David A. Priestman

Background: Parkinson’s disease (PD) is a prevalent neurodegenerative disorder characterized by progressive motor dysfunction and broad cellular impairment, including significant disruptions in lysosomal function, lipid metabolism, and intracellular trafficking. Glycosphingolipids (GSLs), critical for various cellular processes, depend on effective lysosomal degradation. Aberrant GSL metabolism has been linked to PD pathology, and glycoprotein non-metastatic melanoma protein B (GPNMB) has emerged as a biomarker associated with lysosomal dysfunction and lipid imbalance in PD Objectives: To assess the relationship between GPNMB and GSL levels in cerebrospinal fluid (CSF) and plasma from PD patients and controls within the BioFIND cohort. We also investigated potential sex differences and associations with PD-related biomarkers such as α-synuclein Methods: GSL species and GPNMB protein levels were quantified using high-performance liquid chromatography (HPLC) and ELISA assays, respectively, in matched CSF and plasma samples from PD patients and controls Results: Levels of the paraglobosides GSL species, alpha-2,3SpG and pGb were significantly elevated in the plasma of PD patients compared to healthy controls, while levels of the ganglioside GD1a and the lacto-series GSL, Le ^b combined (GD1a + Le ^b ), were significantly reduced in PD. GPNMB levels positively correlated with several GSL species in both plasma and CSF. Plasma GSLs and GPNMB concentrations were significantly higher in females compared to males, independent of PD diagnosis. CSF GPNMB correlated positively with age and α-synuclein concentrations Interpretation: Our findings confirm that GSL metabolism is altered in PD. They also highlight significant sex-based biochemical variations in GSL and GPNMB levels, emphasizing the need for sex-specific analyses in PD biomarker research. The relationship between GSLs and GPNMB supports their potential as interconnected biomarkers of lipid pathology in PD.

Gangliosides are abundant neuronal glycosphingolipids, yet their roles in organizing the plasma membrane and supporting neuronal function remain poorly defined. Mutations in the biosynthetic enzymes ST3GAL5 or B4GALNT1 cause severe neurodevelopmental disorders, yet their cellular consequences are unclear. Using isogenic human iPSC-derived cortical neurons, we show that loss of these enzymes eliminates major neuronal gangliosides but produces strikingly divergent outcomes. ST3GAL5 deficiency reprograms the glycosphingolipid repertoire toward non-neuronal species and abolishes network-level electrical activity. In contrast, B4GALNT1-deficient neurons retain near-normal excitability, supported by accumulation of simple sialylated precursors (GM3/GD3). Quantitative proteomics reveals a profound loss of plasma membrane proteins, including ion channels and synaptic organizers, only in ST3GAL5-deficient neurons. These findings identify sialylated glycosphingolipids as essential scaffolds for plasma membrane organization and neuronal excitability, providing a mechanistic basis for the severe phenotype caused by loss of GM3 synthase in humans. TEASER: Human neuronal models reveal why loss of GM3 synthase causes severe neurodevelopmental disease

Glycosphingolipids (GSL) are important bioactive components of cellular membranes. Complex GSLs, containing sialic acid residues are known as gangliosides and are highly abundant in the brain. Diseases of ganglioside metabolism often result in severe, early-onset neurodegeneration. The ganglioside GM2 is the substrate of the hydrolytic lysosomal β- hexosaminidase A (HexA) enzyme and when subunits of this enzyme are non-functional, GM2 lipid accumulates in cells leading to the GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases. We have developed high-quality i3Neuron-based models of Tay-Sachs and Sandhoff diseases, that demonstrate storage of GM2, formation of membrane whorls and accumulation of endolysosomal proteins consistent with disease phenotypes. Importantly, in addition to lysosomal dysfunction, the composition of the plasma membrane (PM) is significantly impacted in these diseases with changes in the abundance of both lipids and proteins. The changes to the PM proteome are driven in part by exocytosis of lysosomal material resulting in the aberrant accumulation of lysosomal proteins and lipids on the cell surface. The altered abundance of GM2 at the PM was striking, bringing the abundance of this precursor lipid up to that of the common neuronal gangliosides. Furthermore, the PM profiling identifies significant changes in synaptic protein abundances with direct functional impact on neuronal activity including rapid electrical firing consistent with neuronal hyperactivity. This work provides mechanistic insights into neuronal dysfunction in the GM2 gangliosidoses and highlights that these are also severe PM disorders. This work has broad implications for other lysosomal storage disorders and late-onset neurodegenerative diseases involving sphingolipid dysregulation.

Identification of genetic modulators of lysosomal enzyme activities and glycosphingolipids (GSLs) may facilitate the development of therapeutics for diseases in which they participate, including Lysosomal Storage Disorders (LSDs). To this end, we used a systems genetics approach: we measured 11 hepatic lysosomal enzymes and many of their natural substrates (GSLs), followed by modifier gene mapping by GWAS and transcriptomics associations in a panel of inbred strains. Unexpectedly, most GSLs showed no association between their levels and the enzyme activity that catabolizes them. Genomic mapping identified 30 shared predicted modifier genes between the enzymes and GSLs, which are clustered in three pathways and are associated with other diseases. Surprisingly, they are regulated by ten common transcription factors, and their majority by miRNA-340p. In conclusion, we have identified novel regulators of GSL metabolism, which may serve as therapeutic targets for LSDs and may suggest the involvement of GSL metabolism in other pathologies.

Background: Alteration in glycosphingolipids (GSLs) in Parkinson's disease (PD) still needs to be determined.Objectives: We evaluated if PD subjects show abnormal GSLs levels compared to healthy controls (HC) and if GSLs correlate with clinical features.Methods: We analyzed GSLs and glucosylceramide (GlcCer) in plasma using two normal‐phase high‐performance liquid chromatography assays; clinico‐demographic data were extracted.Results: Eighty PD subjects and 25 HCs were analyzed. Levels of GlcCer, GD1b, Gb4, GalNAcGA1, and b‐series were higher in PD patients than in HCs; total GSLs, GT1b, GM1a, GM3, GM2, and a‐series levels were lower in PD patients than in HCs. Changes in GSLs were present in PD subjects, with GlcCer levels similar to those in HCs. The results were similar after excluding certain GBA1 mutation carriers. Movement Disorder Society Unified Parkinson's Disease Rating Scale, Part III, correlated with Gb4 and Montreal Cognitive Assessment with GD1b levels.Conclusions: Multiple GSL abnormalities in plasma were detected in patients with and without GlcCer changes, indicating a broader shift in lipid homeostasis. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

The majority of mucopolysaccharidosis IIIC (MPS IIIC) patients have missense variants causing misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), which are potentially treatable with pharmacological chaperones. To test this approach, we generated a novel HgsnatP304L mouse model expressing misfolded HGSNAT Pro304Leu variant. HgsnatP304L mice present deficits in short-term and working/spatial memory 2–4 mo earlier than previously described constitutive knockout Hgsnat-Geo mice. HgsnatP304L mice also show augmented severity of neuroimmune response, synaptic deficits, and neuronal storage of misfolded proteins and gangliosides compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons further reduced levels of synaptic proteins. Memory deficits and majority of brain pathology were rescued in mice receiving HGSNAT chaperone, glucosamine. Our data for the first time demonstrate dominant-negative effects of misfolded HGSNAT Pro304Leu variant and show that they are treatable by oral administration of glucosamine. This suggests that patients affected with mutations preventing normal folding of the enzyme can benefit from chaperone therapy.

It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson’s disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.

Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2 ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutral glycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers are similar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice.