Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

Biological systems are highly sensitive to changes in their environment. Indeed, the molecular basis of the environmental stress response suggests that the specialized stress responses share more commonalities than previously believed. Here, we used the nematode C. elegans to gain insight into the role of Rho signaling during two common environmental challenges, oxidative and thermal stress. In response to heat shock (HS), wild type (N2) worms demonstrated reduced viability which was rescued by genetic suppression of CDC42 and RHO-1. Visualization of F-actin by phalloidin-rhodamine underscored a strict correlation between the levels of F-actin following GTPase suppression and survival. Additionally, genetic ablation of OSG-1, a Guanine Nucleotide Exchange Factor (GEF) previously implicated in oxidative stress, was associated with constitutively lower levels of F-actin and increased mortality. However, upon an oxidative insult F-actin stability decreased in N2 worms, a rescue of this affect was observed in OSG-1 null worms, consistent with the resistance exhibited by these worms to oxidative stress (OS). Together these data suggest that during conditions of thermal or oxidative stress Rho signaling promotes vulnerability by altering actin dynamics. Thus, the stability of the actin cytoskeleton, in part through a conserved mechanism mediated by Rho signaling, is a crucial factor for the cell’s survival to environmental challenges.

A common cause of amyotrophic lateral sclerosis and frontotemporal dementia is the presence of a G4C2 intronic expansion in the C9orf72 gene. This expansion is translated by a non-AUG-dependent mechanism into five different dipeptide repeat proteins (DPRs), including the aggregation-prone poly glycine-arginine (GR), which is neurotoxic. Poly(GR) was found to interact with the nuclear importin Kapβ2 in non-neuronal cell lines. However, whether this interaction also occurs in neurons impacting their survival has not been studied. Here, we demonstrated that Kapβ2 and poly(GR) co-aggregate in neurons in-vitro and in-vivo in CNS tissue. Moreover, we showed that Kapβ2 mitigates poly(GR) neurotoxicity. Indeed, overexpression of Kapβ2 relieved poly(GR)-mediated neurotoxicity and restored nuclear TDP-43 levels, whereas silencing Kapβ2 increased the risk of death of neurons expressing poly(GR), suggesting that Kapβ2 plays a critical role in neurodegeneration. These findings open a new therapeutic avenue in C9-linked ALS/FTD focused on modulating Kapβ2 levels.