Cytoplasmic Accumulation of Histones Induced by BET Inhibition Protects Cells from C9orf72 Poly(PR)-Induced Cell Death.

Repeat dipeptides such as poly(proline-arginine) (polyPR) are generated from the hexanucleotide GGGGCC repeat expansions in the C9orf72 gene. These dipeptides are often considered as the genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In the study, fluorescein isothiocyanate (FITC) labeled PR20 is used to investigate PR20-induced cell death. The findings reveal that the cell death induced by PR20 is dependent on its nuclear distribution and can be blocked by a nuclear import inhibitor called importazole. Further investigation reveals that BRD4 inhibitors, such as JQ-1 and I-BET762, restrict cytoplasmic localization of PR20, thereby reducing its cytotoxic effect. Mechanistically, the inhibition of BRD4 leads to an increase in the expression of numerous histones, resulting in the accumulation of histones in the cytoplasm. These cytoplasmic histones associate with PR20 and limit its distribution within the nucleus. Notably, the ectopic expression of histones alone is enough to confer protection to cells treated with PR20. In addition, phenylephrine (PE) induces cellular hypertrophy and cytoplasmic distribution of histone, which also helps protect cells from PR20-induced cell death. The research suggests that temporarily inducing the presence of cytoplasmic histones may alleviate the neurotoxic effects of dipeptide repeat proteins.

Overexpression of C9orf72 exacerbates Aß25­35­induced oxidative stress and apoptosis in PC12 cells.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is manifested by memory loss and spatial disorientation. There is currently no effective treatment for AD. Abnormalities of the chromosome 9 open reading frame 72 (C9ORF72) gene have been associated with various neurodegenerative diseases. However, its intrinsic roles in AD remain to be elucidated. Here we found that Aß25­35 increased the expression of C9orf72 in PC12 cells at both mRNA and protein levels. In Aß25­35­treated PC12 cells, C9orf72 overexpression induced an abnormally condensed and fragmented nucleus and apoptosis, as well as significantly enhanced reactive oxygen species (ROS) levels. Mechanistically, an Aß25­35­induced decrease of superoxide dismutase activity was augmented by C9orf72 overexpression, which in contrast increased malondialdehyde content. Consistently, further apoptotic analysis revealed significant downregulation of Bcl­2 and Bcl­xL expression and enhanced cleavage of caspase­3 with Aß25­35 treatment, all of which were exacerbated by C9orf72 overexpression. In addition, tau phosphorylation, another hallmark of AD pathology, was induced by Aß25­35 and was remarkably enhanced by C9orf72 overexpression. Our data indicate that C9orf72 plays important roles in intracellular ROS signaling and Aß25­35­induced neuronal apoptosis in AD. These findings provide insights into C9orf72 function in the pathogenesis of many related neurodegenerative diseases and provide a basis for potential therapeutic interventions.

Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.

The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-ß polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS.