Probes for C9ORF72

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) with synaptic dysfunction identified as an early pathological hallmark. Although TDP-43 pathology and overt neurodegeneration are largely absent from the cerebellum, the pathological hallmarks of RNA foci and dipeptide repeat protein (DPR) inclusions are most abundant. Here, we present a systematic literature search in the databases of PubMed, Scopus, Embase, Web of Science and Science Direct up until March 5, 2021, which yielded 19,515 publications. Following the exclusion criteria, 72 articles were included having referred to C9orf72, synapses and the cerebellum. Meta-analyses were conducted on studies which reported experimental and control groups with means and standard deviations extracted from figures using the online tool PlotDigitizer. This revealed dendritic defects (P = 0.03), reduced C9orf72 in human patients (P = 0.005) and DPR-related neuronal loss (P = 0.0006) but no neuromuscular junction abnormalities (P = 0.29) or cerebellar neuronal loss (P = 0.23). Our results suggest that dendritic arborisation defects, synaptic gene dysregulation and altered synaptic neurotransmission may drive cerebellar synaptic dysfunction in C9-ALS/FTD. In this review, we discuss how the chronological appearance of the different pathological hallmarks alters synaptic integrity which may have profound implications for disease progression. We conclude that a reduction in C9orf72 protein levels combined with the accumulation of RNA foci and DPRs act synergistically to drive C9 synaptopathy in the cerebellum of C9-ALS/FTD patients.

Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), where the key pathological cell-type, the motor neuron (MN), has an axon 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. To address this, we undertook cellular and molecular interrogation of multiple patient-derived induced pluripotent stem cell lines and patient autopsy samples harbouring the most common ALS causing mutation, C9orf72. Using paired mutant and isogenic expansion-corrected controls, we show that C9orf72 MNs have shorter axons, impaired fast axonal transport of mitochondrial cargo, and altered mitochondrial bioenergetic function. RNAseq revealed reduced gene expression of mitochondrially encoded electron transport chain transcripts, with neuropathological analysis of C9orf72-ALS post-mortem tissue importantly confirming selective dysregulation of the mitochondrially encoded transcripts in ventral horn spinal MNs, but not in corresponding dorsal horn sensory neurons, with findings reflected at the protein level. Mitochondrial DNA copy number was unaltered, both in vitro and in human post-mortem tissue. Genetic manipulation of mitochondrial biogenesis in C9orf72 MNs corrected the bioenergetic deficit and also rescued the axonal length and transport phenotypes. Collectively, our 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.

Amyotrophic lateral sclerosis (ALS) is caused by selective degeneration of motor neurons in the brain and spinal cord; however, the primary cell death pathway(s) mediating motor neuron demise remain elusive. We recently established that necroptosis, an inflammatory form of regulated cell death, was dispensable for motor neuron death in a mouse model of ALS, implicating other forms of cell death. Here, we confirm these findings in ALS patients, showing a lack of expression of key necroptotic effector proteins in spinal cords. Rather, we uncover evidence for ferroptosis, a recently discovered iron-dependent form of regulated cell death, in ALS. Depletion of glutathione peroxidase 4 (GPX4), an anti-oxidant enzyme and central repressor of ferroptosis, occurred in post-mortem spinal cords of both sporadic and familial ALS patients. GPX4 depletion was also an early and universal feature of spinal cords and brains of transgenic mutant superoxide dismutase 1 (SOD1G93A), TDP-43 and C9orf72 mouse models of ALS. GPX4 depletion and ferroptosis were linked to impaired NRF2 signalling and dysregulation of glutathione synthesis and iron-binding proteins. Novel BAC transgenic mice overexpressing human GPX4 exhibited high GPX4 expression localised to spinal motor neurons. Human GPX4 overexpression in SOD1G93A mice significantly delayed disease onset, improved locomotor function and prolonged lifespan, which was attributed to attenuated lipid peroxidation and motor neuron preservation. Our study discovers a new role for ferroptosis in mediating motor neuron death in ALS, supporting the use of anti-ferroptotic therapeutic strategies, such as GPX4 pathway induction and upregulation, for ALS treatment.