated endocytosis was unaltered. Levels of clathrin heavy chain were increased, which was due to decreased levels of Hsc70 (also known as HSPA8), a protein involved in clathrin uncoating. Hsc70 levels were decreased because of lower levels of binding of Sp1 to position -68 in the Hsc70 promoter. Levels of Sp1 were downregulated due to oxidative stress, which was elevated fourfold in CerS2 null astrocytes. Furthermore, induction of oxidative stress in wild-type astrocytes decreased the rate of CME, whereas amelioration of oxidative stress in CerS2 null astrocytes reversed the decrease. Our data are consistent with the notion that sphingolipids not only change membrane biophysical properties but also that changes in their composition can result in downstream effects that indirectly impinge upon a number of cellular pathways, such as CME.

The lysosomal acid β-glucosidase GBA1 and the non-lysosomal β-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of β-glucosidase activity in general.

In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD.

Defining the mechanisms of action of the antipsychotic drug (APD), clozapine, is of great importance, as clozapine is more effective and has therapeutic benefits in a broader range of psychiatric disorders compared with other APDs. Its range of actions have not been fully characterized. Exposure to APDs early in development causes dose-dependent developmental delay and lethality in Caenorhabditis elegans. A previous genome-wide RNAi screen for suppressors of clozapine-induced developmental delay and lethality revealed 40 candidate genes, including sms-1, which encodes a sphingomyelin synthase. One sms-1 isoform is expressed in the C. elegans pharynx, and its transgene rescues the sms-1 mutant phenotype. We examined pharyngeal pumping and observed that clozapine-induced inhibition of pharyngeal pumping requires sms-1, a finding that may explain the role of the gene in mediating clozapine-induced developmental delay/lethality. By analyzing multiple enzymes involved in sphingolipid metabolism, and by observing the effect of addition of various lipids directly to the worms, we suggest that glucosylceramide may be a key mediator of the effects of clozapine. We further observed that clozapine clears protein aggregates, such as α-synuclein, PolyQ protein, and α-1-antitrypsin mutant protein. In addition, it enhances ATG8/LC3. We conclude that clozapine appears to affect the development and induce lethality of worms, in part, through modulating glucosylceramide. We discuss the possible connections among glucosylceramide, protein aggregate clearance, and autophagy. Interactions, including mechanistic pathways involving these elements, may underlie some of the clinical effects of clozapine.

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.

Gaucher disease is caused by the defective catabolism of the simple glycosphingolipid, glucosylceramide (GlcCer), due to mutations in the GBA1 gene which encodes for acid p-glucosidase (GCase), the lysosomal enzyme that degrades GIcCer. Today, Gaucher disease patients are routinely treated with recombinant GCase, in a treatment regimen known as enzyme replacement therapy (ERT). We now review the biochemical basis of ERT and discuss how this treatment has advanced since it was first pioneered by Dr. Roscoe Brady in the 1960s. We will place particular emphasis on the three dimensional structure of GCase, and subsequently discuss a relatively new treatment paradigm, substrate reduction therapy (SRT), in which GlcCer synthesis is partially inhibited, thus reducing its accumulation. Both of these approaches are based on studies and concepts developed by Dr. Brady over his remarkable research career spanning six decades.

Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin−/− mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin−/− mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin−/− hepatic stellate cells. Sortilin−/− hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin−/− hepatocytes, had increased aSMase-dependent susceptibility to bile acidinduced apoptosis in vitro. Mechanistically, short-term IL-6 neutralization in bile ductligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile ductligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6.

Glucosylceramide (GlcCer) plays an active role in the regulation of various cellular events. Moreover, GlcCer is also a key modulator of membrane biophysical properties, which might be linked to the mechanism of its biological action. In order to understand the biophysical implications of GlcCer on membranes of living cells, we first studied the effect of GlcCer on artificial membranes containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). Using an array of biophysical methods, we demonstrate that at lower GlcCer/Chol ratios, GlcCer stabilizes SM/Chol-enriched liquid-ordered domains. However, upon decreasing the Chol content, GlcCer significantly increased membrane order through the formation of gel domains. Changes in pH disturbed the packing properties of GlcCer-containing membranes, leading to an increase in membrane fluidity and reduced membrane electronegativity. To address the biophysical impact of GlcCer in biological membranes, studies were performed in wild type and in fibroblasts treated with conduritol-B-epoxide (CBE), which causes intracellular GlcCer accumulation, and in fibroblasts from patients with type I Gaucher disease (GD). Decreased membrane fluidity was observed in cells containing higher levels of GlcCer, such as in CBE-treated and GD cells. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathology of diseases, such as GD, in which GlcCer accumulates at high levels.

Sphingolipids (SL) and their metabolites play key roles both as structural components of membranes and as signaling molecules. Many of the key enzymes and regulators of SL, metabolism were discovered using the yeast Saccharomyces cerevisiae, and based on the high degree of conservation, a number of mammalian homologs were identified. Although yeast continues to be an important tool for SL research, the complexity of SL structure and nomenclature often hampers the ability of new researchers to grasp the subtleties of yeast SL biology and discover new modulators of this intricate pathway. Moreover, the emergence of lipidomics by mass spectrometry has enabled the rapid identification of SL species in yeast and rendered the analysis of SL composition under various physiological and pathophysiolbgical conditions readily amenable. However, the complex nomenclature of the identified species renders much of the data inaccessible to non-specialists. In this review, we focus on parsing both the classical SL nomenclature and the nomenclature normally used during mass spectrometry analysis, which should facilitate the understanding of yeast SL data and might shed light on biological processes in which SLs are involved. Finally, we discuss a number of putative roles of various yeast SL species.

Endothelial oxidative stress develops with aging and reactive oxygen species impair endotheliumdependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial KCa3.1, which contributes to EDR, is upregulated by H2O2. We investigated whether KCa3.1 upregulation compensates for diminished EDR to NO during agingrelated oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1phosphate were increased in aged wildtype and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wildtype and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and agematched wildtype mice. Increased H2O2 levels induced Fyn and extracellular signalregulated kinases (ERKs) phosphorylation and KCa3.1 upregulation. Catalase/GPX1 double knockout (catalase−/−/GPX1−/−) upregulated KCa3.1 in MAECs. NO production was decreased in aged wildtype, CerS2 null, and catalase−/−/GPX1−/− MAECs. However, KCa3.1 activationinduced, NGnitrolarginine, and indomethacinresistant EDR was increased without a change in acetylcholineinduced EDR in aortic rings from aged wildtype, CerS2 null, and catalase−/−/GPX1−/− mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1phosphate induced similar changes in levels of the antioxidant enzymes and upregulated KCa3.1. Our findings suggest that, during agingrelated oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H2O2 and thereby upregulate KCa3.1 expression and function via a H2O2/Fynmediated pathway. Altogether, enhanced KCa3.1 activity may compensate for decreased NO signaling during vascular aging.

Diseases caused by single-gene mutations can display substantial phenotypic variability, which may be due to genetic, environmental, or epigenetic modifiers. Here, we induce Gaucher disease (GD), a rare inherited metabolic disorder, by injecting 15 inbred mouse strains with a low dose of a chemical inhibitor of acid beta-glucosidase, the enzyme defective in GD. Different mouse strains exhibit widely different lifespans, which is unrelated to levels of acid beta-glucosidase's substrate accumulation. Genome-wide association reveals a number of candidate risk loci, including a marker within Grin2b, which in combination with another marker allows us to predict the lifespan of additional mouse strains. An antagonist of the NMDA receptor (encoded by Grin2b) significantly increases the lifespan of GD mice that would otherwise have lived for a short time. Our data identify putative modifier genes that may be involved in determining GD severity, which might help elucidate phenotypic variability between patients with similar GD mutations.

Recent studies have demonstrated that the expression of sphingosine kinase 1, the enzyme that catalyses formation of the bioactive lipid, sphingosine 1-phosphate, is increased in lungs from patients with pulmonary arterial hypertension. In addition, Sk1(-/-) mice are protected from hypoxic-induced pulmonary arterial hypertension. Therefore, we assessed the effect of the sphingosine kinase 1 selective inhibitor, PF-543 and a sphingosine kinase 1/ceramide synthase inhibitor, RB-005 on pulmonary and cardiac remodelling in a mouse hypoxic model of pulmonary arterial hypertension. Administration of the potent sphingosine kinase 1 inhibitor, PF-543 in a mouse hypoxic model of pulmonary hypertension had no effect on vascular remodelling but reduced right ventricular hypertrophy. The latter was associated with a significant reduction in cardiomyocyte death. The protection involves a reduction in the expression of p53 (that promotes cardiomyocyte death) and an increase in the expression of anti-oxidant nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). In contrast, RB-005 lacked effects on right ventricular hypertrophy, suggesting that sphingosine kinase 1 inhibition might be nullified by concurrent inhibition of ceramide synthase. Therefore, our findings with PF-543 suggest an important role for sphingosine kinase 1 in the development of hypertrophy in pulmonary arterial hypertension.

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid βglucosidase (GCase) using the irreversible inhibitor conduritol Bepoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBEtreated mice and a genetic GD mouse model, the Gbaflox/flox;nestinCre mouse, with 120 of the 144 genes upregulated in CBEtreated mice also upregulated in Gbaflox/flox;nestinCre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for timeconsuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland.

Background: Neuroinflammation is a key phenomenon in the pathogenesis of many neurodegenerative diseases. Understanding the mechanisms by which brain inflammation is engaged and delineating the key players in the immune response and their contribution to brain pathology is of great importance for the identification of novel therapeutic targets for these devastating diseases. Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the GBA1 gene and is a significant risk factor for Parkinson's disease; in some forms of Gaucher disease, neuroinflammation is observed. Methods: An unbiased gene profile analysis was performed on a severely affected brain area of a neurological form of a Gaucher disease mouse at a pre-symptomatic stage; the mouse used for this study, the Gba(flox/flox); nestin-Cre mouse, was engineered such that GBA1 deficiency is restricted to cells of neuronal lineage, i.e., neurons and macroglia. Results: The 10 most up-regulated genes in the ventral posteromedial/posterolateral region of the thalamus were inflammatory genes, with the gene expression signature significantly enriched in interferon signaling genes. Interferon beta levels were elevated in neurons, and interferon-stimulated genes were elevated mainly in microglia. Interferon signaling pathways were elevated to a small extent in the brain of another lysosomal storage disease mouse model, Krabbe disease, but not in Niemann-Pick C or Sandhoff mouse brain. Ablation of the type I interferon receptor attenuated neuroinflammation but had no effect on GD mouse viability. Conclusions: Our results imply that the type I interferon response is involved in the development of nGD pathology, and possibly in other lysosomal storage diseases in which simple glycosphingolipids accumulate, and support the notion that interferon signaling pathways play a vital role in the sterile inflammation that often occurs during chronic neurodegenerative diseases in which neuroinflammation is present.

Glucosylceramide (GlcCer), one of the simplest glycosphingolipids, plays key roles in physiology and pathophysiology. It has been suggested that GlcCer modulates cellular events by forming specialized domains. In this study, we investigated the interplay between GlcCer and cholesterol (Chol), an important lipid involved in the formation of liquid-ordered (l_o) phases. Using fluorescence microscopy and spectroscopy, and dynamic and electrophoretic light scattering, we characterized the interaction between these lipids in different pH environments. A quantitative description of the phase behavior of the ternary unsaturated phospholipid/Chol/GlcCer mixture is presented. The results demonstrate coexistence between l_o and liquid-disordered (l_d) phases. However, the extent of l_o/l_d phase separation is sparse, mainly due to the ability of GlcCer to segregate into tightly packed gel domains. As a result, the phase diagram of these mixtures is characterized by an extensive three-phase coexistence region of fluid (l_d-phospholipid enriched)/l_o (Chol enriched)/gel (GlcCer enriched). Moreover, the results show that upon acidification, GlcCer solubility in the l_o phase is increased, leading to a larger l_o/l_d coexistence region. Quantitative analyses allowed us to determine the differences in the composition of the phases at neutral and acidic pH. These results predict the impact of GlcCer on domain formation and membrane organization in complex biological membranes, and provide a background for unraveling the relationship between the biophysical properties of GlcCer and its biological action.

Sphingolipids are ubiquitous components of eukaryotic cell membranes and have been widely studied over the past couple of decades due to the discovery of their involvement in a wide range of biological processes. This chapter focuses on the structure, biosynthesis and degradation of sphingolipids, with particular emphasis on recent studies documenting the huge numbers of individual sphingolipid structures that are based on variation in both the sphingolipid backbone and the head group. Attention is also paid to the roles of sphingolipids in signalling pathways with emphasis on sphingosine 1-phosphate and on ceramide. The significance of sphingolipids in human diseases is highlighted, as sphingolipids are now known to play a role in conditions ranging in diversity from inflammation and infection, to cancer, psychiatric disorders and neurological and immunological diseases. Thus, sphingolipids have been repositioned as fundamental players in the regulation of human health and disease.

K_Ca1.1 regulates smooth muscle contractility by modulating membrane potential, and age-associated changes in K_Ca1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect K_Ca1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2-null mice. The levels of C16- and C18-ceramides, sphinganine, sphingosine, and sphingosine 1-phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild-type and young CerS2-null mice. The altered SL composition upregulated K_Ca1.1 and increased K_Ca1.1 currents, while no change was observed in K_Ca1.1 channel activity. The upregulation of K_Ca1.1 impaired intracellular Ca²⁺ mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3-kinase, protein kinase C_ζ, c-Jun N-terminal kinases, and nuclear factor kappa-B were found to be involved in K_Ca1.1 upregulation. Our findings suggest that age-associated changes in SL composition or CerS2 ablation upregulate K_Ca1.1 via the phosphoinositide 3-kinase/protein kinase C_ζ/c-Jun N-terminal kinases/nuclear factor kappa-B-mediated pathway and impair Ca²⁺ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2-null mice exhibited similar effects to aged wild-type mice; therefore, CerS2-null mouse models may be utilized for investigating the pathogenesis of aging-associated motility disorders.

Tumor necrosis factor alpha (TNF alpha) is an inflammatory cytokine that plays an intimate role in septic shock. Injection of high levels of lipopolysaccharide induces septic shock and death in mice within 30 h, whereas ceramide synthase 2 (CerS2) null mice, defective in the synthesis of very-long acyl chain ceramides, die within similar to 10 h. The augmented rate of death of CerS2 null mice is due to elevated levels of TNF alpha secretion as a result of enhanced activity of TNF alpha-converting enzyme (TACE). We discuss the relationship between the sphingolipid acyl chain length and TACE activity and the relevance of this data to septic shock.

Background: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection ortreatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia. Methods: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and \fnarh mice under the influence of LCMV or poly(I:C). Results: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well. Conclusion: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed.

Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ∼15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes ceramides with very-long (C22-C24) chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullary tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ∼13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by 6 months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.

Introduction: Gaucher disease (GD) is an inherited metabolic disorder caused by mutations in the glucocerebrosidase (GBA1) gene. Although infusions of recombinant GBA ameliorate the systemic effects of GD, this therapy has no effect on the neurological manifestations. Patients with the neuronopathic forms of GD (nGD) are often severely disabled and die prematurely. The search for innovative drugs is thus urgent for the neuronopathic forms.Areas covered: Here we briefly summarize the available treatments for GD. We then review recent studies of the molecular pathogenesis of GD, which suggest new avenues for therapeutic development.Expert opinion: Existing treatments for GD are designed to target the primary consequence of the inborn defects of sphingolipid metabolism, that is, lysosomal accumulation of glucosylceramide (GlcCer). Here we suggest that targeting other pathways, such as those that are activated as a consequence of GlcCer accumulation, may also have salutary clinical effects irrespective of whether excess substrate persists. These pathways include those implicated in neuroinflammation, and specifically, receptor-interacting protein kinase-3 (RIP3) and related components of this pathway, which appear to play a vital role in the pathogenesis of nGD. Once available, inhibitors to components of the RIP kinase pathway will hopefully offer new therapeutic opportunities in GD.

Lysosomal storage diseases (LSDs) are mainly caused by the defective activity of lysosomal hydrolases. A sub-class of LSDs are the sphingolipidoses, in which sphingolipids accumulate intra-cellularly. We here discuss the role of innate immunity in the sphingolipidoses, and compare the pathways of activation in two classical sphingolipidoses, namely Gaucher disease and Sandhoff disease, and in Niemann-Pick C disease, in which the main storage material is cholesterol but sphingolipids also accumulate. We discuss the mechanisms leading to neuroinflammation, and the different pathways of neuroinflammation in the different diseases, and suggest that intervention in these pathways may be a useful therapeutic approach to address these devastating human diseases.

Background & Aims: Sortilin traffics newly synthesized molecules from the trans- Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). Methods: DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. Results: Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. Conclusions: Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.

Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [³H]-palmitate was also abrogated in the hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.

Inhibition of ceramide synthesis prevents diabetes, steatosis, and cardiovascular disease in rodents. Six different ceramide synthases (CerS) that differ in tissue distribution and substrate specificity account for the diversity in acyl-chain composition of distinct ceramide species. Haploinsufficiency for ceramide synthase 2 (CerS2), the dominant isoform in the liver that preferentially makes very-long-chain (C22/C24/C24:1) ceramides, led to compensatory increases in long-chain C16-ceramides and conferred susceptibility to diet-induced steatohepatitis and insulin resistance. Mechanistic studies revealed that these metabolic effects were likely due to impaired β-oxidation resulting from inactivation of electron transport chain components. Inhibiting global ceramide synthesis negated the effects of CerS2 haploinsufficiency in vivo, and increasing C16-ceramides by overexpressing CerS6 recapitulated the phenotype in isolated, primary hepatocytes. Collectively, these studies reveal that altering sphingolipid acylation patterns impacts hepatic steatosis and insulin sensitivity and identify CerS6 as a possible therapeutic target for treating metabolic diseases associated with obesity.

HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.

