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Phenotypic characterisation of human iPSC neuronal models of GM2 gangliosidoses https://doi.org/10.17863/CAM.122830 Nicholson, Alex 0000000254754874 Deane, Janet Gangliosides are crucial components on the outer leaflet of the plasma membrane of many cells, especially neurons. Their functions are broad and varied but their high abundance in neurons leaves these cells especially vulnerable to the effects of their accumulation in ganglioside lysosomal storage disorders (LSD). The GM2 gangliosidoses Tay-Sachs and Sandhoff disease are a type of LSD, resulting from the inability of the lysosome to catabolise the breakdown of the ganglioside GM2. This is due to a loss or mutation of either the HEXA or HEXB genes which form the two subunits of the heterodimeric β-Hexosaminidase A enzyme (βHexA). The pathology of this disease involves a period of normal growth and development, followed by a period of neurodegeneration, resulting in premature death. However, on a cellular level, how the lysosomal accumulation of GM2 leads to neuronal cell death is not well understood. I have generated a model of these diseases using an inducible, human stem cell-based neuronal cell line (i3N). This isogenic cellular system allows for rapid, large-scale growth of stem cell-derived cortical glutamatergic neurons, enabling experiments that require large amounts of input material such as mass spectrometry-based proteomic analysis. Utilising CRISPRi, I have knocked down the expression of HEXA or HEXB to disease relevant levels. Analysis of gene expression and enzyme activity validates the loss of βHexA, whilst profiling of the ganglioside repertoire indicates massive accumulation of the GM2 ganglioside, increasing over time. Further validation of these cell lines using fluorescence and electron microscopy confirms an abundance of enlarged lysosomes containing the GM2 ganglioside with multilamellar lysosomal substructures typical of these diseases. Furthermore, proteomic analysis of these cells reveals that accumulation of GM2 hugely increases abundance of a subset of lysosomal proteins, especially those involved in lysosomal exocytosis and lipid transport. Importantly, I have identified that in addition to intracellular GM2 accumulation, the ganglioside profile of the plasma membrane of these neurons also shows accumulation of GM2, likely due to fusion of the lysosomal compartment with the PM. Proteomic analysis of changes specifically at the plasma membrane has identified significant changes in abundances of synaptic proteins. Synaptic signalling deficits or changes have been implicated in other lysosomal storage disorders and may be the root cause for the neurodegeneration seen in this disease. To address this, I have measured the electrical signalling of these cells V using a Multi Electrode Array and show, for the first time, synchronous network signalling in i 3Neurons and an alteration of this signalling in GM2 gangliosidosis neurons. To address which disease phenotypes are due to specific accumulation of GM2, I have also generated a GM1 gangliosidosis i3N line and identify many shared changes between these two closely related diseases. Finally, to exemplify the usefulness of using human neurons to correctly test potential treatments for these diseases, I trial the use of ML-SA5, a drug shown to induce lysosomal exocytosis in non-neuronal cell types and proposed as a treatment for neurodegenerative lysosomal storage disorders. I show the deleterious effects that this drug has on synaptic signalling, without materially alleviating lysosomal burden, further highlighting the need to use the right model to test treatments for disease. Overall, I provide novel insights into the mechanisms of cellular dysfunction in the GM2 gangliosidoses and provide an exciting platform to test drug treatments for these diseases. University of Cambridge 2025-04-22 Thesis Doctoral Doctor of Philosophy (PhD) eng Wellcome Trust Funding https://www.repository.cam.ac.uk/handle/1810/391861 https://www.repository.cam.ac.uk/bitstreams/ad7597a3-057f-4ebb-b12e-9ded8ed8eccd/download e15b2646835288277de5a1b7a5f09aca https://www.repository.cam.ac.uk/bitstreams/6ac0e6aa-0d90-4898-83d3-198027a7507c/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Disease Lysosomal storage disorder Lysosome Neuron