Abstract
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, causing progressive muscle wasting and eventual paralysis. A number of the cellular functions that are disrupted in ALS are mediated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical contacts between areas of ER and mitochondrial membranes. The ER membrane protein, VAPB, and the outer mitochondrial membrane protein, PTPIP51, interact to act as tethers, mediating the formation of ER-mitochondria contacts. Transgenic mouse and cellular studies have shown that ER-mitochondria contacts are disrupted by ALS insults, some of which disrupt the VAPB-PTPIP51 interaction via activation of the kinase GSK-3β. However, ER-mitochondria contacts have not been studied in ALS in humans and the mechanism by which GSK-3β disrupts the VAPB-PTPIP51 interaction is yet to be elucidated.The first hypothesis that underlies this thesis is that damage to ER-mitochondria signalling, involving disruption of the VAPB-PTPIP51 tethers, is a feature of disease in ALS in humans. This was investigated by quantifying the VAPB-PTPIP51 interaction using proximity ligation assays in post-mortem spinal cord tissues from ALS and control cases, and in iPSC-derived motor neurons from control donors and ALS donors carrying familial pathogenic TDP-43 mutations. The VAPB-PTPIP51 interaction was reduced in both ALS post-mortem spinal cord and iPSC-derived motor neurons. VAPB protein levels were reduced in ALS, but other ER-mitochondria proteins investigated were unchanged.
The second hypothesis of this thesis is that GSK-3β phosphorylates VAPB or PTPIP51, reducing their interaction as ER-mitochondria tethers. Recombinant VAPB and PTPIP51 were phosphorylated in vitro by GSK-3β and then analysed by mass spectrometry. Novel potential GSK-3β phosphorylation sites were identified in both proteins. Using phospho-mimicking mutants in a cellular GAL4 based 2-hybrid binding assay, one phosphorylation site in VAPB was shown to alter its binding to PTPIP51. To complement these in vitro studies, cellular phosphorylation of VAPB and PTPIP51 was also investigated. This involved immunoprecipitation of VAPB and PTPIP51 from cells in which GSK-3β or AMP kinase activities were increased, and mass spectrometry. These studies identified novel phosphorylation sites in both proteins, which may facilitate future studies on the role of the VAPB-PTPIP51 interaction in ER-mitochondria signalling.
In summary, the studies presented in this thesis identify disruption of the VAPB-PTPIP51 ER-mitochondria tethers as a novel feature of ALS in humans and provide insight into a mechanism for this disruption. These studies also provide further support for correcting damage to the VAPB-PTPIP51 interaction as a novel therapeutic target for ALS.
| Date of Award | 30 Apr 2020 |
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| Original language | English |
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| Supervisor | Christopher Miller (Supervisor), Wendy Noble (Supervisor) & Patricia Gomez Suaga (Supervisor) |