Therapeutic reduction of GGGGCC repeat RNA levels by hnRNPA3 suppresses neurodegeneration in Drosophila models of C9orf72-linked ALS/FTD

Abstract

The abnormal expansion of GGGGCC hexanucleotide repeats within the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of GGGGCC repeat-containing RNAs as RNA foci, and the deposition of dipeptide repeat proteins (DPR) produced from these repeat RNAs by unconventional translation are major pathological hallmarks of C9orf72-linked ALS/FTD (C9-ALS/FTD), and are both thought to play a crucial role in the pathogenesis of these diseases. Because GGGGCC repeat RNA is likely to be the most upstream therapeutic target in the pathogenic cascade of C9-ALS/FTD, lowering the cellular level of GGGGCC repeat RNA is expected to mitigate repeat RNA toxicity, and will therefore be a disease-modifying therapeutic strategy for the treatment of C9-ALS/FTD. In this study, we demonstrated using a Drosophila model of C9-ALS/FTD that elevated expression of a subset of human RNA-binding proteins that bind to GGGGCC repeat RNA, including hnRNPA3, IGF2BP1, hnRNPA2B1, hnRNPR, and SF3B3, reduces the level of GGGGCC repeat RNA, resulting in the suppression of neurodegeneration. We further showed that hnRNPA3-mediated reduction of GGGGCC repeat RNA suppresses disease pathology, such as RNA foci and DPR accumulation. These results demonstrate that hnRNPA3 and other RNA-binding proteins negatively regulate the level of GGGGCC repeat RNA, and mitigate repeat RNA toxicity in vivo, indicating the therapeutic potential of the repeat RNA-lowering approach mediated by endogenous RNA-binding proteins for the treatment of C9-ALS/FTD.

PABPC1 mediates degradation of C9orf72-FTLD/ALS GGGGCC repeat RNA

“…Next, we tested whether a reduction in PABPC1 promotes the formation of repeat RNA foci as a disease-associated phenotype of repeat RNA accumulation. 19 , 39 Fluorescence in situ hybridization (FISH) analysis of (G 4 C 2 ) 80 expressing HeLa cells revealed that siRNA-mediated knockdown of PABPC1 increased the number of (G 4 C 2 ) 80 repeat RNA foci positive cells, which is comparable to cells with hnRNPA3 knockdown ( Figures 3 F and 3G). These results suggest that PABPC1 is involved in the degradation pathway of G 4 C 2 repeat RNA as well as hnRNPA3.…”

“… 15 , 16 Interestingly, overexpression of wild-type hnRNPA3 reduced poly-GA expression, but RNA-binding ability deficient mutant hnRNPA3 overexpression failed to suppress DPR expression levels in the cellular model or the Drosophila model. 15 , 19 From these observations, we hypothesized that hnRNPA3 that binds to G 4 C 2 repeat RNA may physically interact with RNA degradation factors and facilitate selective RNA degradation in cells.…”

Sporadic Amyotrophic Lateral Sclerosis Skeletal Muscle Transcriptome Analysis: A Comprehensive Examination of Differentially Expressed Genes

“…Among the hub genes derived from the up-regulated DEGs, no ALS-related literature was found for the EEF1A1 , RPLP0 , EEF2 , CCT2 , RPL12 and RPL15 genes. However, studies related to ALS were identified for the EIF4A1 [ 44 ], HNRNPR [ 45 ], HNRNPA1 [ 45 , 46 ] and PABPC1 genes [ 47 ]. In the case of hub genes obtained from the down-regulated DEGs, only the NDUFA4 gene was associated with ALS-related studies [ 48 ].…”

Aspects of degradation and translation of the expanded C9orf72 hexanucleotide repeat RNA

“…We have previously shown that hnRNPA3 preferentially binds to G 4 C 2 and C 4 G 2 repeat RNAs (Mori, Lammich, et al, 2013; Nihei et al, 2020); hnRNPA3 was observed to frequently mislocalize to the cytoplasm, deviating from its physiological nuclear predominant distribution (Boeynaems et al, 2016; Davidson et al, 2017; Fifita et al, 2017; Mori, Lammich, et al, 2013), but hnRNPA3 does not seem to colocalize with RNA foci (Lee et al, 2013). Interestingly, hnRNPA3 suppresses the expression levels of the C9orf72 repeat RNA, through its RNA recognition motifs, and corresponding suppression of DPR expression was observed (Mori et al, 2016; Nihei et al, 2020; Taminato et al, 2023). Moreover, similar to hnRNPA3, expression of IGF2BP1, hnRNPA2B1, and SF3B3, which bind to G 4 C 2 repeat RNA, was found to reduce G 4 C 2 repeat RNA expression in a Drosophila model (Taminato et al, 2023) (Figure 3).…”

“…Interestingly, hnRNPA3 suppresses the expression levels of the C9orf72 repeat RNA, through its RNA recognition motifs, and corresponding suppression of DPR expression was observed (Mori et al, 2016; Nihei et al, 2020; Taminato et al, 2023). Moreover, similar to hnRNPA3, expression of IGF2BP1, hnRNPA2B1, and SF3B3, which bind to G 4 C 2 repeat RNA, was found to reduce G 4 C 2 repeat RNA expression in a Drosophila model (Taminato et al, 2023) (Figure 3). We speculate that these RNA‐binding proteins may exert their effect by promoting the degradation of the repeat RNA.…”