Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS

Abstract

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (c9FTD/ALS). Recently, it was reported that an unconventional mechanism of repeat-associated non-ATG (RAN) translation arises from C9orf72 expansion. Sense and anti-sense transcripts of the expanded C9orf72 repeat, i.e. the dipeptide repeat protein (DRP) of glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA), are deposited in the brains of patients with c9FTD/ALS. However, the pathological significance of RAN-translated peptides remains unknown. We generated synthetic cDNAs encoding 100 repeats of DRP without a GGGGCC repeat and evaluated the effects of these proteins on cultured cells and cortical neurons in vivo. Our results revealed that the poly-GA protein formed highly aggregated ubiquitin/p62-positive inclusion bodies in neuronal cells. In contrast, the highly basic proteins poly-GR and PR also formed unique ubiquitin/p62-negative cytoplasmic inclusions, which co-localized with the components of RNA granules. The evaluation of cytotoxicity revealed that overexpressed poly-GA, poly-GP and poly-GR increased the substrates of the ubiquitin-proteasome system (UPS), including TDP-43, and enhanced the sensitivity to a proteasome inhibitor, indicating that these DRPs are cytotoxic, possibly via UPS dysfunction. The present data indicate that a gain-of-function mechanism of toxic DRPs possibly contributes to pathogenesis in c9FTD/ALS and that DRPs may serve as novel therapeutic targets in c9FTD/ALS.

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism

“…The construct containing 50 GP repeats was obtained through spontaneous contraction of a construct containing 100 GP repeats [kind gift from M. Yamakawa (Keio University School of Medicine, Tokyo, Japan)] [52]. Sequences from all constructs can be provided upon request.…”

Cerebellar neuronal loss in amyotrophic lateral sclerosis cases with ATXN2 intermediate repeat expansions

“…Of the distinct p62-positive, TDP43-negative histopathological inclusions in the cerebellum, the dipeptide protein, poly-GA, is the main prevalent constituent. [7][8][9] All slides were counterstained with hematoxylin to visualize neurons. Severity of pathological inclusions in Purkinje and granule cells in each section was semigraded quantitatively using an Olympus microscope at 2003 magnification and a 4-point severity scale (0 5 none; 1 5 mild; 2 5 moderate; 3 5 severe).…”

“…5 These pathological inclusions were found to contain dipeptide repeat proteins, 6 the poly-GA protein in particular. [7][8][9] Another finding focusing attention on the cerebellum in ALS is the discovery that intermediate repeat expansions (27-33 repeats) in the ataxin-2 gene (ATXN2) is a major risk factor for ALS, with a prevalence of 2%. ; 10 Large expansions in the ATXN2 gene (>33 glutamine repeats) give rise to spinocerebellar ataxia type 2 (SCA2), 11 a cerebellar syndrome characterized by early and pronounced Purkinje cell degeneration in the lateral cerebellar hemispheres and vermis.…”

Elevated Global DNA Methylation Is Not Exclusive to Amyotrophic Lateral Sclerosis and Is Also Observed in Spinocerebellar Ataxia Types 1 and 2

“…Alongside holistic methylation studies, others have focused on the methylation status of the C9orf72 gene promoter, shown to be hypermethylated in expansion carrier ALS and FTD patients [19-22]. Such aberrant methylation has been suggested to be a disease modifier acting through a loss-of-function [23] rather than a toxic gain-of-function mechanism [12, 24]. …”