Expansion of a GGGGCC repeat in a non-coding region of
C9orf72 is the most common cause of ALS/FTLD. Recently, RAN translation from the sense and antisense GGGGCC repeat transcript was observed in
C9orf72 repeat expansion carriers generating 5 different DPR species (poly-GA, −GR, −GP, −PA and -PR) [
12‐
14,
30]. These DPRs coaggregate with p62 and form the characteristic star shaped inclusions in
C9orf72 repeat expansion carriers [
13].
The relative contribution of RNA and DPR toxicity is still under debate since many conflicting results have been obtained in a variety of different model systems (reviewed in [
31]). The GGGGCC repeat RNA forms foci in cells, animal models and patients and has been shown to be able to induce neuronal cell death and to sequester RNA binding proteins [
8,
15,
20,
32]. However, there is only a weak correlation between RNA foci and neurodegeneration in patients [
33‐
35]. In zebrafish RNA injection of 8x, 38x, and 72x GGGGCC repeats has been shown to cause RNA foci and cell death by apoptosis in a repeat length dependent manner [
9]. This study did not report on RAN translation products upon repeat RNA injections in zebrafish. In line with these studies we observe RNA foci in two independent transgenic ggggcc80-GFP lines and RNA toxicity. In the ggggcc80-GFP fish we were not able to detect GA, GP, and GR species, most likely due to the relatively short repeat length or inefficient or even lack of RAN translation in early zebrafish that preclude detection of DPR species by Western blotting. This is in contrast to fly and mouse models in which repeat expression leads to DPR translation in the absence of a start codon [
18,
20]. Whether the mild toxic effects seen in ggggcc80-GFP fish is due to RNA toxicity or low level DPRs remains to be determined. To further address DPR toxicity we focused on poly-GA since it is the most abundant species found in
C9orf72 repeat carriers and induced the neuronal cell death in primary cultured cell model as well as in animal models [
16‐
18,
20]. We generated several transgenic zebrafish lines and demonstrated that poly-GA is toxic in zebrafish. In primary neurons poly-GA toxicity has been attributed to sequestration of Unc119 (a trafficking factor for myristylated proteins), interference with the ubiquitin proteasome system, and endoplasmic reticulum stress [
16,
17]. Recently, poly-GR and poly-PR were shown to be the most toxic DPR species in
Drosophila [
18,
36]. Moreover, the arginine-rich DPR species are also toxic in primary neurons, potentially affecting RNA synthesis [
18,
19]. Interestingly, DPRs interfere with nucleocytoplasmic shuttling in
Drosophila, cells, and yeast [
21,
22,
24,
25]. Two independently generated BAC transgenic mouse models recapitulate
C9orf72 repeat associated pathology, however they lack neurodegeneration [
34,
35]. In contrast, another BAC transgenic mouse model shows neurodegeneration and TDP-43 pathology [
37]. Expression of high levels of
C9orf72 repeats by adeno associated virus in mouse brain also generate neurodegeneration and TDP-43 pathology [
20]. These differences might reflect that sufficiently high expression levels are required to induce neurodegeneration. It remains unclear which DPR proteins contribute to ALS/FTLD pathogenesis in patients under physiological conditions. There is currently little evidence for a regional correlation of DPR aggregates in humans and neurodegeneration [
38]. These animal models will be valuable tools to further dissect the relative contribution and synergistic effects of repeat RNA and DPRs to toxicity.
GA80-GFP fish showed a circulation defect at 2 dpf and a severe pericardial edema phenotype at 4 dpf. Interestingly, double knockout zebrafish (
tardbp−/−, tardbpl−/−) also showed circulation defects at 2 dpf and vascular mispatterning, resulting in a pericardial edema phenotype reminiscent of the GGGGCC repeat induced phenotype [
39]. Considering that partial loss of TDP-43 function could be linked to the pathogenesis of ALS/FTLD-TDP-43, including
C9orf72 repeat expansion carriers [
40], we analyzed expression of Tardbp and Tardbpl_tv1 in GA80-GFP fish. However, no apparent changes in Tardbp and Tardbpl_tv1 protein level were observed upon transgene expression, indicating that neither RNA foci nor poly-GA lead to a loss of TDP-43 function in our zebrafish model. We speculate that potentially a common downstream pathway is affected in double knockout zebrafish (
tardbp−/−, tardbpl−/−) and GA80-GFP zebrafish, resulting in a similar circulation defect. However, since pericardial edemas are a common phenotype, which can be caused by a variety of defects, we cannot exclude the possibility that these similar phenotypes have distinct causes. Unfortunately, the larval lethality precludes analysis of possible neurodegenerative phenotypes of the repeat expressing transgenic zebrafish during adulthood.