Casey, Jacqueline; (2023) PINK1/Parkin mitophagy in iPSC models of Frontotemporal dementia caused by GRN haploinsufficiency mutations. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Introduction: We examined mitophagy in frontotemporal dementia (FTD) caused by heterozygous GRN mutations, which result in haploinsufficiency of the progranulin protein. Impairments in mitophagy, the selective autophagy of damaged mitochondria, have been identified in a number of neurodegenerative diseases, with a number of FTD genes known to play a role in mitophagy (e.g, TBK1 and OPTN). Reduced xenophagy, the selective clearance of non-host pathogens, has been identified in GRN-/- mice and is reliant on some proteins involved in mitophagy (TBK1 and Parkin). We therefore hypothesised that progranulin might affect mitophagy. Methodology: We examined PINK1/Parkin mitophagy in neuroblastoma Parkin overexpressing SHSY5Y cells (POE-5Ys) and neuroglioma H4 cells +/- GRN siRNA. We also investigated induced pluripotent stem cells (iPSCs) differentiated to cortical neurons, astrocytes and microglia from 4 controls, 3 GRN FTD patient and an isogenic R493X GRN mutation CRISPR series (control, heterozygous and homozygous) from the human iPSC Neurodegenerative Disease Initiative (iNDI). Mitophagy was induced using oligomycin and antimycin A (O/A). PINK1 accumulation, levels of S65 phosphorylated ubiquitin (pUb) and other proteins involved in PINK1/Parkin mitophagy were examined using western blotting and immunofluorescence (ICC). Results: Lower levels of pUb were detected in POE-5Ys following GRN knockdown and O/A treatment in an imaging experiment on the Opera Phenix. We also detected a significant reduction in mitophagy in GRN siRNA treated H4 cells by ICC and western blotting of mitophagy markers. There was no significant difference in mitophagy between control and patient iPSC neurons, but there was variability in mitophagy and progranulin levels between inductions. We found a significant reduction in mitophagy in the homozygous R493X mutation neurons. There was no significant difference in mitophagy in the heterozygous line, potentially due to compensatory upregulation of progranulin protein levels from the wild type allele. CDC37, which traffics PINK1 to the mitochondria and affects its stability at the mitochondria, was significantly reduced in homozygous R493X mutation neurons. Preliminary experiments imaging experiment in iPSC-derived astrocytes and microglia on the Opera Phenix indicate that there is accumulation of damaged mitochondria at the lysosome in these cells, we will confirm this with electron microscopy. Conclusions: We have shown that loss of progranulin leads to impairments in PINK1/Parkin mitophagy. Current work aims to further understand the mechanisms of this process. Work is also ongoing in iPSC derived astrocytes and microglia to dissect cell-type specific contributions of progranulin to mitophagy.

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