The role of endoplasmic reticulum-mitochondria contacts in neurodegeneration

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Background. Neurodegenerative disorders are complex diseases and a significant public health concern. Multiple molecular mechanisms underpin neuronal dysfunction, but many of them converge on altered function of mitochondria and/or endoplasmic reticulum (ER). ER and mitochondria have interdependent functions and physically interact with each other at specialised contact sites. Mutations in genes that regulate ER-mitochondria contact cause genetic forms of neurodegeneration, but the exact mechanisms by which defects in ER-mitochondria communication underpin neurodegeneration remain poorly understood. Here, we propose to investigate the role of ER-mitochondria contacts in Charcot-Marie-Tooth disease type 2A (CMT2A), a motor and sensory neuropathy caused by mutations in a mitochondrial fusion factor, mitofusin 2 (MFN2). MNF2 was recently shown to act as a possible tether protein regulating ER-mitochondria contacts. Our hypothesis is that CMT2A mutations in MFN2 affect ER-mitochondria contacts causing disruption of mitochondrial dynamics and function, and consequently neuronal death. We will test this hypothesis using a novel human pluripotent stem cell (hPSC) model of CMT2A, namely MFN2R94Q/+ motor and sensory neurons developed in the laboratory of the first supervisor and the cellular and molecular ER-mitochondria and neurobiology assays established in the laboratory of the second supervisor.Objectives.1: Determine the impact of altering ER-mitochondria contacts on mitochondrial dynamics and the functional behaviour of motor and sensory neurons.We will manipulate ER-mitochondria contacts (e.g. by knocking down MNF2 or the PTPIP51/VAPB tether or by overexpression of synthetic tethers) and analyse the impact of such manipulations on mitochondrial morphology and dynamics and neuronal function.2: Characterise ER-mitochondria contacts in motor and sensory neurons derived from MFN2R94Q/+ hPSCs and their isogenic wild type counterparts. We will quantify ER-mitochondria contacts using proximity-ligation assays and electron microscopy, and assay the contact functionally by calcium imaging.3: Determine the role of ER-mitochondria contacts in the phenotype of from MFN2R94Q/+. We will assess the impact of manipulating ER-mitochondria contacts in MFN2R94Q/+ neurons on the mitochondrial trafficking defects characteristic of these cells and on their behaviour (electrophysiology, survival/death).Novelty and timeliness. ER-mitochondria contacts have been implicated in CMT and several other neurodegenerative diseases only recently. The mechanistic understanding of ER-mitochondria contacts uncovered here will directly inform novel approaches for CMT treatment and may be relevant to other neurodegenerative diseases.Experimental approach. This project uses stem cell technology, super-resolution microscopy, electrophysiology, biochemistry and molecular biology approaches, including the use of CRISPR/Cas9.