Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy

Johnathan Cooper-Knock,Pamela J Shaw,Guillaume M Hautbergue,Adrian Higginbottom,Paul G Ince,Stuart Pickering-Brown,Stephen B Wharton,Janine Kirby,Matthew J Stopford,J Robin Highley

doi:10.1007/s00401-015-1429-9

Johnathan Cooper-Knock, Pamela J Shaw + Show 8 more

Open Access

https://doi.org/10.1007/s00401-015-1429-9

Copy DOI

Abstract

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ2, p < 0.00001) but not sense foci (χ2, p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1429-9) contains supplementary material, which is available to authorized users.

Highlights

GGGGCC hexanucleotide repeat expansions in C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [7, 27]
In the cerebellar populations and motor neurons but not hippocampal neurons, there was a difference between the frequency of antisense and sense foci which was consistent between cases. To determine whether this difference was statistically significant, the foci count was modelled as a Poisson distribution and performing a likelihood-ratio test revealed that, within each individual case, the frequency of antisense compared to sense RNA foci was significantly higher in Purkinje neurons and motor neurons, but significantly lower in cerebellar granule neurons
The precise mechanisms of neuronal injury in C9ORF72disease appear complex, and are likely to involve RNA gain-of-function toxicity mediated by sense and antisense transcription of the GGGGCC repeat expansion

Summary

Introduction

GGGGCC hexanucleotide repeat expansions in C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [7, 27]. It has been observed that RNA foci are formed, from sense, and from antisense transcription of the repeat expansion [7, 16, 21]. Work by Haeusler et al [11] recently suggested that, with a small number of exceptions, the protein binding partners of the two species of RNA foci are similar. Another suggested mechanism of pathogenesis is direct toxicity of one or more of five dipeptide repeat proteins (DPRs) translated in different reading frames from either the sense [23] or antisense [24] RNA molecules. We have previously identified interactions between sense RNA repeat sequences and mRNA export adaptor proteins which might have a role in inappropriate licencing for nuclear export [3]

Methods

Results

Conclusion