Biology subjects

Priestman, D. A.

Publications and source records attributed to Priestman, D. A..

5 recordsLinked to original sources

bioRxiv2026

Human iPSC Models of Ganglioside Deficiency Reveal a Sialylated Lipid Requirement for Plasma-Membrane Organization and Neuronal Activity

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. TEASERHuman neuronal models reveal why loss of GM3 synthase causes severe neurodevelopmental disease

cell biology↗

bioRxiv2024

Molecular Insights into Neuronal Dysfunction in GM2 Gangliosidoses

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 {beta}- 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.

neuroscience↗

bioRxiv2023

Compartmentalisation proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL help facilitate elimination of damaged membrane and cytosolic proteins, protein aggregates, membranous organelles and also play an important role in calcium signalling. Despite the importance of EL, their specific role in cardiovascular disease is not well understood. In particular, its unclear how EL contribute to atrial pathology over longer time frames. To shed light on this question, we conducted a comprehensive analysis that involved proteomics, transcriptomics, integrated analysis, electron tomography, western blotting, and enzyme assays. To identify the role of EL in atrial fibrillation (AF), we applied a recently published organelle protein isolation method. We used this method to study biopsies from AF goat model and analyse the EL-specific proteins and pathways involved in this condition. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates (TL), including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophago-vacuole formation, irregularly shaped mitochondria, and glycogen deposition, which provide insights into the ELs contribution to AF and related pathways and molecular mechanisms. Overall, our findings suggest that EL play an important role in the development of AF over longer time frames, and provide a more detailed understanding of the underlying molecular processes involved.

molecular biology↗

bioRxiv2021

Oral Glucosamine Ameliorates Aggravated Neurological Phenotype in Mucopolysaccharidosis III Type C Mouse Model Expressing Misfolded HGSNAT Variant

ObjectiveOver 55% of mucopolysaccharidosis IIIC (MPS IIIC) patients have at least one allelic missense variant responsible for misfolding of heparan sulfate acetyl-CoA:-glucosaminide N- acetyltransferase (HGSNAT). These variants are potentially treatable with pharmacological chaperones, such as a competitive HGSNAT inhibitor, glucosamine. Since the constitutive HGSNAT knockout mice, we generated previously cannot be used to test such strategy in vivo, we generated a novel model, the HgsnatP304L strain, expressing misfolded mutant HGSNAT with human missense mutation Pro311Leu (Pro304Leu in the mouse enzyme). ResultsHgsnatP304L mice present deficits in short-term (novel object recognition test) and working/spatial (Y-maze test) memory at 4 months of age, 2-4 months earlier than previously described gene-targeted Hgsnat-Geo mice, which lack HGSNAT protein. HgsnatP304L mice also show increased severity of synaptic deficits in CA1 neurons, and accelerated course of CNS pathology including neuronal storage of heparan sulfate, accumulation of misfolded proteins, increase of simple gangliosides, and neuroinflammation as compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat- Geo neurons aggravated reduction of synaptic proteins. Memory deficits and majority of pathological changes in the brain were rescued in mice receiving daily doses of oral glucosamine. InterpretationAltogether, our data for the first time demonstrate dominant-negative effects of the misfolded HGSNAT Pro304Leu variant and show that these effects are treatable by oral administration of glucosamine, suggesting that patients, affected with missense mutations preventing normal folding of the enzyme, could benefit from chaperone therapy.

animal behavior and cognition↗

bioRxiv2021

Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

The importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

molecular biology↗