Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity

INTRODUCTION Frontotemporal dementia(FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative diseases that share clinical and neuropathological features. Furthermore, the most common genetic cause of both FTD and ALS is a GGGGCC (G 4 C 2 ) repeat expansion in the C9orf72gene. This repeat expansion leads to several abnormalities, including C9orf72 haploinsufficiency, the accumulation of repeat RNA, and the production of five aggregation-prone proteins composed of repeating dipeptides. However, the contribution of these abnormalities to disease pathogenesis remains unresolved. RATIONALE Among the five dipeptide repeat proteins nonconventionally translated from expanded G 4 C 2 repeats, proline-arginine (PR) repeat proteins [poly(PR) proteins] have proven especially toxic in various model systems. Their involvement in C9orf72-associated FTD and ALS (c9FTD/ALS) has nevertheless been questioned because poly(PR) pathology is relatively infrequent in c9FTD/ALS patient brains. Postmortem tissues, however, represent end-stage disease and do not necessarily reflect early events in the disease process. Therefore, we generated mice that express poly(PR) in the brain to evaluate the temporal consequences of its expression in a mammalian in vivo model. More specifically, we engineered mice to express green fluorescent protein (GFP)–conjugated (PR) 50 (a 50-repeat PR protein) or GFP via intracerebroventricular administration of adeno-associated viral vectorsand then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice. RESULTS We found that ~60% of poly(PR)-expressing mice died by 4 weeks of age and had significantly decreased brain and body weights at death compared with age-matched GFP control mice. Poly(PR) mice that escaped premature death developed motor and memory impairments, likely as a consequence of their progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis. In investigating the mechanisms by which poly(PR) caused neurodegeneration and functional deficits, we found that poly(PR) localized to heterochromatin(highly condensed regions of transcriptionally silent chromatin) and caused abnormal histone H3 methylation, features that we also detected in brain tissues from patients with c9FTD/ALS. Additionally, we observed aberrations in nuclear lamins and heterochromatinprotein 1α (HP1α), key proteins that maintain heterochromatinstructure and regulate gene silencing. Nuclear laminainvaginations and decreased HP1α protein expression were seen in poly(PR)-positive cells in poly(PR) mice, and in vitro studies demonstrated that poly(PR) disrupted HP1α liquid phases. Because poly(PR)-induced histone H3 posttranslational modifications, lamin invaginations, and decreased HP1α levels could profoundly affect gene expression, we compared transcriptome profiles between control and poly(PR) mice. As well as analyzing differentially expressed genes, we examined repetitive element expression given that repetitive DNA sequences make up a large portion of heterochromatinand that repetitive elements are substantially up-regulated in the brains of c9FTD/ALS patients. Whereas the majority of differentially expressed genes in poly(PR) mice were down-regulated, repetitive elements were markedly up-regulated, and this up-regulation was accompanied by the accumulation of double-stranded RNA. Furthermore, we confirmed that HP1α depletion caused double-stranded RNA accumulation in human induced pluripotent stem cell–derived neurons and decreased their survival. CONCLUSION Our studies provide compelling evidence that, by disrupting HP1α liquid phases, interacting with heterochromatin, and eliciting aberrant histone posttranslational modifications, poly(PR) adversely influences heterochromatinstructure. Consequently, repetitive element expression is induced and double-stranded RNA accumulates, contributing to the neurodegeneration seen in patients with c9FTD/ALS. Rescuing histone methylation, lamin, and HP1α abnormalities and/or inhibiting abnormal repetitive element expression may represent promising therapeutic strategies for treating c9FTD/ALS.