Etude de nouvelles mutations dans le gene C9ORF72 responsables de Sclérose Latérale Amyotrophique – NewMutALS

Using various biochemical approaches, we found that C9ORF72 forms a tight complex with the Smith-Magenis syndrome chromosome region candidate 8 (SMCR8) protein. Bioinformatic analyzes indicate that both C9ORF72 and SMCR8 possess DENN and longin domains, which are characteristic of GDP/GTP exchange factors. Indeed, we found that the C9ORF72/SMCR8 complex regulates the initiation of autophagy by acting as a GDP/GTP exchange factor for specific RAB GTPases (Sellier et al., 2016). These results demonstrate that the C9ORF72/SMCR8 complex regulates autophagy and that alteration of this pathway may play fundamental pathogenic roles in ALS. Importantly and in support of that model, we identified novel missense mutations of C9ORF72 or SMCR8 in ALS-FTD patients. Our preliminary results indicate that these mutations are pathogenic in cells models. Thus, it was crucial to confirm these results and explore further the pathogenic importance of these mutations. Moreover, the relationship, if any exist, between these missense mutations and the G4C2 repeat expansion in C9ORF72 is unclear. Finally, there is no treatment for this devastating disease. Thus, we proposed to:

1 - Develop state-of-the-art cell and animal models of C9ORF72 and SMCR8 mutations.
2 - Explore by which mechanisms C9ORF72 and SMCR8 mutations alter autophagy.
3 - Explore whether these mutations can help to clarify the pathogenic effect of the G4C2 expansion.
4 - Identify compounds correcting autophagy alterations due to C9ORF72 or SMCR8 mutations.

We developed novel cell models to study the pathogenic effects of the C9ORF72 mutation, which helped us to confirm that the C9ORF72 protein is a regulator of autophagy, which is a catabolic cellular mechanism indispensable for neurons to eliminate toxic proteins prone to aggregation. Consequently, we found that reduce expression of C9ORF72 leads to sub-optimal autophagy. As DPR proteins translated from expanded G4C2 repeats are prone to aggregation, we tested whether DPR expression could constitute a stress regulated by C9ORF72-mediated autophagy. Importantly, our results demonstrate that accumulation of DPR under their natural sequences is prevented by autophagy, resulting in little accumulation of DPR proteins in normal conditions. In contrast, suboptimal autophagy due to reduce expression of C9ORF72 enhances accumulations of DPR aggregates, ultimately resulting into neuronal cell death. Furthermore, our data also indicate that expanded G4C2 repeats are translated through initiation to near cognate start codons. Near cognate initiation codons are codons differing from the initiation AUG codons by one base but that can still initiate translation provided they are embedded in a correct Kozac consensus sequence.

Importantly, this work is now accepted for publication: Boivin et al., Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders. EMBO J. 2020 Feb 17;39(4):e100574. doi: 10.15252/embj.2018100574.

In conclusion, our work provides a novel mechanism to the neuronal cell death in ALS that may result from a double hit mechanism, where the sub-optimal autophagy due to C9ORF72 reduced expression may synergize the toxicity of a second stress: the near-cognate codon-initiated translation of expanded G4C2 repeats into toxic DPR proteins prone to aggregation.

However, these data were obtained in immortalized cell models and we are now in the process of developing state-of-the-art human induced pluripotent cells (iPS) and animal (Zebrafish and mouse) models to confirm and validate these results. We will also test various chemical compounds to boost autophagy and try to rescue neuronal cell death in these novel cell and animal models. If positive, this work may open novel routes for therapeutic options in Amyotrophic Lateral Sclerosis.

Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders.
Boivin M, Pfister V, Gaucherot A, Ruffenach F, Negroni L, Sellier C, Charlet-Berguerand N. EMBO J. 2020 Feb 17;39(4):e100574. doi: 10.15252/embj.2018100574.