Cellular life depends on continuous transport of lipids and small molecules between mitochondria and the endomembrane system. Recently, endoplasmic reticulum-mitochondrial encounter structure(ERMES) was identified as an important yet nonessential contact for such transport. Using a high-contentscreen in yeast, we found a contact site, marked by Vam6/Vps39, between vacuoles (the yeast lysosomal compartment) and mitochondria, named vCLAMP (va. cuo. le and mitochondria patch). vCLAMP is enriched with ion and amino-acid transporters and has a role in lipid relay between the endomembrane system and mitochondria. Critically, we show that mitochondria are dependent on having one of two contact sites, ERMES or vCLAMP. The absence of one causes expansion of the other,and elimination of both is lethal. Identification of vCLAMP adds to our ability to understand the complexity of interorganellar crosstalk.

Ceramide is located at a key hub in the sphingolipid metabolic pathway and also acts as an important cellular signaling molecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amide bond, with the acyl chain varying in length and degree of saturation. The identification of a family of six mammalian ceramide synthases (CerS) that synthesize ceramide with distinct acyl chains, has led to significant advances in our understanding of ceramide biology, including further delineation of the role of ceramide in various pathophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from ceramide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in the diseases in which the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-Associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-Associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-Aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

Glucosylceramide (GlcCer) is a signaling lipid involved in the regulation of several cellular processes. It is present in different organelles, including the plasma membrane, Golgi apparatus, endoplasmic reticulum, and lysosomes. Accordingly, GlcCer is exposed to different pH environments in each organelle, which may lead to alterations in its properties and lateral organization and subsequent biological outcome. In this study, we addressed the effect of pH on the biophysical behavior of this lipid and other structurally related sphingolipids (SLs). Membranes composed of POPC (1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine) and C16-GlcCer, sphingomyelin, and different acyl chain ceramides were characterized by fluorescence spectroscopy, confocal microscopy, and surface pressure-area measurements under neutral and acidic conditions. The results show that changing the pH from 7.4 to 5.5 has a larger impact on C16-GlcCer-containing membranes compared to other SLs. In addition, acidification mainly affects the organization and packing properties of the GlcCer-enriched gel phase, suggesting that the interactions established by the glucose moiety, in the GlcCer molecule, are those most affected by the increase in the acidity. These results further highlight the role of GlcCer as a modulator of membrane biophysical properties and will possibly contribute to the understanding of its biological function in different organelles.

Gaucher's disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinson's disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.

Gaucher disease has recently received wide attention due to the unexpected discovery that it is a genetic risk factor forParkinson's disease.Gaucher disease is causedby the defective activity of the lysosomal enzyme, glucocerebrosidase (GCase; GBA1), resulting in intracellular accumulation of the glycosphingolipids, glucosylceramide and psychosine. The rare neuronopathic forms of GD (nGD) are characterized by profound neurological impairment and neuronal cell death. We have previously described the progression of neuropathological changes in amouse model of nGD.We now examine the relationship between glycosphingolipid accumulation and initiation of pathology at two pre-symptomatic stages of the disease in four different brain areas which display differential degrees of susceptibility to GCase deficiency. Liquid chromatography electrospray ionization tandem mass spectrometry demonstrated glucosylceramide and psychosine accumulation in nGD brains prior to the appearance of neuroinflammation, although only glucosylceramide accumulation correlated with neuroinflammation and neuron loss. Levels of other sphingolipids, including the pro-apoptotic lipid, ceramide, were mostly unaltered. Transmission electron microscopy revealed that glucosylceramide accumulation occurs in neurons, mostly in the form of membrane-delimited pseudo-tubules located near the nucleus. Highly disrupted glucosylceramide-storing cells, which are likely degenerating neurons containing massive inclusions, numerous autophagosomes and unique ultrastructural features, were also observed. Together, our results indicate that a certain level of neuronal glucosylceramide storage is required to trigger neuropathological changes in affected brain areas, while other brain areas containing similar glucosylceramide levels are unaltered, presumably because of intrinsic differences in neuronal properties, or in the neuronal environment, between various brain regions.

Objective: Ceramides are precursors of complex sphingolipids (SLs), which are important for normal functioning of both the developing and mature brain. Altered SL levels have been associated with many neurodegenerative disorders, including epilepsy, although few direct links have been identified between genes involved in SL metabolism and epilepsy. Methods: We used quantitative realtime PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. Results: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ~50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL composition was also reflected in a reduction in cholera toxin B immunofluorescence, indicating that membrane composition and function are altered. Interpretation: We propose that reduced levels of CERS2, and consequently diminished levels of ceramides and SLs containing very long-chain fatty acids, lead to development of PME.

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N - acyl chain composition differentially impact cell physiology, which may in part be due to specifi c alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N -acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs ). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fl uid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the signifi cance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specifi c biological actions.

Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.

Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol:C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol:C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn²⁺ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn²⁺ in Δspf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn ²⁺ we could observe concurrent reduction in many Mn ²⁺-related process in the ER lumen. Conversely, cytosolic Mn ²⁺-dependent processes were increased. Together, these data support a role for Spf1p in Mn²⁺ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn²⁺-dependent neurological disorders.

Unlike the synthesis of other membrane lipids, sphingolipid synthesis is compartmentalized between the endoplasmic reticulum and the Golgi apparatus. The initial steps of sphingolipid synthesis, from the activity of serine palmitoyltransferase through to dihydroceramide desaturase, take place in the endoplasmic reticulum, but the further metabolism of ceramide to sphingomyelin and complex glycosphingolipids takes place mostly in the Golgi apparatus. Studies over the last decade or so have revealed unexpected levels of complexity in the sphingolipid biosynthetic pathway, mainly due to either the promiscuity of some enzymes towards their substrates, or the tight selectivity of others towards specific substrates. We now discuss two enzymes in this pathway, namely serine palmitoyltransferase (SPT) and ceramide synthase (CerS), and one lipid transport protein, CERT. For SPT and CERT, significant structural information is available, and for CerS, significant information has recently been obtained that sheds light of the roles of the specific ceramide species that are produced by each of the CerS. We consider the mechanisms by which specificity is generated and speculate on the reasons that sphingolipid biosynthesis is so complex. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

The involvement of ceramide in death receptor-mediated apoptosis has been widely examined with most studies focusing on the role of ceramide generated from sphingomyelin hydrolysis. We now analyze the effect of the ceramide acyl chain length by studying tumor necrosis factor a receptor-1 (TNFR1)-mediated apoptosis in a ceramide synthase 2 (CerS2) null mouse, which cannot synthesize very-long acyl chain ceramides. CerS2 null mice were resistant to lipopolysaccharide/galactosaminemediated fulminant hepatic failure even though TNFα secretion from macrophages was unaffected. Cultured hepatocytes were also insensitive to TNFα-mediated apoptosis. In addition, in both liver and in hepatocytes, caspase activities were not elevated, consistent with inhibition of TNFR1 pro-apoptotic signaling. In contrast, Fas receptor activation resulted in the death of CerS2 null mice. Caspase activation was blocked because of the inability of CerS2 null mice to internalize the TNFR1; whereas Fc-TNFα was internalized to a perinuclear region in hepatocytes from wild-type mice, no internalization was detected in CerS2 null mice. Our results indicate that altering the acyl chain composition of sphingolipids inhibits TNFR1 internalization and inhibits selective pro-apoptotic downstream signaling for apoptosis.

Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as D-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.

The barrier function of the human epidermis is supposed to be governed by lipid composition and organization in the stratum corneum. Disorders of keratinization, namely ichthyoses, are typically associated with disturbed barrier activity. Using autozygosity mapping and exome sequencing, we have identified a homozygous missense mutation in CERS3 in patients with congenital ichthyosis characterized by collodion membranes at birth, generalized scaling of the skin, and mild erythroderma. We demonstrate that the mutation inactivates ceramide synthase 3 (CerS3), which is synthesized in skin and testis, in an assay of N-acylation with C26-CoA, both in patient keratinocytes and using recombinant mutant proteins. Moreover, we show a specific loss of ceramides with very long acyl chains from C26 up to C34 in terminally differentiating patient keratinocytes, which is in line with findings from a recent CerS3-deficient mouse model. Analysis of reconstructed patient skin reveals disturbance of epidermal differentiation with an earlier maturation and an impairment of epidermal barrier function. Our findings demonstrate that synthesis of very long chain ceramides by CerS3 is a crucial early step for the skin barrier formation and link disorders presenting with congenital ichthyosis to defects in sphingolipid metabolism and the epidermal lipid architecture.

Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC) are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs). These OxPLs are factors contributing to proatherogenic effects of oxidized LDLs (OxLDLs), including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer) which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C-16-, C-22-, and C-24.0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.

Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.

Background: Myristate is a novel potential substrate for sphingoid base synthesis. Results: Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases. Conclusion: d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles. Significance: This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection.

Gaucher disease is an inherited metabolic disease caused by the defective activity of the lysosomal enzyme, glucosylceramidase (GlcCerase), which is responsible for the last step in the degradation of complex glycosphingolipids. As a result, glucosylceramide (GlcCer) accumulates intracellularly. Little is known about the mechanisms by which GlcCer accumulation leads to Gaucher disease, particularly for the types of the disease in which severe neuropathology occurs. We now summarize recent advances in this area and in particular focus in the biochemical and cellular pathways that may cause neuronal defects. Most recent work has taken advantage of newly available mouse models, which mimic to a large extent human disease progression. Finally, we discuss observations of a genetic link between Gaucher disease and Parkinson's disease and discuss how this link has stimulated research into the basic biology of the previously underappreciated glycosphingolipid, GlcCer.

Glucosylceramide (GlcCer), a relevant intermediate in the pathways of glycosphingolipid metabolism, plays key roles in the regulation of cell physiology. The molecular mechanisms by which GlcCer regulates cellular processes are unknown, but might involve changes in membrane biophysical properties and formation of lipid domains. In the present study, fluorescence spectroscopy, confocal microscopy and surface pressure-area (π-A) measurements were used to characterize the effect of GlcCer on the biophysical properties of model membranes. We show that C16:0-GlcCer has a high tendency to segregate into highly ordered gel domains and to increase the order of the fluid phase. Monolayer studies support the aggregation propensity of C16:0-GlcCer. π-A isotherms of single C16:0-GlcCer indicate that bilayer domains, or crystal-like structures, coexist within monolayer domains at the air-water interface. Mixtures with POPC exhibit partial miscibility with expansion of the mean molecular areas relative to the additive behavior of the components. Moreover, C16:0-GlcCer promotes morphological alterations in lipid vesicles leading to formation of flexible tubule-like structures that protrude from the fluid region of the bilayer. These results support the hypothesis that alterations in membrane biophysical properties induced by GlcCer might be involved in its mechanism of action.

Scope: Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new fumonisin metabolites and to evaluate their cytotoxic potential. Methods and results: MS, molecular biology, and in vitro enzyme assays were used to investigate fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl fumonisins depends on the CerS isoform acylating them. The N-acyl fumonisins are more cytotoxic than the parent fumonisin B1. Conclusion: The identification of N-acyl fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of fumonisins.

Background: Ceramide synthase 2 null mice, which cannot synthesize very-long chain ceramides, display severe hepatopathy. Results: These mice have elevated sphinganine and altered N-acyl chain ceramides that disrupt mitochondrial function by modifying respiratory chain activity. Conclusion: Alteration of mitochondrial sphingolipids results in formation of reaction oxygen species in liver. Significance: Ceramides with defined acyl chains influence oxidative stress signaling pathways.

Sphingolipids are important structural components of cell membranes and act as critical regulators of cell function by modulating intracellular signaling pathways. Specific sphingolipids, such as ceramide, glucosylceramide, and ganglioside GM3, have been implicated in various aspects of insulin resistance, because they have been shown to modify several steps in the insulin signaling pathway, such as phosphorylation of either protein kinase B (Akt) or of the insulin receptor. We now explore the role of the ceramide acyl chain length in insulin signaling by using a ceramide synthase 2 (CerS2) null mouse, which is unable to synthesize very long acyl chain (C22-C24) ceramides. CerS2 null mice exhibited glucose intolerance despite normal insulin secretion from the pancreas. Both insulin receptor and Akt phosphorylation were abrogated in liver, but not in adipose tissue or in skeletal muscle. The lack of insulin receptor phosphorylation in liver correlated with its inability to translocate into detergent-resistant membranes (DRMs). Moreover, DRMs in CerS2 null mice displayed properties significantly different from those in wild-type mice, suggesting that the altered sphingolipid acyl chain length directly affects insulin receptor translocation and subsequent signaling. Conclusion: We conclude that the sphingolipid acyl chain composition of liver regulates insulin signaling by modifying insulin receptor translocation into membrane microdomains.

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM.We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin- 3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cerrich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.

PURPOSE. CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina. METHODS. CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay. RESULTS. We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cationbinding domains (EF-hands 2-4). CONCLUSIONS. This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca²⁺ signaling, providing important clues for future research required in this direction.

Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS) that each use acyl-CoAs of defined chain length for N-acylation of the sphingoid long chain base. CerS mRNA expression and enzymatic activity do not always correlate with the sphingolipid acyl chain composition of a particular tissue, suggesting post-translational mechanism(s) of regulation of CerS activity. We now demonstrate that CerS activity can be modulated by dimer formation. Under suitable conditions, high M_r CerS complexes can be detected by Western blotting, and various CerS co-immunoprecipitate. CerS5 activity is inhibited in a dominant-negative fashion by co-expression with catalytically inactive CerS5, and CerS2 activity is enhanced by co-expression with a catalytically active form of CerS5 or CerS6. In a constitutive heterodimer comprising CerS5 and CerS2, the activity of CerS2 depends on the catalytic activity of CerS5. Finally, CerS dimers are formed upon rapid stimulation of ceramide synthesis by curcumin. Together, these data demonstrate that ceramide synthesis can be regulated by the formation of CerS dimers and suggest a novel way to generate the acyl chain composition of ceramide (and downstream sphingolipids), which may depend on the interaction of CerS with each other.

Gaucher's disease, the most common lysosomal storage disorder, is caused by the defective activity of glucocerebrosidase, the lysosomal hydrolase that degrades glucosylceramide. The neuronopathic forms of Gaucher's disease are characterized by severe neuronal loss, astrocytosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. In the current study, we delineate the role of neuroinflammation in the pathogenesis of neuronopathic Gaucher's disease and show significant changes in levels of inflammatory mediators in the brain of a neuronopathic Gaucher's disease mouse model. Levels of messenger RNA expression of interleukin-1β, tumour necrosis factor-α, tumour necrosis factor-α receptor, macrophage colony-stimulating factor and transforming growth factor-β were elevated by up to ∼30-fold, with the time-course of the increase correlating with the progression of disease severity. The most significant elevation was detected for the chemokines CCL2, CCL3 and CCL5. Blood-brain barrier disruption was also evident in mice with neuronopathic Gaucher's disease. Finally, extensive elevation of nitrotyrosine, a hallmark of peroxynitrite (ONOO ^-) formation, was observed, consistent with oxidative damage caused by macrophage/microglia activation. Together, our results suggest a cytotoxic role for activated microglia in neuronopathic Gaucher's disease. We suggest that once a critical threshold of glucosylceramide storage is reached in neurons, a signalling cascade is triggered that activates microglia, which in turn releases inflammatory cytokines that amplify the inflammatory response, contributing to neuronal death.

In previous studies we showed that the replication of Cryptococcus neoformans in the lung environment is controlled by the glucosylceramide (GlcCer) synthase gene (GCS1), which synthesizes the membrane sphingolipid GlcCer from the C9-methyl ceramide. Here, we studied the effect of the mutation of the sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position 9 of the sphingosine backbone of ceramide. The C.neoformansΔsmt1 mutant does not make C9-methyl ceramide and, thus, any methylated GlcCer. However, it accumulates demethylated ceramide and demethylated GlcCer. The Δsmt1 mutant loses more than 80% of its virulence compared with the wild type and the reconstituted strain. Interestingly, growth of C.neoformansΔsmt1 in the lung was decreased and C.neoformans cells were contained in lung granulomas, which significantly reduced the rate of their dissemination to the brain reducing the onset of meningoencephalitis. Thus, using fluorescent spectroscopy and atomic force microscopy we compared the wild type and Δsmt1 mutant and found that the altered membrane composition and GlcCer structure affects fungal membrane rigidity, suggesting that specific sphingolipid structures are required for proper fungal membrane organization and integrity. Therefore, we propose that the physical structure of the plasma membrane imparted by specific classes of sphingolipids represents a critical factor for the ability of the fungus to establish virulence.

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22-C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by the defective activity of the lysosomal hydrolase glucocerebrosidase, which is encoded by the GBA gene. Generation of animal models that faithfully recapitulate the three clinical subtypes of GD has proved to be more of a challenge than first anticipated. The first mouse to be produced died within hours after birth owing to skin permeability problems, and mice with point mutations in Gba did not display symptoms correlating with human disease and also died soon after birth. Recently, conditional knockout mice that mimic some features of the human disease have become available. Here, we review the contribution of all currently available animal models to examining pathological pathways underlying GD and to testing the efficacy of new treatment modalities, and propose a number of criteria for the generation of more appropriate animal models of GD.

Ceramide is an important bioactive sphingolipid involved in a variety of biological processes. The mechanisms by which ceramide regulates biological events are not fully understood, but may involve alterations in the biophysical properties of membranes. We now examine the properties of ceramide with different acyl chains including long chain (C16- and C18-), very long chain (C24-) and unsaturated (C18:1- and C24:1-) ceramides, in phosphatidylcholine model membranes. Our results show that i) saturated ceramides have a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (C24:1-) or no (C18:1-) ability to form gel domains at 37 °C; ii) differences between saturated species are smaller and are mainly related to the morphology and size of the gel domains, and iii) very long chain ceramides form tubular structures likely due to their ability to form interdigitated phases. These results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology.

Recently, two studies were published that examined the structure of the acid-β-glucosidase N370S mutant, the most common mutant that causes Gaucher disease. One study used the experimental tool of X-ray crystallography, and the other utilized molecular dynamics (MD). The two studies reinforced each other through the similarities in their findings, but each approach also added some unique information. Both studies report that the conformation of active site loop 3 changes, due to an altered hydrogen bonding network; however, the MD study produced additional data concerning the flexibility of loop 1 and the catalytic residues that are not observed in the other study.

