Research Article

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

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Science  15 Feb 2019:
Vol. 363, Issue 6428, eaav2606
DOI: 10.1126/science.aav2606

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How dipeptide repeats cause pathology

A repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene is the most common known cause of two neurodegenerative diseases: frontotemporal dementia and amyotrophic lateral sclerosis. This expansion leads to the abnormal production of proteins of repeating dipeptides, but their contribution to disease pathogenesis remains unclear. Zhang et al. engineered a mouse model to study the consequences of one of these dipeptides—prolinearginine dipeptide repeat protein, poly(PR)—in the brain. They found that poly(PR) caused neuron loss as well as motor and memory impairments. These detrimental effects resulted from poly(PR)-induced perturbation of heterochromatin function, a tightly packed form of DNA that represses gene expression.

Science, this issue p. eaav2606

Structured Abstract


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 (G4C2) repeat expansion in the C9orf72 gene. 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.


Among the five dipeptide repeat proteins nonconventionally translated from expanded G4C2 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 vectors and then performed behavioral, pathological, and transcriptomic characterizations of poly(PR) mice in comparison with control GFP mice.


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 heterochromatin protein 1α (HP1α), key proteins that maintain heterochromatin structure and regulate gene silencing. Nuclear lamina invaginations 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 heterochromatin and 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.


Our studies provide compelling evidence that, by disrupting HP1α liquid phases, interacting with heterochromatin, and eliciting aberrant histone posttranslational modifications, poly(PR) adversely influences heterochromatin structure. 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.

Poly(PR) interactions with heterochromatin cause repetitive element expression.

Heterochromatin consists of tightly packed nucleosomes, DNA segments wound around histones. The C9orf72-associated dipeptide repeat protein poly(PR) disrupts HP1α liquid compartments on heterochromatin, thus evicting HP1α from heterochromatin and causing its degradation. Poly(PR) also binds heterochromatin and causes abnormal histone H3 methylation. These events alter heterochromatin structure and ultimately increase repetitive element expression and double-stranded RNA accumulation.


How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein–conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

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