Large-scale proteomics analysis of five brain regions from Parkinson's disease patients with a GBA1 mutation.

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-ß-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.

The Sphinx and the egg: Evolutionary enigmas of the (glyco)sphingolipid biosynthetic pathway.

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.

Proteomics analysis of the brain from a Gaucher disease mouse identifies pathological pathways including a possible role for transglutaminase 1.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid ß-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.

Q-RAI data-independent acquisition for lipidomic quantitative profiling.

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 (LC-MS) 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 fats of the matter: Lipids in prebiotic chemistry and in origin of life 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.

Cell lipid droplet heterogeneity and altered biophysical properties induced by cell stress and metabolic imbalance.

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.

Computational design and molecular dynamics simulations suggest the mode of substrate binding in ceramide synthases.

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.

Design of a stable human acid-ß-glucosidase: towards improved Gaucher disease therapy and mutation classification.

Acid-ß-glucosidase (GCase, EC3.2.1.45), 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 toward 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.

Elevation of gangliosides in four brain regions from Parkinson's disease patients with a GBA mutation.

A number of genetic risk factors have been identified over the past decade for Parkinson's 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 sphingolipid anteome: implications for evolution of the sphingolipid metabolic pathway.

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.

Generation of a ceramide synthase 6 mouse lacking the DDRSDIE C-terminal motif.

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 CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA 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.

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

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.

Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.

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.

16pdel lipid changes in iPSC-derived neurons and function of FAM57B in lipid metabolism and synaptogenesis.

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.

GBA mutations, glucosylceramide and Parkinson's disease.

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.

Dependence of ABCB1 transporter expression and function on distinct sphingolipids generated by ceramide synthases-2 and -6 in chemoresistant renal cancer.

Oncogenic multidrug resistance 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 (SM)-rich domains. As ceramides are the sole source for SMs, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-Seq databases, we found that 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 (approximately twofold) in chemoresistant ABCB1high (A498, Caki-1) compared with chemosensitive ABCB1low (ACHN, normal human proximal convoluted tubule cell) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias toward CerS2-associated C20:0/C20:1-ceramides compared with CerS5/6-associated C14:0/C16:0-ceramides (2:1). SMs 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. 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 SM composition to ultra long-chain species (C22-C26). Inhibitors of endoplasmic reticulum-associated degradation (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/SM species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse multidrug resistance in renal cancers.

A novel C-terminal DxRSDxE motif in ceramide synthases involved in dimer formation.

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-Lag-CLN8 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 CerS6Δ338-344 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.

Silencing of ceramide synthase 2 in hepatocytes modulates plasma ceramide biomarkers predictive of cardiovascular death.

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.

The Complex Tail of Circulating Sphingolipids in Atherosclerosis and Cardiovascular Disease.

Sphingolipids (SLs) are critical players in a number of cellular processes and have recently been implicated in a large number of human diseases, including atherosclerosis and cardiovascular disease (CVD). SLs are generated intracellularly in a stepwise manner, starting with the generation of the sphingoid long chain base (LCB), followed by N-acylation of the LCB to form ceramide, which can be subsequently metabolized to sphingomyelin and glycosphingolipids. Fatty acids, which are taken up by cells prior to their activation to fatty acyl-CoAs, are used in 2 of these enzymatic steps, including by ceramide synthases, which use fatty acyl-CoAs of different chain lengths to generate ceramides with different N-acyl chain lengths. Recently, alterations in plasma ceramides with specific N-acyl chain lengths and degrees of saturation have emerged as novel biomarkers for the prediction of atherosclerosis and overall cardiovascular risk in the general population. We briefly review the sources of plasma SLs in atherosclerosis, the roles of SLs in CVD, and the possible use of the "ceramide score" as a prognostic marker for CVD.