Sphingolipids (SLs) act as signaling molecules and as structural components in both neuronal cells and myelin. We now characterize the biochemical, histological, and behavioral abnormalities in the brain of a mouse lacking very long acyl (C22-C24) chain SLs. This mouse, which is defective in the ability to synthesize C22-C24-SLs due to ablation of ceramide synthase 2, has reduced levels of galactosylceramide (GalCer), a major component of myelin, and in particular reduced levels of non-hydroxy-C22-C24-GalCer and 2-hydroxy-C22-C24- GalCer. Noteworthy brain lesions develop with a time course consistent with a vital role for C22-C24-GalCer in myelin stability. Myelin degeneration and detachment was observed as was abnormal motor behavior originating from a subcortical region. Additional abnormalities included bilateral and symmetrical vacuolization and gliosis in specific brain areas, which corresponded to some extent to the pattern of ceramide synthase 2 expression, with astrogliosis considerably more pronounced than microglial activation. Unexpectedly, unidentified storage materials were detected in lysosomes of astrocytes, reminiscent of the accumulation that occurs in lysosomal storage disorders. Together, our data demonstrate a key role in the brain for SLs containing very long acyl chains and in particular GalCer with a reduction in their levels leading to distinctive morphological abnormalities in defined brain regions.

Parkinson's disease is associated with mutations in the glucocerebrosidase gene, which result in the enzyme deficiency causing Gaucher disease, the most common lysosomal storage disorder. We have performed an exhaustive literature search and found that additional lysosomal storage disorders might be associated with Parkinson's disease, based on case reports, the appearance of pathological features such as alpha-synuclein deposits in the brain, and substantia nigra pathology. Our findings suggest that the search for biochemical and cellular pathways that link Parkinson's disease with lysosomal storage disorders should not be limited exclusively to changes that occur in Gaucher disease, such as changes in glucocerebrosidase activity or in glucosylceramide levels, but rather include changes that might be common to a wide variety of lysosomal storage disorders. Moreover, we propose that additional genetic, epidemiological, and clinical studies should be performed to check the precise incidence of mutations in genes encoding lysosomal proteins in patients displaying Parkinson's symptoms. (C) 2011 Movement Disorder Society

Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40. mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.

Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of recombinant human acid β-glucosidase (β-glucocerebrosidase, GlcCerase) with amphiphilic bicyclic nojirimycin analogues of the sp²-iminosugar type. Attempts to co-crystallize GlcCerase with 5-N,6-O-[N-(n-octyl)iminomethylidene]nojirimycin (NOI-NJ) or with 5-N,6-S-[N-(n-octyl)iminomethylidene]-6-thionojirimycin (6S-NOI-NJ), two potent inhibitors of the enzyme with promising pharmacological chaperone activity for several Gaucher disease-associated mutations, were unsuccessful probably due to the formation of aggregates that increase the heterogeneity of the sample and affect nucleation and growth of crystals. Cyclomaltoheptaose (β-cyclodextrin, βCD) efficiently captures NOI-NJ and 6S-NOI-NJ in aqueous media to form inclusion complexes in which the lipophilic tail is accommodated in the hydrophobic cavity of the cyclooligosaccharide. The dissociation constant of the complex of the amphiphilic sp²-iminosugars with βCD is two orders of magnitude higher than that of the corresponding complex with GlcCerase, allowing the efficient transfer of the inhibitor from the βCD cavity to the GlcCerase active site. Enzyme-inhibitor complexes suitable for X-ray analysis were thus grown in the presence of βCD. In contrast to what was previously observed for the complex of GlcCerase with the more basic derivative, 6-amino-6-deoxy-5-N,6-N-[N-(n-octyl)iminomethylidene]nojirimycin (6N-NOI-NJ), the β-anomers of both NOI-NJ and 6S-NOI-NJ were seen in the active site, even though the α-anomer was exclusively detected both in aqueous solution and in the corresponding βCD:sp²-iminosugar complexes. Our results further suggest that cyclodextrin derivatives might serve as suitable delivery systems of amphiphilic glycosidase inhibitors in a biomedical context.

Gaucher disease (GD), the most common lysosomal storage disorder, is caused by a deficiency in the lysosomal enzyme glucocerebrosidase (GlcCerase), which results in intracellular accumulation of glucosylceramide (GlcCer). The rare neuronopathic forms of GD are characterized by profound neurological impairment and neuronal cell death, but little is known about the neuropathological changes that underlie these events. We now systematically examine the onset and progression of various neuropathological changes (including microglial activation, astrogliosis and neuron loss) in a mouse model of neuronopathic GD, and document the brain areas that are first affected, which may reflect vulnerability of these areas to GlcCerase deficiency. We also identify neuropathological changes in several brain areas and pathways, such as the substantia nigra reticulata, reticulotegmental nucleus of the pons, cochlear nucleus and the somatosensory system, which could be responsible for some of the neurological manifestations of the human disease. In addition, we establish that microglial activation and astrogliosis are spatially and temporally correlated with selective neuron loss.

Gaucher disease is caused by the defective activity of the lysosomal hydrolase, glucosylceramidase. Although the x-ray structure of wild type glucosylceramidase has been resolved, little is known about the structural features of any of the >200 mutations. Various treatments for Gaucher disease are available, including enzyme replacement and chaperone therapies. The latter involves binding of competitive inhibitors at the active site to enable correct folding and transport of the mutant enzyme to the lysosome. We now use molecular dynamics, a set of structural analysis tools, and several statistical methods to determine the flexible behavior of the N370S Gaucher mutant at various pH values, with and without binding the chaperone, N-butyl- deoxynojirimycin. We focus on the effect of the chaperone on the whole protein, on the active site, and on three important structural loops, and we demonstrate how the chaperone modifies the behavior of N370S in such a way that it becomes more active at lysosomal pH. Our results suggest a mechanism whereby the binding of N-butyl-deoxynojirimycin helps target correctly folded glucosylceramidase to the lysosome, contributes to binding with saposin C, and explains the initiation of the substrate-enzyme complex. Such analysis provides a new framework for determination of the structure of other Gaucher disease mutants and suggests new approaches for rational drug design.

Ceramide forms the backbone of all complex sphingolipids and has been the focus of considerable attention in the past few years due to the discovery that ceramide plays vital roles as an intracellular messenger. Ceramide, which consists of a sphingoid long chain base to which a fatty acid is N-acylated, is synthesized in mammals by a family of ceramide synthases (CerS), each of which uses a restricted subset of fatty acyl CoAs for N-acylation. Sphingolipids are found at high levels in nervous tissue, where they perform a variety of important functions in both the adult and the maturing brain. We now review what is known about the role of the acyl chain composition of ceramides and sphingolipids in normal brain development and in neurological diseases. Specifically, we attempt to integrate the information that is available about CerS expression and activity in the brain with the changes in the acyl chain composition of ceramide and complex sphingolipids in a number of neurodegenerative diseases and conditions, such as metachromatic leukodystrophy, neuronal ceroid lipofuscinoses, HIV infection, aging, Alzheimer's disease, ischemia, and epilepsy. We conclude that understanding the direct relationship between the CerS proteins and neurological conditions will be of great importance for delineating the precise roles of sphingolipids in the brain and is likely to be the subject of intense research activity in the years ahead.

The neuronopathic forms of the human inherited metabolic disorder, Gaucher disease (GD), are characterized by severe neuronal loss, astrogliosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. Recently, a mouse model of neuronopathic GD was generated in which glucocerebrosidase deficiency is limited to neural and glial progenitor cells. We now show significant changes in the levels and in the distribution of cathepsins in the brain of this mouse model. Cathepsin mRNA expression was significantly elevated by up to ~10-fold, with the time-course of the increase correlating with the progression of disease severity. Cathepsin activity and protein levels were also elevated. Significant changes in cathepsin D distribution in the brain were detected, with cathepsin D elevated in areas where neuronal loss, astrogliosis and microgliosis were observed, such as in layer V of the cerebral cortex, the lateral globus pallidus and in various nuclei in the thalamus, brain regions known to be affected in the disease. Cathepsin D elevation was greatest in microglia and also noticeable in astrocytes. The distribution of cathepsin D was altered in neurons in a manner consistent with its release from the lysosome to the cytosol. Remarkably, ibubrofen treatment significantly reduced cathepsin D mRNA levels in the cortex of Gaucher mice. Finally, cathepsin levels were also altered in mouse models of a number of other sphingolipidoses. Our findings suggest the involvement of cathepsins in the neuropathology of neuronal forms of GD and of other lysosomal storage diseases, and are consistent with a crucial role for reactive microglia in neuronal degeneration in these diseases.

Lysosomal storage diseases (LSDs), of which about 50 are known, are caused by the defective activity of lysosomal proteins, resulting in accumulation of unmetabolized substrates. As a result, a variety of pathogenic cascades are activated such as altered calcium homeostasis, oxidative stress, inflammation, altered lipid trafficking, autophagy, endoplasmic reticulum stress, and autoimmune responses. Some of these pathways are common to many LSDs, whereas others are only altered in a subset of LSDs.Wenow review how these cascades impact upon LSD pathology and suggest how intervention in the pathways may lead to novel therapeutic approaches.

In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their structure and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases. (C) 2010 IUBMB IUBMB Life, 62(5): 347-356, 2010

In mammals, ceramide, a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), each of which synthesizes ceramides with distinct acyl chain lengths. There are a number of common biochemical features between the CerS, such as their catalytic mechanism, and their structure and intracellular localization. Different CerS also display remarkable differences in their biological properties, with each of them playing distinct roles in processes as diverse as cancer and tumor suppression, in the response to chemotherapeutic drugs, in apoptosis, and in neurodegenerative diseases.

We have generated a mouse that cannot synthesize very long acyl chain (C22-C24) ceramides (Pewzner-Jung, Y., Park, H., Laviad, E. L., Silva, L. C., Lahiri, S., Stiban, J., Erez-Roman, R., Brugger, B., Sachsenheimer, T., Wieland, F. T., Prieto, M., Merrill, A. H., and Futerman, A. H. (2010) J. Biol. Chem. 285, 10902-10910) due to ablation of ceramide synthase 2 (CerS2). As a result, significant changes were observed in the sphingolipid profile of livers from these mice, including elevated C16-ceramide and sphinganine levels. We now examine the functional consequences of these changes. CerS2 null mice develop severe nonzonal hepatopathy from about 30 days of age, the age at which CerS2 expression peaks in wild type mice, and display increased rates of hepatocyte apoptosis and proliferation. In older mice there is extensive and pronounced hepatocellular anisocytosis with widespread formation of nodules of regenerative hepatocellular hyperplasia. Progressive hepatomegaly and noninvasive hepatocellular carcinoma are also seen from -10 months of age. Even though CerS2 is found at equally high mRNA levels in kidney and liver, there are no changes in renal function and no pathological changes in the kidney. High throughput analysis of RNA expression in liver revealed up-regulation of genes associated with cell cycle regulation, protein transport, cell-cell interactions and apoptosis, and down-regulation of genes associated with intermediary metabolism, such as lipid and steroid metabolism, adipocyte signaling, and amino acid metabolism. In addition, levels of the cell cycle regulator, the cyclin dependent-kinase inhibitor p21^WAF1/CIP1, were highly elevated, which occurs by at least two mechanisms, one of which may involve p53. We propose a functional rationale for the synthesis of sphingolipids with very long acyl chains in liver homeostasis and in cell physiology.

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22-C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

Lysosomal storage disorders are caused by the defective activity of lysosomal proteins, which results in the lysosomal accumulation of undegraded metabolites. Sphingolipid storage disorders are a subgroup of lysosomal storage disorders, in which unmetabolized sphingolipids and glycosphingolipids accumulate. Sphingolipid storage disorders are normally associated with devastating neurodegeneration and death at an early age. Despite years of study of the genetic and molecular bases of sphingolipid storage disorders, little is known about the events that lead from lipid accumulation to pathology. Research over the past few years has demonstrated that sphingolipid storage can result in multiple direct or indirect effects on various cellular compartments and on biochemical pathways. This article offers a guided tour through the cellular scenes that may be of relevance to sphingolipid storage disorder pathogenesis and to the development of new therapeutic approaches.

The ceramide synthase (CerS) enzymes are key regulators of ceramide homeostasis. CerS1 is central to regulating C18 ceramide which has been shown to be important in cancer and the response to chemotherapeutic drugs. Previous work indicated that some drugs induced a novel and specific translocation of CerS1 from the endoplasmic reticulum to the Golgi apparatus. We now show that diverse stresses such as UV light, DTT, as well as drugs with different mechanisms of action induce CerS1 translocation. The stresses cause a specific cleavage of the CerS1 enzyme, and the cleavage is dependent on the action of the proteasome. Inhibition of proteasome function inhibits stress-induced CerS1 translocation, indicating that this proteolytic cleavage precedes the translocation. Modulation of protein kinase C activity shows that it plays a central role in regulating CerS1 translocation. Analysis of the C-terminus of the CerS1 protein shows that several KxKxx motifs are not involved in regulating stress induced translocation. The study suggests that diverse stresses initiate responses through different signaling pathways, which ultimately converge to regulate CerS1 localization. The data provide an increasingly detailed understanding of the regulation of this important enzyme in normal and stressed cells and support the idea that it is uniquely regulated with respect to the other CerS enzymes.

Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

Ceramide synthases (CerS) are integral membrane proteins of the endoplasmic reticulum. Six mammalian CerS have been described, with each utilizing fatty acyl CoAs of relatively defined chain lengths for N-acylation of t N he sphingoid long chain base. In this chapter, we review the main functional features of the CerS proteins, discuss their fatty acid specificity, kinetics, tissue distribution and mode of inhibition, as well as possible posttranslational modifications. We then address the reason that mammals contain six distinct CerS, whereas most other enzymes in the sphingolipid biosynthetic pathway only occur in one or two isoforms. Finally, we discuss the putative roles of CerS and the ceramide derived from the CerS, in signaling pathways and in development of disease.

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated T™ human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K_m and V_max) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Keywords: Biochemistry & Molecular Biology; Biophysics

Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis.

A number of studies have shown altered cytokine levels in serum from Gaucher disease patients, including changes in levels of the anti-inflammatory cytokine, interleukin-10 (IL-10). However, the source of IL-10, or the mechanisms leading to changes in IL-10 serum levels are not known. We now show that mouse macrophages treated with an active site-directed inhibitor of glucocerebrosidase, or macrophages from a mouse model of Gaucher disease, the L444P mouse, release significantly less IL-10 than their untreated counterparts, but that TNFα release is unaffected. These changes are due to reduced transcription of IL-10 mRNA in macrophages. The reduction in IL-10 secretion observed in animal models of Gaucher disease macrophages may be of relevance to explain the increase in inflammation that is often observed in Gaucher disease.

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nM to 5 μM) but not low (

Plasma cells (PCs) are terminally differentiated B lymphocytes responsible for the synthesis and secretion of Igs. The differentiation of B cells into PCs involves a remarkable expansion of both lipid and protein components of the endoplasmic reticulum. Despite their importance in many signal transduction pathways, the role of ceramides, and of complex sphingolipids that are derived from ceramide, in PC differentiation has never been directly studied. To assess their putative role in PC differentiation, we blocked ceramide synthesis with fumonisin B1, a specific inhibitor of ceramide synthase. Under fumonisin B1 treatment, N-linked glycosylation was severely impaired in LPS-activated, but not in naive, B cells. We also show that ceramide synthesis is strongly induced by XBP-1 (X box-binding protein-1). In the absence of ceramide synthesis, ER expansion was dramatically diminished. Our results underscore ceramide biosynthesis as a key metabolic pathway in the process of PC differentiation and reveal a previously unknown functional link between sphingolipids and N-linked glycosylation in PCs.

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay among SMase activity, initial membrane properties (i.e., phase behavior and lipid lateral organization) and lipid composition, and the amount of product (Cer) generated, and how it modulates membrane properties were studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that 1), the enzyme activity and extent of hydrolysis are strongly dependent on membrane physical properties but not on substrate content, and are higher in raft-like mixtures, i.e., mixtures with liquid-disordered/liquid-ordered phase separation; and 2), Cer-induced alterations are also dependent on membrane composition, specifically the cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small (∼8.5 nm) Cer/SM-gel domains. With increasing Chol, the ability of Cer to recruit SM and form gel domains strongly decreases. In the high-Chol range, a Chol-enriched/SM-depleted liquid-ordered phase predominates. Together, these data suggest that in biological membranes, Chol in particular and raft domains in general play an important role in modulating SMase activity and regulating membrane physical properties by restraining Cer-induced alterations.

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase, resulting in intracellular accumulation of glucosylceramide (GlcCer). Neuronopathic forms, which comprise only a small percent of GD patients, are characterized by neurological impairment and neuronal cell death. Little is known about the pathways leading from GlcCer accumulation to neuronal death or dysfunction but defective calcium homeostasis appears to be one of the pathways involved. Recently, endoplasmic reticulum stress together with activation of the unfolded protein response (UPR) has been suggested to play a key role in cell death in neuronopathic forms of GD, and moreover, the UPR was proposed to be a common mediator of apoptosis in LSDs (Wei et al. (2008) Hum. Mol. Genet. 17, 469-477). We now systematically examine whether the UPR is activated in neuronal forms of GD using a selection of neuronal disease models and a combination of western blotting and semi-quantitative and quantitative real-time polymerase chain reaction. We do not find any changes in either protein or mRNA levels of a number of typical UPR markers including BiP, CHOP, XBP1, Herp and GRP58, in either cultured Gaucher neurons or astrocytes, or in brain regions from mouse models, even at late symptomatic stages. We conclude that the proposition that the UPR is a common mediator for apoptosis in all neurodegenerative LSDs needs to be re-evaluated.

