Deciphering the function and mechanisms of C9ORF72 in ALS

author: Hortense de Calbiac, Institut du Cerveau et de la Moelle épinière - ICM
published: July 21, 2017,   recorded: May 2017,   views: 7
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ALS has a major genetic contribution, the most common genetic abnormality being the GGGGCC hexanucleotide repeat expansion (HRE) in the C9ORF72 gene. Proposed mechanisms concerning C9ORF72 mutation pathogenicity are loss and gain of function including aggregation of dipeptide repeats (DPRs) translated through a non- ATG-initiated translation. One of the hypotheses would be a role in autophagy for C9ORF72. Interestingly, autophagy is also linked with other ALS causative genes such as VCP, UBQLN2, OPTN and TBK1. SQSTM1 is another gene mutated in ALS patients with a well-known cellular function. SQSTM1/p62, is an essential actor of the initiation of the autophagy pathway. p62+ inclusions have been detected in patients carrying the C9ORF72 HRE leading to the hypothesis of a functional common purpose between these genes. To investigate the pathogenic mechanisms induced by C9ORF72 and SQSTM1 mutations in ALS, we developed zebrafish models for these genes. Loss of function of the Sqstm1 and C9orf72 zebrafish orthologues leads to specific motor phenotypes associated with shorter motor neuron axons and reduced swimming capacity. To elucidate the common cellular mechanisms underlying autophagy dysregulation in motor neuron degeneration, C9orf72 and Sqstm1 zebrafish models were used to analyze their epistatic interactions. We found that C9orf72 and Sqstm1 partial inhibitions have an additive effect and that C9orf72 can rescue the phenotype obtained with Sqstm1 knockdown. These results indicate that both proteins belong to the same pathway and that C9orf72 is downstream of Sqstm1. Also, we found that C9orf72 depletion in mouse motor neurons primary cultures leads to the early death of motor neurons associated with their inability to product autophagosomes. To develop a vertebrate model of DPR pathogenicity, we expressed plasmids containing GFP-tagged expanded repeats for all DPRs: poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA). We found out that the loss of function of C9orf72 is essential to trigger DPR accumulation in zebrafish. Inclusions of DPR happening under C9orf72 knockdown are associated with motor abnormalities - with fish losing their capability to swim, and with shortened motor neuron axons. These results indicate that DPR inclusions and C9orf72 knockdown act in a common pathogenic mechanism, suggesting that both gain and loss of function synergize in the C9ORF72 HRE pathogenicity and thus opening novel avenues for potential treatment of ALS.

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