Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS

Abstract

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes “c9FTD/ALS” are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1192-8) contains supplementary material, which is available to authorized users.

Timing and significance of pathological features inC9orf72expansion-associated frontotemporal dementia

“…Dipeptide repeat protein and TDP-43 inclusion formation was assessed using immunohistochemistry with the following antibodies: TDP-43 (anti-rabbit, 1:2500, Proteintech), phospho-TDP-43 (pTDP-43, anti-rat, 1:500, courtesy of Prof. Manuela Neumann, University Hospital Tü bingen, Germany), and dipeptide repeat protein antibodies, developed by one of the authors (L.P.), that recognize GA (anti-rabbit, 1:20 000), GP (Gly-Pro) (anti-rabbit, 1:20 000), GR (Gly-Arg) (anti-rabbit, 1:5000), PA (Pro-Ala) (anti-rabbit, 1:5000) and PR (Pro-Arg) (anti-rabbit, 1:2000) Gendron et al, 2013). Immunostained sections were counterstained with Harris haematoxylin.…”

“…These candidates include loss of C9orf72 gene product function and toxicity through repeat RNA foci and protein aggregation. RNA foci can be composed of sense or antisense repeat mRNA strands (DeJesus-Hernandez et al, 2011;Gendron et al, 2013;Mizielinska et al, 2013) and have been shown to sequester RNA-binding proteins, leading to transcriptome abnormalities (Donnelly et al, 2013;Lee et al, 2013;Mori et al, 2013a;Sareen et al, 2013). Expanded repeats also undergo abnormal repeat associated non-ATG (RAN) translation (Zu et al, 2011).…”

C9ORF72: grabbing a tiger by the tail

“…Therefore, at present we have three plausible, and indeed not mutually exclusive, hypotheses for neurotoxicity: in fact studies have shown that cells accumulating toxic RNA foci again do not overlap with those accumulating DPRs [21,43], suggesting these are complimentary and not competing events. So where does this leave us?…”

“…This too occurs in C9ALS and C9FTLD with translation of the expansion occurring in both a sense and an antisense direction, leading to the formation and brain aggregation of all five possible dipeptide repeat proteins (DPRs) [3,21,45,47,67,69]. Indeed, DPR can consist of any or all of the five possible species [3,21,39,40,45,47,67,69].…”

“…This too occurs in C9ALS and C9FTLD with translation of the expansion occurring in both a sense and an antisense direction, leading to the formation and brain aggregation of all five possible dipeptide repeat proteins (DPRs) [3,21,45,47,67,69]. Indeed, DPR can consist of any or all of the five possible species [3,21,39,40,45,47,67,69]. These DPR proteins seem to be entirely specific to the expansion, not being seen in FTLD and ALS cases without expansions [39,40], though curiously are not specific for FTLD and ALS per se with rare appearances in cases with pathologically confirmed corticobasal degeneration [40,58], AD [12,29,35] but see [15], Creutzfeldt-Jakob disease [6], and even in apparently healthy controls [6,13].…”