Sphingolipids are important constituents of biological membranes. Ceramide, the major metabolite of this family, is involved in many cellular processes, ranging from differentiation to senescence and apoptosis. Ceramide is an amphipathic molecule with a small head group that allows it to be more promiscuous within membranes than other lipids. Ceramide has a strong ability to change the physical properties of membranes through the formation of ceramide-rich domains, whose physical and morphological characteristics can be studied by a variety of biophysical techniques. While the existence of lipid domains is widely accepted, data from the literature is not consistent concerning many of their properties. We now discuss the biophysical and biological significance of two types of membrane domains (lipid rafts and ceramide-domains). In addition, we discuss other properties of ceramide, such as its ability to permeabilize the outer membrane of mitochondria. Finally, we attempt to integrate these various issues from biochemical and biophysical perspectives.

In mammalian cells, glucosylceramide (GlcCer), the simplest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid β-glucosidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantly decreased owing to one of approximately 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme replacement therapy (ERT), in which patients are given intravenous injections of recombinant human GlcCerase; the Genzyme product Cerezyme® has been used clinically for more than 15 years and is administered to approximately 4000 patients worldwide. Here we review the crystal structure of Cerezyme® and other recombinant forms of GlcCerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme®, which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease.

Ceramide is an important lipid signaling molecule and a key intermediate in sphingolipid biosynthesis. Recent studies have implied a previously unappreciated role for the ceramide N-acyl chain length, in as much as ceramides containing specific fatty acids appear to play defined roles in cell physiology. The discovery of a family of mammalian ceramide synthases (CerS), each of which utilizes a restricted subset of acyl-CoAs for ceramide synthesis, strengthens this notion. We now report the characterization of mammalian CerS2. qPCR analysis reveals that CerS2 mRNA is found at the highest level of all CerS and has the broadest tissue distribution. CerS2 has a remarkable acyl-CoA specificity, showing no activity using C16:0-CoA and very low activity using C18:0, rather utilizing longer acyl-chain CoAs (C20-C26) for ceramide synthesis. There is a good correlation between CerS2 mRNA levels and levels of ceramide and sphingomyelin containing long acyl chains, at least in tissues where CerS2 mRNA is expressed at high levels. Interestingly, the activity of CerS2 can be regulated by another bioactive sphingolipid, sphingosine 1-phosphate (S1P), via interaction of S1P with two residues that are part of an S1P receptor-like motif found only in CerS2. These findings provide insight into the biochemical basis for the ceramide N-acyl chain composition of cells, and also reveal a novel and potentially important interplay between two bioactive sphingolipids that could be relevant to the regulation of sphingolipid metabolism and the opposing functions that these lipids play in signaling pathways.

We previously observed that gangliosides GM2, GM1, and GM3 inhibit Ca ²⁺-uptake via the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) in neurons and in brain microsomes. We now systematically examine the effect of various gangliosides and their analogs on Ca²⁺-uptake via SERCA and demonstrate that an exposed carboxyl group on the ganglioside sialic acid residue is required for inhibition. Thus, asialo-GM2 and asialo-GM1 have no inhibitory effect, and modifications of the carboxyl group of GM1 and GM2 into a hydroxymethyl residue (CH₂OH), a methyl ester (COOCH₃) or a taurine-conjugated amide (CONHCH₂CH₂SO₃H) drastically diminish their inhibitory activities. We also demonstrate that the saccharides must be attached to a ceramide backbone in order to inhibit SERCA as the ceramide-free ganglioside saccharides only inhibit SERCA to a minimal extent. Finally, we attempted to use the ceramide-free ganglioside saccharides to antagonize the effects of the gangliosides on SERCA; although some reversal was observed, the inhibitory effects of the gangliosides were not completely abolished.

Microbatch crystallization under oil is a powerful procedure for obtaining protein crystals. Using this method, aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil, with a concomitant increase in the concentrations of both protein and precipitant until the nucleation point is reached. A technique is presented for regulating the rate of water evaporation, which permits fine tuning of the crystallization conditions as well as preventing complete desiccation of the drops in the microbatch crystallization trays.

A monoclonal antibody raised against mixed monolayers of 60:40 mol% cholesterol/C16-ceramide of known structure was used to label cholesterol/ceramide-rich domains in cell membranes. The antibody, Cer-Chol 405F specifically recognizes the mixed crystalline and homogeneous phase in monolayers, but it does not interact with either of the components separately. It interacts differently with mixed monolayers that contain ceramides of different acyl chain length. When used on cells, the antibody labeling is sensitive to changes in cholesterol and ceramide levels, as well as to over-expression of specific ceramides; this is in agreement with the results that were obtained on lipid monolayers. This represents a proof of concept of the applicability of a new approach to the structural characterization of lipid microdomains in cell membranes. The approach consists of raising antibodies that recognize specific structural organizations of lipids in artificial mixtures, characterizing the antibody/ordered domain complexes in vitro, and subsequently using them to detect the presence of the same (or similar) domains in cell membranes.

Keywords: Biochemistry & Molecular Biology; Genetics & Heredity; Medicine, Research & Experimental

Ceramide is a key metabolite in the pathway of sphingolipid biosynthesis. In mammals, ceramide is synthesized by N-acylation of a sphingoid long-chain base by a family of ceramide synthases (CerS), each of which displays a high specificity towards acyl CoAs of different chain lengths. We now optimize a previously-described assay for measuring CerS activity for use upon over-expression of mammalian CerS, and using these conditions, establish the K_m value of each CerS towards sphinganine. Remarkably, the K_m values towards sphinganine are all similar, ranging from 2 to 5 μM, even for CerS proteins that are able to use more than one acyl CoA for ceramide synthesis (i.e. CerS4). The availability of this assay will permit further accurate characterization of the kinetic parameters of mammalian CerS proteins.

With its role in intracellular protein transport already known, the FAPP2 protein has now also been implicated in lipid transfer and synthesis. What is more, these two FAPP2-mediated events seem to be linked.

Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2 Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars.

Ceramide is synthesized in mammals by a family of ceramide synthases (CerS) each of which uses a relatively restricted set of fatty acyl-CoAs for N-acylation of the sphingoid long chain base (Pewzner-Jung, Y., Ben-Dor, S., and Futerman, A. H. (2006) J. Biol. Chem. 281, 25001-25005). CerS are characterized by two functional domains, the Tram-Lag-CLN8 (TLC) domain and the homeobox (Hox) domain, which is found in all mammalian CerS except CerS1. We now demonstrate that the majority of the Hox domain is not required for CerS activity since its deletion in CerS5 does not affect activity. Subsequently, we define a highly conserved new motif of 12 amino acid residues that flanks the Hox and TLC domains but is not part of the TLC domain, which is essential for CerS5 and CerS6 activity. Two positively charged residues in this domain, one of which is conserved in all putative CerS in all organisms, are essential for activity since site-directed mutagenesis of either (Lys-134 and Lys-140 in CerS5) results in an ∼50% loss of activity, whereas mutation of both leads to a complete loss of activity. Because this region is conserved across species, we propose that it plays a previously unidentified and essential role in CerS activity and can be used as a new motif to define Hox domain-containing CerS.

Sphingolipids and glycosphingolipids are emerging as major players in many facets of cell physiology and pathophysiology. We now present an overview of sphingolipid biochemistry and physiology, followed by a brief presentation of recent advances in translational research related to sphingolipids. In discussing sphingolipid biochemistry, we focus on the structure of sphingolipids, and their biosynthetic pathways - the recent identification of most of the enzymes in this pathway has led to significant advances and better characterization of a number of the biosynthetic steps, and the relationship between them. We then discuss some roles of sphingolipids in cell physiology, particularly those of ceramide and sphingosine-1-phosphate, and mention current views about how these lipids act in signal transduction pathways. We end with a discussion of sphingolipids and glycosphingolipids in the etiology and pathology of a number of diseases, such as cancer, immunity, cystic fibrosis, emphysema, diabetes, and sepsis, areas in which sphingolipids are beginning to take a central position, even though many of the details remain to be elucidated.

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme®) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme®. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme®. prGCD also displays a level of biological activity similar to that of Cerezyme® produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.

We have recently shown that phosphatidylcholine (PC) metabolism is altered in a macrophage model of Gaucher disease. We now demonstrate that treatment of macrophages with conduritol-B-epoxide (CBE), a glucocerebrosidase inhibitor, results in elevated activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in the pathway of PC biosynthesis. Furthermore, we provide evidence for a role for CCT in Gaucher macrophage growth by using macrophages derived from a genetically modified mouse which lacks a specific CCT isoform, CCTα, in macrophages. Upon CBE-treatment, macrophage size, analyzed by microscopy and by FACS, was significantly increased in macrophages from control mice, but did not increase, or increased to a much lower extent, in CCTα-/- macrophages. Together, these results suggest that the increase in PC biosynthesis is mediated via CCTα, and suggests a possible role for macrophage CCTα in Gaucher disease pathology.

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1- phosphate (S-1-P) in regulating sensitivity to cisplatin [cis- diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

The intracellular routes of sphingolipid trafficking are related to the compartmentalized nature of sphingolipid metabolism, with synthesis beginning in the endoplasmic reticulum, continuing in the Golgi apparatus, and degradation occurring mainly in lysosomes. Whereas bulk sphingolipid transport between subcellular organelles occurs primarily via vesicle-mediated pathways, evidence is accumulating that sphingolipids are found in subcellular organelles that are not connected to each other by vesicular flow, implying additional trafficking routes. After discussing how sphingolipids are transported through the secretory pathway, I will review evidence for sphingolipid metabolism in organelles such as the mitochondria, and then discuss how this impacts upon our current understanding of the regulation of intracellular sphingolipid transport.

Gaucher disease is caused by mutations in the gene encoding acid-beta-glucosidase. A recombinant form of this enzyme, Cerezyme((R)), is used to treat Gaucher disease patients by 'enzyme- replacement therapy'. Crystals of Cerezyme((R)) after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 angstrom resolution [Dvir et al. (2003), EMBO Rep. 4, 704 - 709]. The crystal structure of unmodified Cerezyme1 is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme((R)) is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-beta-glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes.

Although diseases in the pathway of sphingolipid degradation have been known for decades, the first disease in the biosynthetic pathway was only reported in 2004, when a form of infantile-onset symptomatic epilepsy was described as a genetic defect in GM3 synthase. Presumably other diseases in the sphingolipid biosynthetic pathway will yet be discovered, although many may remain undetected due to their putative lethal phenotypes. In contrast, diseases are known for essentially every step in the pathway of SL degradation, caused by the defective activity of one or other of the lysosomal hydrolases in this pathway. Despite the fact that some of these storage disorders were first discovered in the 19th century, the cellular and biochemical events that cause pathology are still poorly delineated. In this review, we focus on recent advances in our understanding of how defects in the pathways of sphingolipid metabolism may lead to pathology. In addition, we discuss currently-available and emerging therapeutic options.

Abstract Sphingolipids are ubiquitous components of cell membranes, and are found in plants, animals, and fungi, as well as in some unicellular organisms and prokaryotes. Sphingolipids consist of a sphingoid long-chain base to which a fatty acid is attached at carbon 2, and a variety of polar and nonpolar head groups at C-1; glycosphingolipids are sphingolipids containing one or more sugar moieties in their head groups. Metabolic pathways have been determined for most sphingolipids, but little is known about how these pathways are regulated. Recent interest in sphingolipids and glycosphingolipids has focused largely on their roles in signaling events, with ceramide and sphingosine-1-phosphate the focus of particular attention. Sphingolipids also play vital roles in the formation and stability of membrane microdomains (rafts), and complex glycosphingolipids play roles in recognition. These lipids are also involved in disease pathology, and the pathways of sphingolipid metabolism, as well as the lipids themselves, have been implicated as possible therapeutic targets and agents.

A new series of hybrid PDMP analogues, based both on PDMP and styryl analogues of natural ceramide, has been synthesized from d-serine. The synthetic route was developed such that future introduction of different aryl groups is straightforward. Biological evaluation, both in vitro on rat liver Golgi fractions as well as in HEK-293 and COS-7 cells, revealed two lead compounds with comparable inhibitory potency as PDMP, which could be elaborated to more potent inhibitors.

In Gaucher disease, 200 different mutations have been described in the gene encoding lysosomal glucocerebrosidase (GlcCerase) (see Chapter 2). Irrespective of the mutation, glucosylceramide (GlcCer) is degraded much more slowly in cells from Gaucher patients than in normal cells and as a consequence, accumulates intracellularly, primarily in cells of mononuclear phagocyte origin. These GlcCerladen macrophages, known as \u201cGaucher cells,\u201d are the classical hallmark of the disease. Since GlcCer is an important constituent of biological membranes and is a key intermediate in the biosynthetic and degradative pathways of complex glycosphingolipids (see Chapter 7), its accumulation in Gaucher disease is likely to have severe pathological consequences. However, and somewhat surprisingly, the pathological mechanism leading from GlcCer accumulation to Gaucher disease has not been established; this is true for most lysosomal storage diseases (LSDs) in which the biochemical and cellular pathways leading from substrate accumulation to disease have not been delineated [1].

Acid β -glucosidase (EC 3.2.1.45, D-glucosyl- N -acylsphingosine glucohydrolase, glucocerebrosidase, GlcCerase), the enzyme defective in Gaucher disease, is a peripheral lysosomal membrane protein that hydrolyzes the β -glucosyl linkage of glucosylceramide (GlcCer). GlcCerase requires the coordinate action of saposin C and negatively charged lipids for maximal activity. ^1,2 Enzyme replacement therapy (ERT) with Cerezyme ®, a recombinant human GlcCerase, ³ is the main treatment for Type 1 Gaucher disease. Although attempts at structural prediction had been made earlier, ^4,5 the lack of an experimental 3-D structure of GlcCerase hampered attempts to establish its catalytic mechanism and to analyze the structural relationships between the mutations, levels of residual enzyme activity, and disease severity. The recent determination in our laboratories of the x-ray structures of GlcCerase at 2.0 Å resolution ⁶ and, subsequently, of a conjugate with an irreversible inhibitor, ⁷ conduritol B epoxide (CBE), has paved the way for detailed structural analysis of GlcCerase. In this chapter we will review the two structures, and discuss the insights that they provide that may help in designing second-generation GlcCerases for ERT.

In September of 2007 Gaucher Disease received a commendation in the Haematology category of the 2007 British Medical Association Medical Book Competition! Although rare in the general population, Gaucher disease is the most prevalent of the lysosomal storage disorders, making research into this particular orphan disorder an invaluable prototype for the diagnosis, research, and treatment of others. Like many rare conditions, Gaucher disease has benefited from the explosion of medical research in the last decade, the amount of new information on this disease is staggering and the rate of new discoveries has left previous books on the subject unable to provide useful, up-to-date information. The most current, fully comprehensive reference to date, Gaucher Disease provides valuable information for academic and industry scientists, and clinicians. Outlining the latest research on the biochemical mechanisms and pathology of lysosomal storage disorders, this book covers diverse topics including animal models, crystallography, imaging and radionuclide evaluation. It not only addresses the developmental basis for current treatments like the now widely available enzyme replacement therapy, but also includes chapters introducing new therapies on the horizon. With contributions from world-renowned experts in substrate reduction therapy, pharmaceutical chaperone therapy, hematopoietic stem cell transplantation and gene therapy, as well as chapters on a second generation of enzyme replacement therapy, this book explores the full spectrum of possibilities offered by the most recent advances in medicine. Some of the most interesting aspects of the book include the discussions on patient management, those touching on the ethics of research, and the societal aspects of treating rare diseases with expensive therapy.

Sandhoff disease, a neurodegenerative disorder characterized by the intracellular accumulation of GM2 ganglioside, is caused by mutations in the hexosaminidase β-chain gene resulting in a hexosaminidase A (αβ) and B (ββ) deficiency. A bicistronic lentiviral vector encoding both the hexosaminidase α and β chains (SIV.ASB) has previously been shown to correct the β-hexosaminidase deficiency and to reduce GM2 levels both in transduced and cross-corrected human Sandhoff fibroblasts. Recent advances in determining the neuropathophysiological mechanisms in Sandhoff disease have shown a mechanistic link between GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity, and axonal outgrowth. To examine the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neurons from embryonic Sandhoff mice were transduced with the lentivector. Normal axonal growth rates were restored, as was the rate of Ca²⁺ uptake via the SERCA and the sensitivity of the neurons to thapsigargin-induced cell death, concomitant with a decrease in GM2 and GA2 levels. Thus, we have demonstrated that the bicistronic vector can reverse the biochemical defects and down-stream consequences in Sandhoff neurons, reinforcing its potential for Sandhoff disease in vivo gene therapy.

Glycosphingolipid (GSL) storage diseases (GSDs) are a group of monogenic disorders among a large family of lysosomal storage diseases, which include more than 40 different human disorders. GSDs are caused by the impaired catalytic activity of the lysosomal hydrolases responsible for GSL degradation, which leads to accumulation of undegraded GSLs in lysosomes. The clinical presentation of GSDs varies between disorders and within each disorder. The reason for this heterogeneity is not known, but almost certainly depends on the level and nature of the accumulating GSL and the down-stream biochemical and cellular pathways activated upon GSL accumulation. We now review what little is known about the correlation of the clinical progression and pathology with the underlying biochemistry of the GSDs.

Ceramide is an important intracellular second messenger, and a key metabolite in the pathway of sphingolipid biosynthesis where it is formed by N-acylation of sphinganine. In this review, we describe the identification and characterization of a gene family consisting of six genes, the LASS genes, that regulate de novo ceramide synthesis in mammalian cells. Unexpectedly, each LASS homolog tested to date synthesizes ceramide with a specific fatty acid composition. We recently purified LASS5 to homogeneity and demonstrated that it is a bona fide ceramide synthase, displaying the same fatty acid specificity as the membrane-bound enzyme. Moreover, unlike in yeast where an additional subunit, Lipl, is required for ceramide synthase activity, LASS5 does not require any additional subunits, consistent with database analyses showing that mammalian cells do not contain Lipl homologs. The reasons that mammalian cells contain six LASS genes are not known, but presumably each gene is involved in the synthesis of ceramides containing specific fatty acids for use in the regulation of different biological processes.

