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

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.

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

“…Second, both sense and antisense repeat RNA were found to be mainly localized to the cytoplasm, both diffusely as well as in RNA foci. While in C9orf72 patients most RNA foci have been found to be located in the nucleus, cytoplasmic RNA foci have also been found in postmortem tissue [11,12,34], as well as in patient-derived cell cultures [17]. These data suggest that RNA toxicity might be mediated by cytoplasmic repeat RNA, which might explain why no correlation between extent of nuclear RNA foci and neurodegeneration has been found in postmortem tissue [15].…”

Timing and significance of pathological features inC9orf72expansion-associated frontotemporal dementia

“…If these neurons reflect C9orf72 disease, nuclear TDP-43 depletion could have resulted from diminished TDP-43 expression or restricted access to the nucleus due to nucleocytoplasmic transport impairment (Freibaum et al, 2015;Jovicic et al, 2015;Zhang et al, 2015). Further work is needed to determine whether loss of nuclear TDP-43 in the absence of a TDP-43 inclusion relates to particular C9orf72-specific inclusions, as suggested in a recent study (Cooper-Knock et al, 2015). Moreover, the absence of a proximal somatodendritic TDP-43 inclusion does not rule out an inclusion somewhere in the distal neuron or composed of a TDP-43 species not recognized by the antibodies used here.…”

Cross-Sectional Survey of Relevant Literatures as to the Current Proposed Disease Mechanisms and Treatments of Amyotrophic Lateral Sclerosis (ALS)

“…79 This expansion itself may not be inherently irregular but instead trigger pathogenicity in differentially methylated states 80,81 and has also demonstrated a concordance with abnormal localization of TDP-43, a hallmark of ALS neuronal pathology. 68 Murine models with induced GGGGCC sequences have also been constructed, evidencing a gain of function relationship theorized elsewhere. 82,83 Although this sequence provides a possible marker for over one third of FALS cases, the ethical and moral implications of how to determine when patients should or should not be screened for this repeat are difficult at best to determine.…”

“…68 In an international cross-sectional study evaluating 4448 ALS patients, 37% of individuals with FALS are in possession of this mutation, having as many as 700-1600 copies of the repeat sequence compared to controls who only have 23 copies. 69 Comparable studies have been conducted in French (46% FALS, 8% SALS), 70 Italian (46% FALS), 71 Greek (50% FALS, 8.2% SALS), 72 Turkish (18.3% FALS, 3.1% SALS), 73 Slavic (5.9% SALS), 74 and Russian (15% FALS, 2.5% SALS) 75 populations testing positive for the GGGGCC repeat.…”