Cellular and molecular insights into neurodegeneration mediated by the C9orf72 repeat expansion mutation
Creators: name: Mehta, Arpan R.
Publisher: name: The University of Edinburgh
Year: 2022
Subjects: motor neuron disease (MND)
Amyotrophic Lateral Sclerosis
C9orf72
Stem Cell
neurodegeneration
Dementia
mitochondria
axon
Bioenergetics
PGC1a
axonal transport
respiration

description: Amyotrophic lateral sclerosis (ALS) is an incurable, rapidly progressive and fatal neurodegenerative disorder, characterised by loss of upper and lower motor neurons (MNs). Approximately 10-20% of ALS cases are familial, of which the C9orf72 (G4C2)n&gt;30 hexanucleotide repeat expansion mutation is the commonest known cause in the western world. The finding that familial ALS is clinically and pathologically indistinguishable from sporadic ALS supports the study of monogenetic causes to better understand common pathogenic mechanisms. Thus, human induced pluripotent stem cell (iPSC) derived MN experimental platforms, combined with paired gene-edited isogenic control lines, provide a powerful approach, both to identify early-stage disease-driving mechanisms, and establish causality between a given mutation and phenotypes. Such in vitro disease modelling is complemented by in vivo approaches, particularly those that harness technologies interrogating molecular neuropathological signatures in human post-mortem tissue. The primary aim of this PhD was to study the MN cell-autonomous pathogenic mechanisms of the C9orf72 repeat expansion against an isogenic control background using a human model, adopting cellular, molecular and bioinformatics techniques. I establish that C9orf72 MNs have dysfunctional axonal homeostasis, with aberrations both in their axonal morphology (reduced neurite length) and function (impaired fast axonal transport of mitochondrial cargo). Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in ALS, where MNs have axons extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. I show that these axonal phenotypes are associated with concomitant metabolic dysfunction, owing to defective mitochondrial respiration. Unbiased RNA-sequencing revealed reduced expression of electron transport chain transcripts, encoded by mitochondrial DNA, the copy number of which was unaltered. Critically, I show, through neuropathological analysis of patient post-mortem tissue, that this transcriptomic dysregulation is selective to anterior horn spinal (motor) neurons, and is absent in dorsal horn spinal (sensory) neurons, with corresponding alterations reflected at the level of protein expression. Through manipulation of this molecular mitochondrial loss-of-function signature, leading to therapeutic rescue of the observed dysfunctional axonal homeostasis, I determine a novel causal relationship between axon dysfunction and contributory metabolic dysfunction in C9orf72-ALS. Collectively, my experimental data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function. These data build on preclinical data from animal models in ALS (albeit largely biased by a preponderance of data from SOD1 models), which I systematically reviewed and meta-analysed, showing that targeting mitochondrial dysfunction may prolong survival, particularly if the intervention is administered early.