We recently demonstrated that calcium homeostasis is altered in mouse models of two sphingolipid storage diseases, Gaucher and Sandhoff diseases, owing to modulation of the activities of a calcium-release channel (the ryanodine receptor) and of the sarco/endoplasmic reticulum Ca ²⁺-ATPase (SERCA) respectively, by the accumulating sphingolipids. We now demonstrate that calcium homeostasis is also altered in a mouse model of Niemann-Pick A disease, the acid sphingomyelinase (A-SMase)-deficient mouse (ASM-/-), with reduced rates of calcium uptake via SERCA in the cerebellum of 6-7-month-old mice. However, the mechanism responsible for defective calcium homeostasis is completely different from that observed in the other two disease models. Thus, levels of SERCA expression are significantly reduced in the ASM-/-cerebellum by 6-7 months of age, immediately before death of the mice, as are levels of the inositol 1,4,5-triphosphate receptor (IP₃R), the major calcium-release channel in the cerebellum. Systematic analyses of the time course of loss of SERCA and IP₃R expression revealed that loss of the IP₃R preceeded that of SERCA, with essentially no IP₃R remaining by 4 months of age, whereas SERCA was still present even after 6 months. Expression of zebrin II (aldolase C), a protein found in about half of the Purkinje cells in the adult mouse cerebellum, was essentially unchanged during development. We discuss possible pathological mechanisms related to calcium dysfunction that may cause Purkinje cell degeneration, and as a result, the onset of neuropathology in Niemann-Pick A disease.

More than 40 lysosomal disorders are known and classified into broad categories based on the nature of the undegraded metabolites. In this review, the defective enzymatic activities are analyzed, and the pathology of the disorders are described. Various treatments are discussed, including enzyme replacement therapy, which is the most successful treatment available today.

We demonstrated recently (Riebeling, C., Allegood, J.C., Wang, E., Merrill, A. H. Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452-43459) that upon over-expression in human embryonic kidney cells, longevity assurance gene homolog 5 (LASS5, previously named TRH4) elevates the synthesis of (dihydro)ceramides selectively enriched in palmitic acid. To determine whether LASS5 is a bona fide dihydroceramide synthase or, alternatively, whether it modifies an endogenous dihydroceramide synthase, we over-expressed LASS5 with a hemagglutinin (HA) tag at the C terminus, solubilized it using digitonin, and purified it by immunoprecipitation. Solubilized LASS5-HA displays the same fatty acid selectivity as the membrane-bound enzyme. After elution from agarose beads, only one band could be detected by SDS-PAGE, and its identity was confirmed to be LASS5 by mass spectrometry. Dihydroceramide synthase activity of the eluted LASS5-HA protein was totally dependent on exogenously added phospholipids. Moreover, eluted LASS5-HA was highly selective toward palmitoyl-Co A as acyl donor and was inhibited by the (dihydro)ceramide synthase inhibitor, fumonisin B1. This study identifies LASS5 as a genuine dihydroceramide synthase and demonstrates that mammalian dihydroceramide synthases do not require additional subunits for their activity.

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-β-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 Å resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep. 4, 704-709) and have now solved the structure of GlcCerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu ³⁴⁰ is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.

Sphingolipids are ubiquitous components of eukaryotic cell membranes, where they play important roles in intracellular signaling and in membrane structure. Even though the biochemical pathway of sphingolipid synthesis and its compartmentalization between the endoplasmic reticulum and Golgi apparatus have been known for many years, the molecular identity of the enzymes in this pathway has only recently been elucidated. Here, we summarize progress in the identification and characterization of the enzymes, the transport of ceramide from the endoplasmic reticulum to the Golgi apparatus, and discuss how regulating the synthesis of sphingolipids might impact upon their functions.

Gaucher disease, the most common lysosomal storage disorder, is caused by the defective activity of the lysosomal enzyme, acid-β-glucosidase (GlcCerase), leading to accumulation of glucosylceramide (GlcCer), particularly in cells of the macrophage lineage. Nearly 200 mutations in GlcCerase have been described, but for the most part, genotype-phenotype correlations are weak, and little is known about the down-stream biochemical changes that occur upon GlcCer accumulation that result in cell and tissue dysfunction. In contrast, the clinical course of Gaucher disease has been well described, and at least one treatment is available, namely enzyme replacement therapy. One other treatment, substrate reduction therapy, has recently been marketed, and others are in early stages of development. This review, after discussing pathological mechanisms, evaluates the advantages and disadvantages of existing therapies.

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids.

Gaucher disease is an inherited metabolic disorder caused by defective activity of the lysosomal enzyme, glucocerebrosidase, resulting in accumulation of the lipids, glucosylceramide (GlcCer), and glucosylsphingosine (GlcSph). Little is known about the mechanism leading from lipid accumulation to disease, particularly in the acute and subacute neuronopathic forms of Gaucher disease, types 2 and 3, respectively. Recent work from our laboratory has shown, in animal models, that GlcCer enhances agonist-induced calcium release from intracellular stores via the ryanodine receptor, which results in neuronal cell death. We now test whether calcium release is altered in human brain tissue obtained post-mortem from Gaucher disease patients. Agonist-induced calcium release via the ryanodine receptor was significantly enhanced (P < 0.05) in brain microsomes from the acute neuronopathic form of Gaucher disease (type 2) (43 ± 6% of the calcium in microsomes) compared to the subacute (type 3) (27 ± 3%) and the non-neuronopathic (type 1) (28 ± 6%) forms, and controls (18 ± 3%), and correlated with levels of GlcCer accumulation. These findings suggest that defective calcium homeostasis may be a mechanism responsible for neuropathophysiology in acute neuronopathic Gaucher disease, and may potentially offer new therapeutic approaches for disease management.

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids - including ceramide, sphingosine and sphingosine-1-phosphate - at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.

Glycosphingolipid storage disorders are inborn errors of metabolism caused by the defective activity of degradative enzymes in lysosomes. In this review, we summarize studies performed over the past few years attempting to define the secondary and down-stream biochemical and cellular pathways affected in GSL storage disorders that are responsible for neuronal dysfunction, a characteristic of most of these disorders. We focus mainly on the regulation of intracellular calcium homeostasis and phospholipid biosynthesis. These studies may help unravel new roles for glycosphingolipids in the regulation of normal cell physiology, as well as suggest potential new therapeutic options in the glycosphingolipid and other lysosomal storage disorders.

Lysosomal storage disorders, of which more than 40 are known, are caused by the defective activity of lysosomal proteins, which results in the intra-lysosomal accumulation of undegraded metabolites. Despite years of study of the genetic and molecular bases of lysosomal storage disorders, little is known about the events that lead from this intra-lysosomal accumulation to pathology. Here, we summarize the biochemistry of lysosomal storage disorders. We then discuss downstream cellular pathways that are potentially affected in these disorders and that might help us to delineate their pathological mechanisms.

Gaucher disease is caused by defective activity of acid-β-glucosidase (GlcCerase), resulting in accumulation of glucosylceramide (GlcCer) mainly in macrophages. We now demonstrate that secondary biochemical pathways regulating levels of phospholipid metabolism are altered in a Gaucher disease macrophage model. Upon treatment of macrophages with the GlcCerase inhibitor, conduritol-B-epoxide, phosphatidylcholine (PC) labeling with the metabolic precursor, [methyl-¹⁴C]choline, was elevated after 6 or 12 days in macrophages but not in lymphocytes. These changes correlated with increases in the cytoplasmic/nuclear ratio and with levels of [³H]GlcCer accumulation. Moreover, metabolic labeling with L-[3-³H]serine and L-[methyl-³H]methionine demonstrated that PC synthesis via the methylation of phosphatidylethanolamine is also increased in CBE-treated macrophages. Since PC is a major structural component of biological membranes and the source of various second messengers, we suggest that changes in its metabolism in macrophages may be relevant for understanding Gaucher disease pathology.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes for the β-subunit of β-hexosaminidase A and B, resulting in ganglioside GM2 accumulation in the brain. We now demonstrate that phospholipid metabolism is altered in both cultured neurons and in brain tissue from a mouse model of Sandhoff disease, the Hexb-/- mouse. Metabolic labelling using [methyl-¹⁴C]choline and L-[3-³H]serine demonstrated reduced incorporation of [methyl- ¹⁴C]choline into phospholipids in brain tissue but not in liver or spleen. Phospholipid mass was also reduced in brain. The activities of CTP: phosphocholine cytidylyltransferase (CCT) and phosphatidylserine synthase were also reduced in brain tissue from Hexb-/- mice, probably because of post-translational modification as no changes were observed in levels of enzyme expression. The relevance of these findings to Sandhoff disease in human patients is strengthened by observations made over 30 years ago on autopsy tissue of Tay Sachs and Sandhoff disease patients, in which reduced phospholipid levels were observed. We suggest that changes in phospholipid metabolism are not simply because of loss of neuronal tissue as a result of degeneration but rather may cause degeneration, and we discuss the possible effects that changes in phospholipid metabolism could play in the neuropathophysiology of Sandhoff disease.

Gaucher disease, an inherited metabolic disorder caused by mutations in the gene encoding acid-β-glucosidase (GlcCerase), is a multi-system disease whose manifestations include anemia, thrombocytopenia, hepatosplenomegaly, bone pathology and, in some cases, neurological signs. Enzyme replacement therapy (ERT) using recombinant GlcCerase (Cerezyme®) alleviates many disease symptoms and is used by ~3000 patients worldwide, and substrate-reduction therapy (SRT) using the glycolipid synthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ (Zavesca®)] has been approved recently for patients for whom ERT is unsuitable. It is our opinion that a multiplicity of treatment strategies is required for the management of Gaucher disease. In this article, we discuss the pros and cons of currently available treatments, and suggest complementary therapies arising from the determination of the X-ray structure of Cerezyme® and from delineation of secondary biochemical pathways affected in Gaucher disease.

Purpose: Elucidating the potential contribution of specific autoantibodies (Ab's) to the etiology and/or pathology of some human epilepsies. Methods: Six epilepsy patients with Rasmussen's encephalitis (RE) and 71 patients with other epilepsies were tested for Ab's to the "B" peptide (amino acids 372-395) of the glutamate/AMPA subtype 3 receptor (GluR3B peptide), double-stranded DNA (dsDNA), and additional autoimmune disease-associated autoantigens, and for the ability of their serum and cerebrospinal-fluid (CSF) to kill neurons. Results: Elevated anti-GluR3B Ab's were found in serum and CSF of most RE patients, and in serum of 17/71 (24%) patients with other epilepsies. In two RE patients, anti-GluR3B Ab's decreased drastically in CSF following functional-hemispherotomy, in association with seizure cessation and neurological improvement. Serum and CSF of two RE patients, and serum of 12/71 (17%) patients with other epilepsies, contained elevated anti-dsDNA Ab's, the hallmark of systemic-lupus-erythematosus. The sera (but not the CSF) of some RE patients contained also clinically elevated levels of "classical" autoimmune Ab's to glutamic-acid-decarboxylase, cardiolipin, β2-glycoprotein-I and nuclear-antigens SS-A and RNP-70. Sera and CSF of some RE patients caused substantial death of hippocampal neurons. Conclusions: Some epilepsy patients harbor Ab's to GluR3 and dsDNA on both sides of the blood-brain barrier, and additional autoimmune Ab's only in serum. Since all these Ab's may be detrimental to the nervous system and/or peripheral organs, we recommend testing for their presence in epilepsy, and silencing their activity in Ab-positive patients.

Sandhoff disease is caused by the defective activity of the lysosomal enzyme beta-hexosaminidase, resulting in accumulation of the glycolipids, GA(2) and GM(2). Niemann-Pick A/B disease is caused by the defective activity of lysosomal acid sphingomyelinase resulting in sphingomyelin accumulation. Pulmonary complications have been observed in both diseases. We now demonstrate changes in phospholipid levels in pulmonary surfactant in mouse models of these diseases. In the Hexb mouse, a model of Sandhoff disease, lipid phosphate levels were elevated in surfactant from 3- and 4-month-old mice, which was mainly due to elevated levels of phosphatidylcholine. In the ASM mouse, a model of Niemann-Pick A disease, levels of the primary storage material, sphingomyelin, were elevated as expected, and levels of phosphatidylcholine and two other phospholipids were also significantly elevated in pulmonary surfactant and in lung tissue from 5-, 6- and 7-month-old mice. These results suggest that changes in phospholipid levels and composition in lung surfactant might be a general feature of sphingolipid storage diseases, which may be in part responsible for the increased susceptibility of these patients to respiratory infections and lung pathology, often the main reason for the death of these patients.

The glycosphingolipid lysosomal storage diseases are a group of monogenic human disorders caused by the impaired catalytic activity of enzymes responsible for glycosphingolipid catabolism. Clinical presentation of the diseases is heterogeneous, with little obvious correlation between the kind of accumulating glycosphingolipid and disease progression or pathogenesis. In this review, we discuss clinical symptoms of this group of diseases, and attempt to link disease progression and pathology with the biochemical and cellular pathways that may be potentially altered in the diseases.

Recently, we demonstrated that the GSL (glycosphingolipid), GlcCer (glucosylceramide), modulates Ca²⁺ release from intracellular stores and from microsomes by sensitizing the RyaR (ryanodine receptor), a major Ca²⁺-release channel of the endoplasmic reticulum, whereas the lyso derivative of GlcCer, namely GlcSph (glucosylsphingosine), induced Ca ²⁺ release via a mechanism independent of the RyaR [Lloyd-Evans, Pelled, Riebeling, Bodennec, de-Morgan, Waller, Schiffmann and Futerman (2003) J. Biol. Chem. 278, 23594-23599]. We now systematically examine the mechanism by which GlcSph and other lyso-GSLs modulate Ca²⁺ mobilization from rat brain cortical and cerebellar microsomes. GlcSph, lactosylsphingosine and galactosylsphingosine all mobilized Ca²⁺, but at significantly higher concentrations than those required for GlcCer-mediated sensitization of the RyaR. GlcSph-induced Ca²⁺ mobilization was partially blocked by heparin, an inhibitor of the Ins(1,4,5)P₃ receptor, and also partially blocked by thapsigargin or ADP, inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase), but completely blocked when both acted together. In contrast, neither lactosylsphingosine nor galactosylsphingosine had any effect on Ca²⁺ release via either the Ins(1,4,5)P₃ receptor or SERCA, but acted as agonists of the RyaR. Finally, and surprisingly, all three lyso-GSLs reversed inhibition of SERCA by thapsigargin. We conclude that different lyso-GSLs modulate Ca²⁺ mobilization via different mechanisms, and discuss the relevance of these findings to the GSL storage diseases in which lyso-GSLs accumulate.

Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [ ³H](dihydro)ceramide synthesis from L-[3-³H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B₁ (FB₁). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB₁. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB ₁-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.

Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease ( one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in V-max. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.

Sandhoff disease is a lysosomal storage disease in which ganglioside GM2 accumulates because of a defective β-subunit of β-hexosaminidase. This disease is characterized by neurological manifestations, although the pathogenic mechanisms leading from GM2 accumulation to neuropathology are largely unknown. We now examine the viability, development and rates of neurite growth of embryonic hippocampal neurones cultured from a mouse model of Sandhoff disease, the Hexb-/- mouse. GM2 was detected by metabolic labelling at low levels in wild type (Hexb+/+) neurones, and increased by approximately three-fold in Hexb-/- neurones. Hexb-/- hippocampal neurones were as viable as their wild type counterparts and, moreover, their developmental programme was unaltered because the formation of axons and of the minor processes which eventually become dendrites was similar in Hexb-/- and Hexb+/+ neurones. In contrast, once formed, a striking difference in the rate of axonal and minor process growth was observed, with changes becoming apparent after 3 days in culture and highly significant after 5 days in culture. Analysis of various parameters of axonal growth suggested that a key reason for the decreased rate of axonal growth was because of a decrease in the formation of collateral axonal branches, the major mechanism by which hippocampal axons elongate in culture. Thus, although the developmental programme with respect to axon and minor process formation and the viability of hippocampal neurones are unaltered, a significant decrease occurs in the rate of axonal and minor process growth in Hexb-/- neurones. These results appear to be in contrast to dorsal root ganglion neurones cultured from 1-month-old Sandhoff mice, in which cell survival is impaired but normal outgrowth of neurones occurs. The possible reasons for these differences are discussed.

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca²⁺ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca²⁺ release from rat brain microsomes. GlcCer had no direct effect on Ca²⁺ release, but rather augmented agonist-stimulated Ca²⁺ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca²⁺ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca²⁺ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca²⁺ release, although only galactosylsphingosine mediated Ca²⁺ release via the RyaR. Finally, we demonstrated that GlcCer levels were ∼10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca²⁺ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ∼200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non-neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α ₈ TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [³H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an ∼40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation. The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

The roles of sphingolipids, and particularly of the complex glycosphingolipids (GSLs), the gangliosides, have been studied for many years in neurons, glia, and cell lines derived from these tissues, due to their abundance in tissues of neuronal origin. More recently, significant attention has been paid to the simple sphingolipids, particularly ceramide, glucosylceramide (GlcCer), and sphingosine-1-phosphate (S1P), each of which appears to be involved in the regulation of specific aspects of neuronal proliferation, differentiation, survival and apoptosis. In this review, we will summarize studies performed in our laboratory over the past few years using cultured hippocampal neurons in an attempt to define the precise roles of these lipids, and to define their mechanisms of action by identifying down-stream targets with which they interact. We will also discuss work suggesting that complex GSLs, such as gangliosides GM2 and GD3, can also regulate neuronal development, although the down-stream targets with which they interact are less well defined. Our work will be reviewed in light of studies from other laboratories, with particular emphasis on the use of models of sphingolipid storage diseases to determine how these lipids affect neuronal function.

