Brian Bertsche

Introduction. Expanded G4C2 repeats in intron 1 of C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis (ALS) and is implicated in non-familial ALS as well.¹ The function of the C9orf72 protein is not definitively known, nor is the mechanism by which it causes ALS. There have been three mechanisms proposed,¹ haploinsufficiency through disrupted expression of expanded allele, RNA mediated gain of function toxicity, and protein mediated gain of function toxicity by dipeptide repeat. Methods. Via immunoprecipitation experiments in cells expressing tagged C9orf72 and tagged RABs, interaction was seen between the protein and all members of the RAB family. This interaction was confirmed in wild-type mouse brain with endogenous molecules.² Using FISH techniques and tissues collected from human samples, researchers visualized RNA foci using high powered microscopes.¹ Phosphorylated eIF2alpha levels were monitored with a specific antibody in HeLa cells transfected with (G4C2)₃₁ repeats. Further testing established that the response was sufficient to cause translation inhibition. Cells were analyzed for their ability to incorporate puromycin into nascent polypeptides as a measure of the rate of mRNA synthesis. Both western blot analysis and immunofluorescence demonstrated that puromycin incorporation was significantly decreased in HeLa and NSC34 cells that expressed (G4C2)₃₁.³ Toxicity of DPR proteins appears to depend on arginine-containing DPR proteins.⁴ Results. C9orf72 interacts and co-localizes with members of the RAB3 protein family. Researchers now hypothesize a role for C9orf72 in regulating synaptic vesicle functions by acting as a guanine exchange factor (GEF) for RAB3. Utilizing RNA FISH (florescence in situ hybridization) methodology, clearly distinguishable RNA foci are present in C9orf72+ patients with ALS compared to C9orf72- ALS patients and control subjects. In C9orf72+ tissues the average proportion of neurons containing RNA foci was 39% compared to 1.6% in C9orf72- and 1.4% in control tissues.¹ Arginine rich DPRs appear to disrupt nucleocytoplasmic transport and RNA processing, allowing for the dysregulation of translation and nuclear stress via the formation of stress granules.⁴ Conclusions. The function of C9orf72 in healthy individuals remains uncertain but is likely related to RAB3 protein family. Each of the 3 proposed mechanisms of pathogenesis has some supporting evidence, but at this time it remains unclear how the C9orf72 mutation leads to the phenotype of ALS.

1.  Cooper-Knock J, Walsh MJ, Higginbottom A, et al. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain. 2014;137(7):2040-2051. doi:10.1093/brain/awu120.
2. Frick P, Sellier C, Mackenzie IRA, et al. Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers. Acta Neuropathologica Communications. 2018;6(1). doi:10.1186/s40478-018-0579-0.
3. Rossi S, Serrano A, Gerbino V, et al. Nuclear accumulation of mRNAs underlies G4C2-repeat-induced translational repression in a cellular model of C9orf72 ALS.Journal of Cell Science. 2015;128(9):1787-1799. doi:10.1242/jcs.165332.
4. Jovičić A, Mertens J, Boeynaems S, et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.Nature Neuroscience. 2015;18(9):1226-1229. doi:10.1038/nn.4085.