Noya, Jorge Alaiz

Abstract Background G 4 C 2 repeat expansions in C9orf72 gene are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mounting evidence indicates that dipeptide repeat (DPR) proteins translated from the expanded repeats, in particular poly(GR) and poly(PR), play a significant role. We and others have shown that these positively‐charged arginine‐rich DPR proteins induce the assembly of stress granules (SGs) and impair their disassembly. While distinct from SGs, processing bodies (PBs) are also cytoplasmic messenger ribonucleoprotein condensates that assemble upon stress. However, the impacts of arginine‐rich DPR proteins on PB assembly and dynamics have yet to be investigated. Method We conducted immunofluorescence staining for arginine‐rich DPR proteins, SG‐ and PB‐associated markers in cultured cells and mouse brain. Additionally, we performed in vitro liquid‐liquid phase separation (LLPS) using recombinant proteins and assessed molecular dynamics of SG and PB through fluorescence recovery (FRAP) after photobleaching in living cells. Result Here, we observed that both GFP‐(GR) 100 and GFP‐(PR) 100 formed cytosolic inclusions immunopositive PB proteins. Unlike GFP‐(GR) 100 and GFP‐(PR) 100 inclusions, cellular cytoplasmic GFP‐(GA) 100 inclusions did not contain PB proteins. We subsequently examined whether the phenomena observed in cultured cells occur in vivo. We observed that PB proteins were recruited to poly(GR) inclusions in cells with aggregated poly(GR). Next, we examined the dynamics of poly(GR)‐induced PBs in cultured cells using the FRAP technique. We observed that the recovery of stress‐induced PBs was more rapid than that of PBs in HEK293T cells expressing GFP‐(GR) 100 . Lastly, mechanistic studies revealed that poly(GR) interacts with PB proteins to form condensates in vitro via LLPS. Conclusion Our results provide both in vitro and in vivo evidence that arginine‐rich DPRs induce the spontaneous formation of PBs via LLPS. Our results also indicate that poly(GR)‐induced PBs are more stable than the transient and dynamic conventional PBs. Given that PBs uniquely enriched with mRNA degradation and decay factors, the consequences of impairing these processes are expected to contribute to abnormal RNA metabolism in c9FTD/ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that share clinical and pathological features, as well as genetic causes. A G4C2 repeat expansion in chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause of ALS and FTD, collectively referred to as c9ALS/FTD. Assembly modulation is a new therapeutic approach which appears to target allosteric sites on aberrant forms of multi-protein complexes and restore them to the healthy state. Recent findings demonstrate that tetrahydroisoquinolone (THIQ)-based protein assembly modulators can ameliorate ALS/FTD-associated phenotypes in cellular and animal models. In the present study, we investigated the effects of PAV-615, a novel and advanced THIQ-based modulator, in a c9ALS/FTD mouse model expressing 149 G4C2 repeat expansions (referred to as 149R mouse model). Specifically, PAV-615 was administered to 5-month-old 149R mice via intraperitoneal injection for one month. Motor function was evaluated using the hang wire test, while anxiety-like behavior and hyperactivity were assessed using the open-field test. Pathological markers, including dipeptide repeat (DPR) proteins, phosphorylated TAR DNA-binding protein 43 (pTDP-43) and ataxin 2-positive stress granules, were quantified by Meso Scale Discovery and immunohistochemistry assays. Compared with vehicle-treated controls, PAV-615 significantly improved motor performance and modestly reduced anxiety-like behavior and hyperactivity in 149R mice. Moreover, PAV-615 treatment significantly decreased cortical DPR, pTDP-43 and ataxin 2-positive stress granule burdens. These results support assembly modulation as a promising therapeutic approach treatment of ALS/FTD.