Methods for isolation of neutral lysoglycosphingolipids (n-lyso-GSLs) such as glucosylsphingosine and galactosylsphingosine normally involve mild alkaline or acid hydrolysis followed by multiple chromatography steps, yielding relatively low recoveries of n-lyso-GSLs and neutral glycosphingolipids (n-GSLs). We now describe a new technique for isolating these compounds using one chromatography step, resulting in quantitative recovery of n-GSLs and n-lyso-GSLs. Lipids are extracted using a modified Folch procedure in which recovery is optimized by reextracting the Folch upper phase with water-saturated butanol. The extract is applied to an aminopropyl solid phase column from which both n-GSLs and n-lyso-GSLs elute in the same fraction. Separation is achieved using a new two-dimensional thin-layer chromatography procedure. The usefulness of this technique for biological samples was tested by examining Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulating at levels of 20 nmol/mg DNA and 40 pmol/mg DNA, respectively. This simple and rapid technique can therefore be used for the analysis of lyso-GSLs and GSLs in the same tissue, which may permit the determination of their metabolic pathways in normal and in pathological tissues, such as those taken from Gaucher and Krabbe's disease patients.

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site-directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDP-choline levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild-type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post-translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200-fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.

A gene family containing a longevity assurance gene (Lag1p) motif is described. Database searches revealed > 40 members of this family of transmembrane proteins, two of which have recently been shown to regulate the synthesis of ceramide, a lipid second messenger involved in a variety of cellular processes. We speculate that other family members, some of which contain a Hox domain, may also be involved in the synthesis of specific ceramide pools, perhaps explaining the role of longevity assurance genes in regulating development.

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containinn-g very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B₁, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-³H]sphinganine or L-3-[³H]serine demonstrated that uog1 conferred fumonisin B₁ resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB₁, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B₁ resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.

The study of sphingolipids has undergone a renaissance over the past decade due to the realization that these lipids are involved in a variety a biological processes, such as differentiation, apoptosis, cell growth, and cell migration. In the nervous system, sphingolipids, particularly gangliosides, have attracted particular attention as they occur at high levels and their levels change in a developmentally regulated program. Despite the fact that a large body of data has accumulated on the expression and metabolism of individual gangliosides within specific brain regions, the role of individual gangliosides in neuronal development is still poorly understood, and their specific functions are only now beginning to be elucidated. In the present article, we discuss various aspects of our current knowledge concerning the involvement of sphingolipids and gangliosides in neuronal development, and then discuss some recent findings that shed light on the role of sphingolipids and gangliosides obtained with animal models of sphingolipid and other lysosomal storage diseases.

Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. NGF application to neuronal cultures from p75^exonIII-/- mice had no effect on ceramide levels and did not affect neuronal viability. The neutral sphingomyelinase inhibitor, scyphostatin, inhibited NGF-induced ceramide generation and neuronal death, whereas hippocampal neurons cultured from acid sphingomyelinase^-/- mice were as susceptible to NGF-induced death as wild type neurons. The acid ceramidase inhibitor, (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.

The synthesis of a new series of D-erythro-homoceramide analogues is described. Several synthetic approaches were investigated. Homoceramides can be successfully synthesized from L-homoserine as chiral building block and a protected Weinreb-amide as a key intermediate. The synthesis of short-chain analogues with a heptyl side chain, as well as with a phenyl residue in the sphingoid part (instead of the naturally occurring tridecyl side chain), was effected. The homoceramides 15-17 and 24 were investigated for their potential to reverse the inhibitory effect of fumonisin B₁ on axonal growth. Unfortunately, none of the tested compounds showed any biological activity due to their lack of metabolism to glucosylhomoceramide.

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C₆-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C₆-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C₆-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C₆-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.

A number of studies have demonstrated that cholera toxin (CT) is found in detergent-insoluble, cholesterol-enriched domains (rafts) in various cells, including neurons. We now demonstrate that even though CT is associated with these domains at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism, at both early and late stages of neuronal development. CT transport to the Golgi apparatus, and its subsequent degradation, is inhibited by hypertonic medium (sucrose), and by chlorpromazine; the former blocks clathrin recruitment, and the latter causes aberrant endosomal accumulation of clathrin. Moreover, both internalization of the transferrin receptor (Tf-R), which occurs via a clathrin-dependent mechanism, and CT internalization, are inhibited to a similar extent by sucrose. In contrast, the cholesterol-binding agents filipin and methyl-β -cyclodextrin have no effect on the rate of CT or Tf-R internalization. Finally, once internalized, CT becomes more detergent-soluble, and chlorpromazine treatment renders internalized CT completely detergent-soluble. We propose two models to explain how, despite being detergent-insoluble at the cell surface, CT is nevertheless internalized via a raft-independent mechanism in hippocampal neurons.

Ceramide (Cer) is a key intermediate in the synthetic and degradative pathways of sphingolipid metabolism, and is also an important second messenger. Natural Cer exists in the D-erythro configuration. Three additional, non-natural stereoisomers exist, but conflicting reports have appeared concerning their metabolism. We now compare the stereospecificity of three enzymes in the sphingolipid biosynthetic pathway, namely dihydroceramide (dihydroCer), sphingomyelin (SM) and glucosylceramide synthases, in subcellular fractions and in cultured cells. The L-erythro enantiomers of sphinganine, dihydroCer and Cer do not act as substrates for any of the three enzymes. In contrast, the diastereoisomer, L-threo-sphinganine, is acylated by dihydroCer synthase, and L-threo-dihydroCer and L-threo-Cer are both metabolized to dihydroSM and SM, respectively, but not to dihydroglucosylceramide and glucosylceramide. No significant difference was detected in the ability of SM synthase to metabolize Cer containing a short (hexanoyl) versus long acyl chain (palmitoyl), demonstrating that short-acyl chain Cers mimic their natural counterparts, at least in the sphingolipid biosynthetic pathway.

We recently demonstrated that although cholera toxin (CT) is found in detergent-insoluble domains/rafts at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism. Thus, cholesterol depletion by methyl-β-cyclodextrin (MβCD) did not affect the rate of CT internalization from the plasma membrane, but did affect the rate of CT degradation, which occurs in lysosomes. In the current study, we analyze which step of CT intracellular transport is inhibited by MβCD. Whereas pre-incubation with MβCD completely blocked CT degradation, it had no effect on the degradation of wheat germ agglutinin (WGA) or bovine serum albumin (BSA), which are internalized by receptor-mediated and fluid phase endocytosis, respectively. Brefeldin A also completely blocked CT degradation but had no effect on WGA or BSA degradation. In contrast, MβCD did not affect CT degradation, or CT-mediated cAMP generation, when added to neurons after CT had been transported to the Golgi apparatus. We conclude that CT transport from endosomes to the Golgi apparatus is cholesterol-dependent, whereas CT transport from the Golgi apparatus to lysosomes is cholesterol-independent.

The biological activity of synthetic ceramide analogues, having modified sphingoid and N-acyl chains, as well as fluorine substituents in the allylic position, was investigated in hippocampal neurons. Their influence on axonal growth was compared to that of C₆-N-acyl analogues of natural ceramides. D-erythro-Ceramides with a phenyl group in the sphingoid moiety and a short N-acyl chain were able to reverse the inhibitory effect of fumonisin B₁ (FB₁), but not of D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), on accelerated axonal growth in hippocampal neurons. Moreover, we demonstrated that a ceramide analogue with an aromatic ring in the sphingoid moiety is recognized as a substrate by glucosylceramide synthase, which suggests that the observed biological effects are mediated by activation of the ceramide analogue via glucosylation. Introduction of a methyl, pentyl, fluoro, or methoxy substituent in the para position of the phenyl ring in the sphingoid moiety yielded partly active compounds. Likewise, substitution of the benzene ring for a thienyl group did not abolish the ability to reverse the inhibition of accelerated axonal growth by FB₁. Both D-erythro- and L-threoceramide analogues, having an allylic fluorine substituent, partly reversed the FB₁ inhibition.

Much discussion has recently centred around the biochemical mechanisms by which ceramide is produced in signalling pathways. Since ceramide is virtually insoluble in aqueous solutions, the biological effects of ceramide should be considered in the context of its generation within the membrane lipid bilayer. To this end, we now summarize recent data describing some biophysical properties of ceramide that are of relevance for understanding the mode of ceramide action as a second messenger, and, as a consequence, how the site(s) of ceramide generation might impact upon its role in signalling. Copyright (C) 2000 Elsevier Science Ltd.

We have previously shown that ongoing glucosylceramide (GlcCer) synthesis is required for basic fibroblast growth factor (bFGF) and laminin to stimulate axonal growth in cultured hippocampal neurons (Boldin, S., and Futerman, A. H. (1997) J, Neurochem. 68, 882-885), We now demonstrate that stimulation of axonal growth by bFGF leads to an increase in the rate of GlcCer synthesis. Within minutes of incubation with bFGF, a significant increase in the rate of metabolism of [C-14]hexanoyl ceramide to [C-14]hexanoyl GlcCer is detected, but there are no changes in the rate of [C-14]hexanoyl sphingomyelin synthesis. lit vitro analysis of GlcCer synthase activity revealed an approximately 2-fold increase in the rate of [C-14]hexanoyl GlcCer synthesis upon incubation with either bFGF or laminin; other growth factors, which did not effect the rate of axon growth, had no effect on the rate of [C-14]hexanoyl GlcCer synthesis. The increased rate of [C-14]hexanoyl GlcCer synthesis was not affected by preincubation with either cycloheximide or actinomycin, and no elevation of GlcCer synthase mRNA levels was detected, suggesting that GlcCer synthase is up-regulated by a post-translational mechanism. The relevance of these results for understanding the regulation of axonal growth is discussed.

Recently a putative mammalian neutral-sphingomyelinase was cloned [Tomiuk et al. (1998) Proc. Natl. Acad. Sci. USA 95, 3638-3643; GenBank accession number AJ222801]. We have overexpressed this enzyme in cultured cells and demonstrate, using four different tagged constructs, that it is localized at the endoplasmic reticulum and not at the plasma membrane. This localization precludes a role for enzyme AJ222801 in the sphingomyelin cycle. Furthermore, a recent publication demonstrated that this enzyme has lyso-platelet activating factor (PAF) phospholipase C activity [Sawai et al. (1999) J. Biol. Chem. 274, 38131-38139]. Together, these data suggest a role for enzyme AJ222801 in the regulation of PAF metabolism. Copyright (C) 2000 Federation of European Biochemical Societies.

We have recently demonstrated that incubation of cultured rat hippocampal neurons with conduritol β-epoxide (CBE), an inhibitor of glucocerebrosidase, the enzyme defective in Gaucher disease, results in changes in intracellular morphology and in functional calcium stores. Changes in levels of functional calcium stores are directly related to neuronal cell death. We now show that neurons incubated with either CBE or a non-hydrolysable analogue of GlcCer (glucosylthioceramide), are more sensitive to the toxic effects of high concentrations of glutamate and of a variety of metabolic inhibitors. A linear relationship exists between level of accumulation of GlcCer and the extent of neuronal cell death. The deleterious effects of elevated GlcCer levels can be completely reversed by addition of human glucocerebrosidase (imiglucerase) to the culture medium. Imiglucerase is internalized to lysosomes, where it presumably degrades excess GlcCer. This suggests that the limited success of enzyme replacement therapy in neuronopathic forms of Gaucher disease is not due to lack of efficacy of glucocerebroside in degrading GlcCer in neurons of the central nervous system, and adds impetus to attempts to develop ways to efficiently deliver glucocerebrosidase to the brains of neurologically compromised Gaucher disease patients.

The signal transduction pathway of the type 1 Fcε receptor (FcεRI) has been proposed to be spatially constrained to plasma membrane microdomains enriched in glycosphingolipids and cholesterol. These domains are proposed to serve as platforms that enhance the efficiency of the antigen-receptor stimulus-response coupling process. Here we describe a monoclonal antibody (mAb) designated 2B5, raised by immunizing mice with rat mucosal-type mast cell (line RBL-2H3) membranes, which binds to glycosphingolipids and causes a dose-dependent secretory response of these cells. This secretory response to mAb 2B5 requires binding of IgE to the FcεRI on these cells, although direct interactions between IgE and mAb 2B5 are excluded. The bound IgE- or FcεRI-specific mAb did not affect binding of mAb 2B5 or its Fab fragments to the RBL-2H3 cells and only a limited interference with the binding of IgE to the FcεRI by mAb 2B5 was observed. Binding of mAb 2B5 to the RBL-2H3 cells induced a distribution of fluorescently labeled IgE similar to that produced by antigen-induced aggregation of the IgE-FcεRI. Thus we suggest that mAb 2B5 binds to cell surface glycosphingolipids that are probably associated with the FcεRI-IgE complexes and causes their aggregation, thereby initiating the cascade leading to the cell's secretory response.

The p75 neurotrophin receptor (p75NTR) binds all known neurotrophins and has been suggested to either function as a coreceptor for the trk receptor tyrosine kinases or be involved in independent signaling leading to cell death. We have analyzed the effects of nerve growth factor (NGF) on the growth of cultured hippocampal pyramidal neurons and examined the possibility that the effects of NGF are mediated via generation of ceramide produced by neutral sphingomyelinase (N-SMase). During the initial hour of culture, the only detectable NGF receptor is p75NTR, which by comparative Western blot is expressed at 50- to 100-fold lower levels than on PC12 cells. At this early stage of culture, NGF accelerates neurite formation and outgrowth and induces ceramide formation in a dose-dependent manner. An NGF mutant that is deficient in p75NTR binding has no effect on neuronal morphology or ceramide formation. Furthermore, two anti-p75NTR antibodies (REX and 9651), which are known to compete with NGF for binding to p75NTR, mimic the effects of NGF, whereas a monoclonal antibody (MC192) targeted against a different epitope does not. Finally, scyphostatin, a specific N-SMase inhibitor, blocks the effects of NGF. We propose that a neurotrophin-p75NTR-ceramide signaling pathway influences outgrowth of hippocampal neurons. This signaling role of p75NTR may be distinct from other signaling pathways that lead to apoptosis.

Analysis of the binding of cholera toxin to ganglioside GM1 in both living and fixed neurons, and comparison with the distribution of defined axonal and dendritic proteins, demonstrates that ganglioside GM1 is distributed in a non-polarized manner over the axonal and dendritic plasma membranes of mature, cultured hippocampal neurons. Likewise, ganglioside GD1b is also distributed in a non-polarized manner. These results suggest that a recent report [Ledesma, M.D. et al. EMBO J. 18 (1999) 1761-1771] proposing that ganglioside GM1 is highly enriched on the axonal versus dendritic membrane of hippocampal neurons may need to be re-evaluated. Copyright (C) 1999 Federation of European Biochemical Societies.

Antibodies to the glutamate/AMPA receptor subunit 3 (GluR3), are found in a human epilepsy, Rasmussen's encephalitis [RE], and were hypothesized as the major cause for the neuronal loss, chronic inflammatory changes and epileptic seizures characteristic of the disease. To establish the pathogenic potential and mechanism of action of such antibodies, we raised murine antibodies against specific peptides of the GluR3 protein and studied their ability to bind, activate, and kill neurons. Mice were immunized with two GluR3 specific peptides: GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395), and with a scrambled GluR3B peptide for control. High levels of antibodies to each of these peptides were obtained, with no cross reactivity between them. Antibodies to the GluR3B peptide were found to bind to cultured neurons, evoke GluR ion channel activity, and kill neurons. In contrast, antibodies against GluR3A peptide bound to neurons but failed to activate the receptor or kill neurons. Anti-scrambled-GluR3B antibodies had no effect. Both the activation of the GluRs and the neuronal death induced by anti- GluR3B antibodies were blocked by CNQX, a specific glutamate/AMPA receptor antagonist; killing was independent of complement. This indicates a mechanism of excitotoxicity-neuronal death due to over-activation of the receptor, a phenomenon known to be caused by excess of glutamate. Purified anti-GluR3B IgGs retained the neuronal killing capacity, and killing was completely and specifically blocked by preincubation with the GluR3B peptide. Excitotoxic neuronal death induced by anti-GluR3B antibodies took place primarily via apoptosis. Taken together, these results show that antibodies to a specific peptide of the GluR can kill neurons by an excitotoxic mechanism, thus mimicking the effects of excess of glutamate. This is the first example that antibodies can lead to neuronal death in a non-classical complement- independent manner, via activation of a membranal neurotransmitter receptor.

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), resulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neurotoxic agent might be responsible for cellular disruption and neuronal death. We now demonstrate that upon incubation with a chemical inhibitor of GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulate GlcCer. Surprisingly, increased levels of tubular endoplasmic reticulum elements, an increase in [Ca²⁺](i) response to glutamate, and a large increase in [Ca²⁺](i) release from the endoplasmic reticulum in response to caffeine were detected in these cells. There was a direct relationship between these effects and GlcCer accumulation since co- incubation with CBE and an inhibitor of glycosphingolipid synthesis, fumonisin B₁, completely antagonized the effects of CBE. Similar effects on endoplasmic reticulum morphology and [Ca²⁺](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C₈- glucosylthioceramide. Finally, neurons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre- incubation with ryanodine, suggesting that [Ca²⁺](i) release from ryanodine-sensitive intracellular stores can induce neuronal cell death, at least in neurons with elevated GlcCer levels. These results may provide a molecular mechanism to explain neuronal dysfunction and cell death in neuronopathic forms of Gaucher disease.

Sphingolipid-rich rafts play an essential role in the posttranslational (Borchelt, D. R., Scott, M., Taraboulos, A., Stahl, N., and Prusiner, S. B. (1990) J. Cell Biol. 110, 743-752)) formation of the scrapie prion protein PrP(Sc) from its normal conformer PrP(c) (Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell Biol. 129, 121-132). We investigated the importance of sphingolipids in the metabolism of the PrP isoforms in scrapie-infected ScN2a cells. The ceramide synthase inhibitor fumonisin B₁ (FB₁) reduced both sphingomyelin (SM) and ganglioside GM1 in cells by up to 50%, whereas PrP(Sc) increased by 3-4- fold. Whereas FB₁ profoundly altered the cell lipid composition, the raft residents PrP(c), PrP(Sc), caveolin 1, and GM1 remained insoluble in Triton X-100. Metabolic radiolabeling demonstrated that PrP(c) production was either unchanged or slightly reduced in FB₁-treated cells, whereas PrP(Sc) formation was augmented by 3-4-fold. To identify the sphingolipid species the decrease of which correlates with increased PrP(Sc), we used two other reagents. When cells were incubated with sphingomyelinase for 3 days, SM levels decreased, GM1 was unaltered, and PrP(Sc) increased by 3-4-fold. In contrast, the glycosphingolipid inhibitor PDMP reduced PrP(Sc) while increasing SM. Thus, PrP(Sc) seems to correlate inversely with SM levels. The effects of SM depletion contrasted with those previously obtained with the cholesterol inhibitor lovastatin, which reduced PrP(Sc) and removed it from detergent-insoluble complexes.

