Investigating modifiers of C9orf72 induced pathology in Amyotrophic Lateral Sclerosis (ALS) using zebrafish.

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Clinically, patients present with painless subacute focal muscle weakness. The disease is rapidly progressive, generally leading to death in three to five years after symptom onset, and is unfortunately still incurable.

While in most patients the cause of the disease is unknown, genetic mutations explain ±15% of ALS patients. A non-coding GGGGCC repeat expansion in the C9ORF72 gene is the most frequent genetic cause of ALS. The pathological underpinnings of this mutation are still unclear and might encompass three possible mechanisms. First, decreased transcription of the C9ORF72 gene might lead to loss-of-function of the C9orf72 protein, which has a presumed function in autophagy and membrane trafficking. Second, repeat RNA might bind to several RNA binding proteins (RBPs) hence disturbing their function. This is coined ‘RNA toxicity’. Third, the repeat sequence itself might undergo unconventional translation into toxic dipeptide repeats (DPRs), called ‘DPR toxicity’. As it is clear that C9orf72 loss-of-function is not the main pathogenic driver, a gain-of-function mechanism seems to be evident. However, at this stage it is still unclear whether this gain-of-function is driven by ‘RNA toxicity’ or ‘DPR toxicity’, or even a combination of both. Whereas a definite answer can only be generated in patient material (e.g. post mortem tissue), extensive in vivo disease models have been generated to disentangle this Gordian knot. Whereas several in vivo models have demonstrated the toxic potential of DPRs, in vivo models supporting RNA toxicity are currently lacking.

Therefore, by generating a new zebrafish model we aimed to assess whether repeat RNA can be toxic independent of DPRs, which would be indicative of RNA toxicity as a potential mechanism. Second, we aimed to identify modifiers of this RNA toxicity in order to unravel the mechanism of RNA toxicity.

We generated a transient zebrafish model by injecting repeat RNA into fertilized oocytes. At 30 hours post fertilization, microscopic analysis an axonopathy of spinal motor neurons upon injection of both sense and antisense repeat RNA. Remarkably, no DPRs were detected using both dot blot and immunoassay approaches. As such, this indicates that the presence of repeat RNA in the absence of DPRs is sufficient to cause neuronal toxicity and hence implicates RNA toxicity in the mechanism of C9orf72 ALS. Nevertheless, expression of individual DPRs through codon-optimized constructs revealed GR and PR to induce an axonopathy as well, hence confirming their toxic potential suggested in other in vivo models.

RNA toxicity is believed to be mediated by the repeat RNA compromising the function of several RBPs. Therefore we overexpressed ten RBPs, known to bind repeat RNA, in our zebrafish model. Interestingly five of them (Purα, hnRNPK, hnRNPA3, ALYREF and SRSF1) alleviated the RNA toxicity, indicating that their dysfunction might contribute to RNA toxicity pathogenesis.

Next we focused on Purα to assess how and if this protein is compromised in C9ORF72 ALS. We found the alleviating effect of Purα to be mediated by an induction of p62, mainly via its PUR2 domain, implicating an unexpected involvement of autophagy in RNA toxicity. In line herewith, Purα was found to reverse the increased LC3 levels in C9ORF72 patient derived fibroblasts. Moreover, Purα protein levels were decreased in C9ORF72 fibroblasts as well as in the RNA toxicity zebrafish model, suggesting it to indeed be compromised in C9ORF72 ALS.

Altogether we generated the first in vivo model demonstrating the toxic potential of C9ORF72 repeat RNA independent of DPR generation, thereby supporting RNA toxicity as a potential pathogenic mechanism of C9ORF72 ALS. Additionally, we identified several repeat RNA binding proteins to mitigate the RNA toxicity upon overexpression, suggesting their dysfunction to contribute to RNA toxicity pathogenesis. The dysfunction of one of these proteins, Purα, suggests RNA toxicity to induce disturbed autophagy.

Future research regarding RNA toxicity will need to be twofold. On the one hand, the true involvement of RNA toxicity in C9ORF72 ALS pathogenesis needs to be proven in humans. On the other hand, the mechanism of RNA toxicity needs to be unraveled further by assessing all RNA binding proteins and their downstream targets.