A series of D-erythro- and L-threo-ceramide analogues was synthesized and investigated for their ability to reverse the inhibitory effects of fumonisin B₁ (FB₁) on axonal growth in hippocampal neurons. The analogues contained either a C₇ side chain or a phenyl group substituted for the C₁₃ residue present in naturally occurring ceramides, while the N-acyl chain length was reduced from C₁₆-C₂₄ to C₂-C₈. D-erythro-Ceramide 18a with a C₇ side chain and an N-acetyl residue and D-erythro-ceramide 20c with an aromatic side chain and an N-hexanoyl residue were most active in reversing the inhibitory effects of FB₁ on axonal growth, although the mechanism remains unclear.

In a previous study we observed that long term (5 days) incubation with fumonisin B₁ (FB₁), an inhibitor of acylation of sphingoid long chain bases to (dihydro)-ceramide, resulted in morphological and biochemical changes in 3T3 fibroblasts (Meivar-Levy, I., Sabanay, H., Bershadsky, A.D., and Futerman, A. H. (1997) J. Biol. Chem. 272, 1558-1564). Among these were changes in the profile of synthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). Whereas [³H]globotriaosylceramide ([³H]Gb₃) comprised 1.9% of the total [³H]SLs and [³H]GSLs synthesized in control cells, it comprised 16.5% in FB₁-treated cells. We now demonstrate by in vitro analysis that inhibition of ceramide synthesis by FB₁ for 5 days results in up-regulation of the activities of three enzymes in the pathway of Gb₃ synthesis, namely glucosylceramide, lactosylceramide, and Gb₃ synthases; up-regulation is due to an increase in V(max), with no change in K(m) values toward lipid substrates. Moreover, molecular analysis (reverse transcriptase-polymerase chain reaction) of glucosylceramide synthase indicated that this enzyme is up-regulated at the transcriptional level. No changes in either the V(max) or K(m) values of sphingomyelin or of GM₃ synthase were detected after FB₁ treatment. Analysis of SL and GSL synthesis in cultured cells using [4,5-³H]sphinganine as a metabolic precursor demonstrated that at low substrate concentrations, Gb₃ synthesis is favored over GM₃ synthesis and glucosylceramide synthesis is favored over sphingomyelin synthesis, whereas the opposite is true at high substrate concentrations. These data demonstrate that GSL synthesis and in particular Gb₃ synthesis are tightly regulated in fibroblasts, presumably so as to maintain constant levels of Gb₃ on the cell surface.

Keywords: CULTURED HIPPOCAMPAL-NEURONS; DENDRITE GROWTH; DISTINCT ROLES; CERAMIDE; INHIBITION; GLUCOSYLCERAMIDE; GANGLIOSIDE; DEGRADATION; SYNTHASE; DISEASE

Readers of this journal may be surprised to read that the development of our thinking about the membrane lipid bilayer parallels the development of the kibbutz, one of Israel's most famous institutions. Whereas the founding fathers of the kibbutz movement (Hebrew word for communal settlement) preached all for one and one for all, today much greater emphasis is placed on the freedom of expression and needs of the individual kibbutz member. Just so our understanding of the membrane lipid bilayer. In the early days, the bilayer was no more than a collective enterprise deemed necessary to keep the good guys in and the bad guys out. But all this has changed, and it is now apparent that some individual lipid members are more equal than others'.Our thinking about biological membranes has come a long way over the past century since Overton first described their lipidic nature, and since Gorter and Grendel suggested that the lipids are arranged in a bilayer. Although it was clear in those early days that there was some heterogeneity in the lipid components of biological membranes, never in their wildest dreams could these early pioneers have imagined the huge number of possible permutations in both the hydrophobic tails and in the hydrophilic heads of the three major lipid classes (glycerolipids, sphingolipids and sterols), that accounts for the existence of the many hundreds of individual lipids known today. If lipids simply act in a communal manner to maintain the structural integrity of the cell, why are there so many different lipid species? It is now becoming apparent that individual lipids are key players in their own right. For instance, a bewildering variety of lipids are known to turn over in response to various extracellular stimuli, and individual lipid species have been implicated to play pivotal roles in regulating intracellular protein and vesicle traffic. A hundred years after Overton, today's challenge is to elucidate the regulatory functions of individual lipid species and to identify their modes of interaction with other membrane components. In this brief report of a recent EMBO workshop (Lipids: Regulatory Functions in Membrane Traffic and Cell Development', Kfar Blum Kibbutz Guest House, Galilee, Israel, 1015 May, 1998), we will summarize progress in these areas. Readers are referred to recent comprehensive reviews for more details.A recurring theme in the workshop was that new methodologies are required to analyze the properties of lipids in cellular membranes, as opposed to artificial lipid bilayers (liposomes). Although the study of liposomes has provided important biophysical information about the dynamic properties of lipids, most studies have been performed on liposomes of relatively simple composition, and with a symmetric rather than an asymmetric distribution of lipids across the bilayer. As a result, analysis of proteinlipid interactions has not addressed regulatory functions that might derive from the complex, asymmetric lipid distribution found in biological membranes. Similarly, no techniques are available to address ways in which the cytoskeleton might modulate cellular membranes. Together, these technical limitations have led to oversimplified views of the structure and dynamics of lipids in biological membranes. The good news is that new methodologies are on the way.

The neuronal cell surface consists of two domains, the somatodendritic and axonal plasma membranes. Each domain serves different functions, and has a different complement of membrane molecules. Since membrane molecules are able to diffuse in the plane of the plasma membrane lipid bilayer, with diffusion coefficients ranging from 10^-8 cm² s^-1 for lipids to 10^-10 cm² s^-1 for proteins, mechanisms must exist to prevent the intermixing of membrane molecules from each domain by diffusion. Presented here is a theoretical analysis of the distribution of axonal molecules in both growing and non-growing axons based on two models for the insertion of these molecules into the axonal membrane, namely insertion exclusively at the distal end of the axon, or insertion with equal probability all along the axon. In all cases, assuming that the molecules have a finite half-life in the axonal membrane, compositional differences between the axonal and somatodendritic membranes can be obtained that are similar to those observed in other polarized cells, even in the absence of a physical barrier to prevent the intermixing of membrane molecules. Moreover, our analyses demonstrate that the diffusion of membrane molecules in the plane of the axonal membrane lipid bilayer is a sufficiently slow process to preclude the possibility that membrane molecules are inserted into axonal membranes at a site remote from their final location, and then move to their final locations by diffusion. Thus, in long axons, for membrane molecules that are localized all along the length of the axon, mechanisms must exist for their insertion in the axonal membrane at sites all along the axon, and not just at the distal end.

Axonal growth can be disrupted by various treatments that inhibit the synthesis of membrane components or their delivery by microtubule-based transport. In cultured hippocampal neurons, a direct correlation exists between the synthesis of sphingolipids, and particularly the simplest glycosphingolipid, glucosylceramide, and the ability of growth factors to stimulate axonal growth [S. Boldin, A.H. Futerman, J. Neurochem. 68 (1997) 882-885]. We now demonstrate that dendritic growth in hippocampal neurons also requires ongoing sphingolipid synthesis. Upon incubation with fumonisin B₁ (FB₁), an inhibitor of acylation of sphingoid long-chain bases, dendritic growth rates are ~ 25% slower than those of control cells, resulting in neurons with shorter dendritic arbors and less dendritic branch points per cell, and readily apparent differences in morphology compared to control cells after 10-14 days in culture. In contrast, FB₁ had no effect on the initial growth of the minor processes, which are destined to become dendrites, even in cells in which FB₁ affected the rate of axon growth. Inhibition of sphingolipid degradation, by incubation with conduritol-B- epoxide (an inhibitor of glucosylceramide degradation) had no effect on dendrite or minor process growth at any stage of development, and no aberrant neurite or ectopic dendrite formation was observed. Together, these data demonstrate that normal dendrite growth in hippocampal neurons requires sphingolipid synthesis, although the molecular requirements for sphingolipid synthesis may differ from those in axons.

1-Methylthiodihydroceramide (10 μM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1- methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 μM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L- threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

Studies on the roles of sphingolipids (SLs) and glycosphingolipids (GSLs) at distinct stages of neuronal development have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. Our data demonstrate that SLs and GSLs play at least three distinct roles in regulating neuronal development, namely: (i) ceramide enhances the formation of minor neuronal processes from lamellipodia and the subsequent stage of axonogenesis; (ii) glucosylceramide synthesis, but not the synthesis of higher-order GSLs, is required for normal axon growth and for accelerated axonal growth upon stimulation by growth factors; and (iii) at both of these stages, ceramide at high concentrations can induce apoptotic cell death. Together, these observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth requires glucosylceramide synthesis, perhaps to support an intracellular transport pathway.

Ceramide can be formed by the activity of two general metabolic pathways, the anabolic pathway, in which ceramide is formed by acylation of a sphingoid long chain base, the catabolic pathway, in which ceramide is formed by the degradation of either glycosphingolipids or of sphingomyelin (SM). The anabolic reactions take place in the early compartments of the secretory pathway (the endoplasmic reticulum and the Golgi apparatus) and the catabolic reactions take place either in lysosomes or at the plasma membrane. Work from our and other laboratories has shown that neuronal growth and development can be regulated by manipulating ceramide metabolism. Thus, synthesis of glucosylceramide from ceramide is required for axonal growth in cultured hippocampal neurons, but the formation of ceramide from SM, by a sphingomyelinase activity, stimulates the earliest stages of development in these cells, namely the formation of minor neuronal processes and the initial formation of the axon. Thus, ceramide and its metabolites play distinct roles in the same neuron, depending on the intracellular site of generation of ceramide and on the stage of neuronal development.

Over the past few years, our laboratory has focused on defining the regulatory roles of sphingolipids at various stages of neuronal development. These studies have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. The data demonstrate that sphingolipids play at least three distinct roles in regulating neuronal development, namely (1) that ceramide enhances the formation of minor neuronal processes from lamellipodia, (2) that glucosylceramide synthesis is required for both normal and accelerated axon growth, and (3) that at both of these stages of development, ceramide induces apoptotic cell death at high concentrations. These observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth may require GlcCer synthesis to support an intracellular transport pathway.

A rapid, simple, and efficient chloroform/methanol-free method of isolating gangliosides is described. The nonionic polyoxyethylene detergent, hexaethyleneglycol mono-n-tetradecyl ether (C14EO6), forms clear micellar solutions in water, but two-phase separation can be achieved by centrifugation in the presence of ammonium sulfate at room temperature. A mixture of pure gangliosides, metabolically labeled gangliosides obtained from cultured hippocampal neurons, and gangliosides from rat cerebellar tissue were quantitatively recovered in the detergent-rich upper (coacervate) phase, with a partition coefficient K > 60. Gangliosides were subsequently separated from the detergent using an Iatrobead column prior to analysis by thin-layer chromatography. The procedure described here is as efficient as other methods of ganglioside extraction, such as that using chloroform/ methanol/water/pyridine, but is less time-consuming inasmuch as extraction, purification, and TLC analysis can be completed within 1 day.

Many studies have examined the localization of gangliosides using anti- ganglioside antibodies, although widely differing conclusions have been reached. We now demonstrate that the apparent localization of gangliosides can be greatly influenced by the fixation method. Using monoclonal antibody (MAb) A2B5 (which reacts with a variety of gangliosides), hippocampal neurons were labeled at the cell surface when incubated with the antibody before fixation, but when incubated after fixation the cells displayed a variety of labeling patterns, depending on the fixation method. Biochemical analysis demonstrated that some of the fixatives (particularly acetone and methanol) significantly reduced or completely depleted cellular gangliosides, implying that the immunoreactivity observed with A2B5, and with other antibodies, was not due to gangliosides. When neurons were incubated with an anti-GD1b antibody prefixation, uniform labeling of the plasma membrane was observed, but after ganglioside depletion using biochemical inhibitors of ganglioside synthesis no cell surface labeling was detected. However, even in cells depleted of gangliosides, labeling of both the cell surface and intracellular compartments was observed when the anti-GD1b antibody was applied after fixation. Moreover, after fixation, antibodies to GM4 and GD2 reacted with hippocampal neurons, although these gangliosides are absent from these neurons. In contrast, the JONES antibody (which reacts with 9-O-acetylated GD3) labeled neurons with a similar pattern, essentially irrespective of the fixation method. These observations demonstrate that great care must be taken in assigning gangliosides to specific cell populations or to intracellular locations solely on the basis of use of anti-ganglioside antibodies, and suggest that optimal fixation conditions must be established for each anti- ganglioside antibody.

Previous studies have shown that hippocampal neurons cultured at high density express g-bungarotoxin binding sites and have α7 nicotinic acetylcholine receptor subunit immunoreactivity [Barrantes, G.E., Rogers, A.T., Lindstrom, J. and Wonnacott, S., Brain Res., 672 (1995) 228-236]. We now examine both of these parameters in well-characterized hippocampal neurons cultured at sufficiently low densities to resolve individual neurons and their processes. The specific binding of [α-¹²⁵I]bungarotoxin is first detectable after 3 days in culture and increases during the next 12 days in culture, reaching a maximum of approximately 30,000 binding sites per cell. This is accompanied, over the same timecourse, by an increase in immunoreactivity for two antibodies that specifically bind to the α7 subunit. Both cell bodies and processes were labelled by 9 days in culture. The timecourse of α7-type nicotinic receptor expression resembles that previously described for synapse formation in hippocampal cultures.

To test the hypothesis that neuronal growth requires the synthesis and supply of new membrane components to the growing neurite, we have examined the relationship between the synthesis of sphingolipids and the ability of two growth factors, basic fibroblast growth factor (bFGF) and laminin, to stimulate axonal growth in cultured hippocampal neurons. Both bFGF and laminin stimulate axonal growth by approximately fourfold, but the stimulatory effects of both factors can be abolished completely by two inhibitors of sphingolipid synthesis, fumonisin B₁ and D-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol. By using these inhibitors, together with two stereoisomers of short acyl chain derivatives of ceramide, only one of which is metabolized to glucosylceramide, we demonstrate that ongoing synthesis of glucosylceramide, the simplest glycosphingolipid, is a prerequisite for both bFGF and laminin to stimulate axon growth. These data imply that the ability of a growth factor to stimulate neuronal growth is dependent on the synthesis of an essential membrane lipid.

Lasalocid (X-537A) is a polyether ionophore compound (Westly et al., 1970) that was isolated from streptomyces lasaliensis in 1951 (Berger et al., 1951). It is a lipid-soluble material which facilitates the passage of divalent and monovalent ions through lipophilic-biological membranes (Westley, 1977; Reed, 1982; Aebi. 1989). Lasalocid is used as a broad-spectrum anticoccidial agent and has been approved for use as a coccidiostat for chickens, and as a growth promoter for cattle (Bergen et al., 1984). However, lasalocid residues in commercial food were found to cause a paralytic syndrome in dogs (Safran et al., 1993a) and overdose in poultry feed caused neuromuscular deficit in chickens

Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B₁ (FB₁) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB₁ affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB₁ resulted in a decrease in synthesis of ganglioside GM₃, the major glycosphingolipld in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB₁-treated cells was 45% less than control cells. FB₁ had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB₁. All the effects of FB₁ on cell morphology could be reversed by addition of ganglioside GM₃ even in the presence of FB₁, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB₁ blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM₃ could not reverse the effects of FB₁ on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.

Sphingolipids (SLs) are important structural and regulatory components of neuronal plasma membranes. Previous studies using fumonisin B₁, an inhibitor of the synthesis of ceramide, the precursor of all SLs, demonstrated that ceramide synthesis is required to sustain axonal growth in hippocampal neurons (Harel and Futerman, 1993; Schwarz et al., 1995) and dendritic growth in cerebellar Purkinje cells (Furuya et al., 1995). We now show that ceramide plays distinct roles at different stages of neuronal development. (1) During axon growth, ceramide must be metabolized to glucosylceramide (GlcCer) to sustain growth. Thus, whereas D-erythro- ceramide, which is metabolized to GlcCer, is able to antagonize the disruptive effects of fumonisin B₁ on axon growth, L-threo-ceramide, which is not metabolized to GlcCer, is ineffective. (2) The formation of minor processes from lamellipodia can be stimulated by incubation with short-acyl chain analogs of ceramide that are active in ceramide-mediated signaling pathways, or by generation of endogenous ceramide by incubation with sphingomyelinase. However, GlcCer synthesis is not required for this initial stage of neuronal development. (3) During minor process formation and during axon growth, incubation with high concentrations of ceramide or sphingomyelinase, but not dihydroceramide, induces apoptosis. Together, these observations are consistent with the possibility that minor process formation and apoptosis can be regulated by ceramide-dependent signaling pathways and that the decision whether to enter these diametrically opposed pathways depends on intracellular ceramide concentrations. In contrast, axonal growth requires the synthesis of GlcCer from ceramide, perhaps to support an intracellular transport pathway.

Various glycolipid-binding toxins are internalized from the cell surface to the Golgi apparatus. Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity. We now demonstrate that rhodamine (Rh)-CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG-2 cells and that the A subunit is essential for retrograde transport to the ER. In addition, the rate of internalization of Rh-CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature. The Golgi apparatus was labeled in almost all 1-day-old neurons after

In order to study the endocytosis of membrane lipids during the development of neuronal polarity, we examined the internalization of a short acyl chain fluorescent derivative of ganglioside GM₁, N-(6-(4-nitrobenz-2-oxa-1,3-diazole-7-yl)-aminohexanoyl)-GM₁ (C₆-NBD-GM₁), in hippocampal neurons cultured at low density, C₆-NBD-GM₁ was internalized by temperature- and energy-dependent mechanisms, and after short times of incubation, accumulated in endosomes in the axon, cell body and dendrites of neurons maintained for up to 4-5 days in culture. C₆-NBD-GM₁ was subsequently transported in a retrograde direction to a pool of recycling endosomes in the cell body, with little transport to lysosomes, as indicated by the lack of degradation of C₆-NBD-GM₁ even after long times, and the re-appearance of intact C₆-NBD-GM₁ at the cell surface after recycling; similarly, little degradation of C₆-NBD-GM₁ was detected in N18TG-2 neuroblastoma cells. In hippocampal neurons maintained for longer than 6 days in culture, there was little internalization of C₆-NBD-GM₁ along the length of axons, but the amount of endocytosis from dendrites was similar to that observed in younger neurons. These results demonstrate that gangliosides turnover rapidly in dendritic membranes at all stages of neuronal development, whereas ganglioside turnover in axons is much less rapid, at least in mature, polarized neurons.

Changes in the levels and types of gangliosides occur during neuronal differentiation and development, but no studies have correlated these changes with defined events in neuronal morphogenesis. Here, we have analyzed the relationship between ganglioside synthesis and the development of axons and dendrites in polarized neurons, using hippocampal neurons cultured in such a way that axons and dendrites are generated by a defined sequence of events and in which there is virtually no contamination by glial cells. Neurons were labeled with [4,5-³H]dihydrosphingosine, which was rapidly incorporated into cells and metabolized to ³H-labeled glycosphingolipids. The rate of ³H- labeled glycosphingolipid synthesis was directly proportional to the initial rate of [4,5-³H]dihydrosphingosine uptake and was linear versus time for up to 9 h of incubation. The major changes in ³H-labeled ganglioside synthesis occurred during the period of axonogenesis and rapid axon growth. During axonogenesis, there was a significant increase in the synthesis of complex gangliosides (i.e. G(M1), G(D1a), G(D1b), and G(T1b)) with a corresponding reduction in the synthesis of glucosylceramide and ganglioside G(D3). During the stage of rapid axon growth, the ratio of a- to b-series gangliosides increased significantly. However, during dendritogenesis, dendrite growth, and synaptogenesis, there was little change in ganglioside synthesis, with a small and gradual increase in the ratio of a- to b-series gangliosides and an increase in the synthesis of gangliosides G(D1a) and G(T1b). These results indicate that despite major changes in neuronal morphology and functionality as neurons mature, changes in ganglioside synthesis are restricted to early stages of neuronal development, namely axonogenesis and rapid axon elongation.

Recent studies have shown that axonal growth is disrupted by treatments that block the synthesis of membrane components or their delivery by microtubule-based transport. This implies that a continuous supply of newly synthesized membrane components is necessary to sustain growth. In contrast, no clear consensus has yet been achieved about the site of insertion of new membrane components in the membrane of the growing axon, despite the application of new and refined biophysical and molecular techniques to the study of this issue. Until the site of insertion of new membrane components is resolved, little progress can be made in defining the feedback mechanisms by which the supply of new membrane components is co-ordinated with the demands of growth, particularly in cases where the dynamics of neurite growth change from minute to minute.

Evidence exists that some newly-synthesized membrane components are inserted into the membrane of the growing axon at the growth cone. We now examine the site of insertion of a glycosylphosphatidylinositol-anchored protein, TAG-1/axonin-1. The protein was cleaved from the plasma membrane by phosphatidylinositol-specific phospholipase C. Newly-synthesized TAG-1/axonin-1 was subsequently detected along the entire length of the axonal membrane, with no evidence for preferential insertion at the distal end. These results raise the possibility that different subsets of proteins are inserted at different locations in axonal membranes.

The idea that the transport and sorting of glycosylphosphatidylinositol (GPI)-anchored proteins depends on their interaction with glycosphingolipids was first proposed five or six years ago. Until recently, only circumstantial evidence was available to support this suggestion. During the past year, compelling support for this hypothesis has been provided by observations that inhibition of sphingolipid synthesis reduces the rate of transport of GPI-anchored proteins in yeast, and abolishes the polarized sorting of a GPI-anchored protein in epithelia.

Sphingolipids, particularly gangliosides, are enriched in neuronal membranes where they have been implicated as mediators of various regulatory events. We recently provided evidence that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons, inhibition of ceramide synthesis by Fumonisin B₁ (FB₁) disrupted axonal outgrowth (Harel, R. and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481). We now analyze further the relationship between neuronal growth and sphingolipid metabolism by examining the effect of an inhibitor of glucosylceramide synthesis, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and by examining the effects of both FB₁ and PDMP at various stages of neuronal development. No effects of FB₁ or PDMP were observed during the first 2 days in culture, but by day 3 axonal morphology was significantly altered, irrespective of the time of addition of the inhibitors to the cultures. Cells incubated with FB₁ or PDMP had a shorter axon plexus and less axonal branches. FB₁ appeared to cause a retraction of axonal branches between days 2 and 3, although long term incubation had no apparent effect on neuronal morphology or on the segregation of axonal or dendritic proteins. In contrast, incubation of neurons with conduritol B-epoxide, an inhibitor of glucosylceramide degradation, caused an increase in the number of axonal branches and a corresponding increase in the length of the axon plexus. A direct correlation was observed between the number of axonal branch points per cell and the extent of inhibition of either sphingolipid synthesis or degradation. These results suggest that sphingolipids play an important role in the formation or stabilization of axonal branches.

Cholera toxin (CT) consists of a pentameric B subunit which binds with high affinity to ganglioside GM₁, and an A subunit which stimulates adenylate cyclase, resulting in the elevation of cAMP. We now examine the effect of cationic amphiphilic drugs (CADs) on the internalization of rhodamine (Rh)-CT in cultured hippocampal neurons. CADs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and on endosomes (Wang, L.-H., Rothberg, K. G., and Anderson, R. G. W. (1993) J. Cell Biol. 123, 1107-1117). Rh-CT was internalized by an energy- and temperature-dependent (presumably vesicular) mechanism to the Golgi apparatus. Internalization to the Golgi apparatus was completely but reversibly blocked by CADs, and the ability of CT to stimulate the elevation of cAMP was significantly reduced. In control cells, cAMP levels were elevated 2.3-fold after 20 min of incubation with CT, but in CAD- treated cells cAMP levels were only elevated 1.3-fold. The effect of CADs on CT internalization was not due to a direct effect of CADs on the Golgi apparatus. Our data demonstrate that CADs inhibit vesicular transport of CT to the Golgi apparatus and imply that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both pathways are inhibited by CADs.

Keywords: BREFELDIN-A; RAT-LIVER; HIPPOCAMPAL-NEURONS; CYTOSOLIC SURFACE; GOLGI-APPARATUS; SPHINGOID BASE; BIOSYNTHESIS; GLUCOSYLCERAMIDE; SPHINGOLIPIDS; SPHINGOMYELIN

The role of the Golgi apparatus in the motile activity of fibroblasts was examined with brefeldin A (BFA), which disrupts the Golgi apparatus in a variety of cells. Upon incubation with BFA, Swiss mouse 3T3 fibroblasts lost their typical polarized morphology, in which the leading edge is characterized by intensive lamellipodia formation. BFA affected cell assymetry as demonstrated by a decrease in the morphometric indices, dispersion, and elongation. After BFA treatment, cells showed little protrusional activity and did not form a dense actin network at the leading edge, and consequently the rate of cell migration into an experimental wound was significantly reduced. In addition, BFA prevented an increase in pseudopodial activity and prevented the formation of long processes induced by phorbol 12-myristate 13-acetate. The effects of BFA on cell shape and protrusional activity were quantitatively similar to those observed with the microtubule-disrupting agent nocodazole, although BFA had no effect on microtubule integrity. These results suggest that the integrity of both the Golgi apparatus and microtubules is necessary for the generation and maintenance of fibroblast asymmetry, which is a prerequisite for directed cell migration.

Sphingolipids have been implied to play important roles in the functioning of the secretory pathway, in which they may be involved in regulating protein transport and retention in the endoplasmic reticulum (ER) and Golgi apparatus, in directing transport, from the tvans-Golgi network to the plasma membrane (PM) of the polarized cells. In this chapter, studies published during the past several years that have demonstrated that the early steps of sphingolipid synthesis are compartmentalized in the ER and Golgi apparatus have been discussed. In particular, the chapter discusses the sites and membrane topology of three enzymes that reside in these compartments-acyl CoA namely, sphinganine N-acyltransferase (dihydroceramide synthase), UDP-glucose:ceramide glucosyltransferase [glucosylceramide (GlcCer) synthase], and phosphatidylcholine (PC) ceramide choline phosphotransferase [sphingomyelin (SM) synthase]. The central molecule in sphingolipid metabolism is ceramide. Ceramide is formed by the condensation of serine and palmitoyl CoA, followed by the reduction of 3-ketosphinganine, acylation of the amide group of sphinganine, and finally dehydrogenation of dihydroceramide. The reactions involved in the process are ceramide synthesis, Sphingomyelin synthesis, and glucosylceramide synthesis. Proteases and membrane impermeable inhibitors have been used to demonstrate that dihydroceramide synthase is oriented with its active site facing the cytosol. After synthesis at the cytosolic surface of the ER, dihydroceramide is dehydrogenated to ceramide. Because the intracellular localization of dihydroceramide dehydrogenase has not been determined, whether dehydrogenation occurs before or after transport out of the ER is not clear.

A number of recent findings concerning the metabolism of sphingolipids (SL) and the role of SLs and glycosphingo-lipids in various aspects of cell function are discussed in this mini-review. Particular attention is paid to three areas, namely i) the intracellular sites and topology of SL synthesis, ii) the transport of SLs by vesicular and non-vesicular mechanisms through the secretory pathway, and iii) the physiological consequences of inhibiting SL synthesis and of depleting SL levels. Experimental strategies are suggested that may yield useful information to advance further our understanding of the roles that sphingolipids may play in cell function.

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[¹⁴C]hexanoyl)-D-erythro-glucosylsphingosine ([¹⁴C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent and was hydrolyzed to N-(1-[¹⁴C]hexanoyl)-D-erythro-sphingosine ([¹⁴C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [¹⁴C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 ± 4.6 nmol of [¹⁴C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 ± 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) J. Biol. Chem. 264, 14354-14360). We now demonstrate that the addition of both Cer-1-phosphate (Cer-1-P) and a short-acyl chain analog of Cer-1-P, N-hexanoylsphingosine-1-phosphate (C₆-Cer-1-P) to cultured cells and a variety of subcellular fractions results in rapid degradation to Cer and C₆-Cer, respectively. The Cer-1-P phosphatase activity is enriched in a rat liver plasma membrane fraction and appears to be distinct from the phosphatase that hydrolyzes phosphatidic acid (PA), PA phosphohydrolase, as shown by the difference in sensitivity of Cer-1-P and PA hydrolysis to propranolol, detergent, and heat treatment. Moreover, the K_m of Cer-1-P hydrolysis is 10-fold lower than the K_m of PA hydrolysis in plasma membrane. PA is a noncompetitive inhibitor of Cer-1-P hydrolysis, with an inhibition constant 1-1.5-fold higher than the K_m of Cer-1-P hydrolysis. In contrast, Cer-1-P does not inhibit PA hydrolysis. Finally, we describe the synthesis of a novel analog of Cer-1-P which is not hydrolyzed in vitro and in vivo and is internalized in cultured cells by endocytosis. These results are discussed in relation to the possible roles of Cer-1-P in regulating intracellular levels of Cer.

Neuronal growth is regulated by both extracellular and cellular determinants and is believed to proceed by the addition of new membrane material at the growth cone. To determine whether lipid synthesis is necessary to maintain neuronal growth, we have examined the effect of Fumonisin B₁, an inhibitor of ceramide synthesis, on the development of cultured hippocampal neurons. Fumonisin B₁ inhibits ceramide synthesis in hippocampal neurons both in vivo and in vitro. Ganglioside synthesis and content was reduced after Fumonisin B₁ treatment, and ganglioside GD_1b was not detectable at the cell surface by immunofluorescence. Inhibition of sphingolipid synthesis by Fumonisin B₁ had a significant effect on axonal growth. Between days 2-3 in culture, mean axon length increased from 170 to 240 μm, but in Fumonisin-treated cells, no increase in axon length was observed. Addition of a fluorescent derivative of ceramide together with Fumonisin B₁ reversed this effect, confirming that Fumonisin B₁ acts via inhibition of ceramide synthase. Further, ceramide by itself caused a significant increase in axon length. We discuss three possible mechanisms by which inhibition of sphingolipid synthesis could disrupt axonal growth, among them the possibility that ongoing sphingolipid synthesis is necessary to provide new membrane material to the growing axon.

Ceramide, a key intermediate in sphingolipid metabolism, is synthesized by acylation of sphinganine followed by dehydrogenation of dihydroceramide to ceramide. Using radioactive sphinganine, we have examined the site and topology of dihydroceramide synthesis in well-characterized subcellular fractions from rat liver. [4,5-H-3]Sphinganine was introduced as a complex with BSA and was metabolized to [4,5-H-3]dihydroceramide upon incubation of rat liver homogenates or microsomes with fatty acyl CoA. Conditions were established in a detergent-free system in which dihydroceramide synthesis was not limited by either substrate availability or by amounts of microsomal protein or reaction time. The distribution of dihydroceramide synthesis was found to exactly parallel that of an endoplasmic reticulum (ER) marker upon subfractionation of microsomes. and no endogenous activity was detected in either purified Golgi apparatus or plasma membrane fractions. Limited protease digestion demonstrated that sphinganine N-acyltransferase is localized at the cytosolic surface of intact ER-derived vesicles. These results are discussed with regard to the subsequent transport of (dihydro)-ceramide from the ER to sites of further metabolism in a pre-Golgi apparatus compartment and in the cis and medial cisternae of the Golgi apparatus.

An advantage of using N-([1-¹⁴C]hexanoyl)sphingolipids to assay sphingolipid metabolism is their ability to rapidly and spontaneously transfer into biological membranes without destroying membrane integrity. This property allows analysis of the activity of enzymes of sphingolipid metabolism under conditions in which the rate of product formation is not limited by availability of substrate, as is often the case with naturally occurring lipids whose rates of spontaneous transfer are extremely slow.^21.22 Thus, the use of N-([1-¹⁴C]hexanoyl)sphingolipids provides an alternative means for studying sphingolipid metabolism in vitro.

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-¹⁴C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.

A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2·0 Å (1 Å = 0·1 nm) resolution. They were found to be orthorhombic, space group P222₁, with a = 163·4(±0·2) A ̊, b = 112·1(±0·2) A ̊, c = 81·3(±0·1) A ̊.

The susceptibility to phosphatidylinositol-specific phospholipase C of the membrane associated acetylcholinesterase (AChE) forms of Xenopus laevis skeletal muscle was examined. This treatment released almost all the detergent-soluble AChE species from muscle homogenates. Sucrose gradient analysis showed that the released acetylcholinesterase form corresponds to a hydrophilic G₂ dimer, indicating that this dimer has a glycolipid anchoring domain which contains phosphatidylinositol.

Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by posttranslational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagenlike fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparinlike proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxylterminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.

The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G₂ form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G₂ form in back muscle is sensitive to PIPLC, whereas the G₄ tetrameter of Torpedo brain is completely resistant.

Acetylcholinesterase (AChE) exists in multiple molecular forms that differ in their quaternary structure and mode of attachment to the cell surface. Attachment is achieved by posttranslational modification of the catalytic subunits. Two such modifications are described. One modification involves the attachment to subunit tetramers, via disulfide bridges, of a collagenlike fibrous anchor that serves to attach the enzyme, by ionic interactions, to the extracellular matrix. A second modification involves the covalent attachment of a single phosphatidylinositol residue, which serves to anchor the enzyme hydrophobically in the lipid bilayer of the plasma membrane. The detailed molecular structure, assembly, and modes of anchoring of these two classes of AChE are discussed.

Our earlier evidence suggested that both acetylcholinesterase and alkaline phosphatase are anchored to the cell surface via covalently-attached phosphatidylinositol. We now present chemical data, based upon a nitrous acid deamination reaction, showing that in both proteins the phosphatidylinositol moiety is attached through glycosidic linkage to a sugar residue bearing a free amino group.

Recently a novel mechanism has been described for the hydrophobic attachment of proteins to membranes, which is shared by membrane proteins of widely differing origin and function. The hydrophobic anchor is the 1,2-diacylglycerol moiety of a phosphatidylinositol molecule which is covalently attached to the polypeptide chain. These findings have important structural and functional implications.

Abstract: Phosphatidylinositolspecific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.OS upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably of soubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K⁺ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a ∼ 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.

The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

The hydrophobic, membrane-bound form of Torpedo acetylcholinesterase is specifically solubilized by a phosphatidylinositol-specific phospholipase C, suggesting that acetylcholinesterase is bound to the membrane via a direct and specific interaction with phosphatidylinositol (Futerman et al., Biochem. J. (1985) 226, 369377). Here we demonstrate the presence of covalently bound inositol in the membrane-anchoring domain of purified Torpedo acetylcholinesterase. Upon removal of this domain, levels of inositol are reduced to only 1520% of those found in the intact enzyme. The results presented strongly support our suggestion that phosphatidylinositol is indeed involved in anchoring acetylcholinesterase to the plasma membrane.

A dimeric form of acetylcholinesterase from the electric organ of Torpedo californica was solubilized by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The solubilized enzyme had a sedimentation coefficient of 7.3S which was not modified by detergents. The high salt-soluble asymmetric forms of acetylcholinesterase were not solubilized by the phospholipase. Our data suggest that the hydrophobic dimer of acetylcholinesterase may be associated with the plasma membrane through a specific interaction involving phosphatidylinositol.