Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration

Abstract

IntroductionCytoplasmic TDP-43 inclusions are the pathological hallmark of amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar dementia (FTLD). The G4C2 repeat mutation in C9ORF72 is the most common cause of ALS and FTLD in which, in addition to TDP-43 inclusions, five different di-peptide repeat (DPR) proteins have been identified. Di-peptide repeat proteins are translated in a non-canonical fashion from sense and antisense transcripts of the G4C2 repeat (GP, GA, GR, PA, PR). DPR inclusions are abundant in the cerebellum, as well as in the frontal and temporal lobes of ALS and FTLD patients and some are neurotoxic in a range of cellular and animal models, implying that DPR aggregation directly contributes to disease pathogenesis. Here we sought to quantify inclusions for each DPR and TDP-43 in ALS cases with and without the C9ORF72 mutation. We characterised the abundance of DPRs and their cellular location and compared this to cytoplasmic TDP-43 inclusions in order to explore the role of each inclusion in lower motor neuron degeneration.ResultsSpinal cord sections from ten cases positive for the C9ORF72 repeat expansion (ALS-C9+ve) and five cases that were not were probed by double immunofluorescence staining for individual DPRs and TDP-43. Inclusions immunoreactive for each of the DPRs were present in the spinal cord but they were rare or very rare in abundance (in descending order of frequency: GA, GP, GR, PA and PR). TDP-43 cytoplasmic inclusions were 45- to 750-fold more frequent than any DPR, and fewer than 4 % of DPR inclusions colocalized with TDP-43 inclusions. In motor neurons, a single cytoplasmic DPR inclusion was detected (0.1 %) in contrast to the 34 % of motor neurons that contained cytoplasmic TDP-43 inclusions. Furthermore, the number of TDP-43 inclusions in ALS cases with and without the C9ORF72 mutation was nearly identical.ConclusionsFor all other neurodegenerative diseases, the neurotoxic protein aggregates are detected in the affected population of neurons. TDP-43 cytoplasmic aggregation is the dominant feature of ALS spinal cord pathology irrespective of C9ORF72 mutation status. The near absence of DPR inclusions in spinal cord motor neurons challenges their contribution to lower motor neuron degeneration in ALS-C9+ve cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0218-y) contains supplementary material, which is available to authorized users.

Modeling ALS and FTD with iPSC-derived neurons

“…In at least one study, the RAN translation protein products and aberrant increase of proteasome marker p62 are not consistently detected (Sareen et al, 2013). These results have also been further extended by another recent study (Gomez-Deza et al, 2015), which shows a distinct lack of RAN translation protein products in spinal motor neurons in FALS patients with C9ORF72 mutations. In the future, it will be important to use the iPSC models to determine how RAN proteins induce p62 pathology and whether p62 pathology is causally linked to neuronal degeneration and functional deficits.…”

“…Similarly, another study indicated that C9ORF72 iPSC-derived neurons show reduced viability when exposed to chloroquine, an inhibitor that blocks the autophagy pathway (Almeida et al, 2013). Finally, C9ORF72 iPSC-derived neurons should also provide a convenient tool to investigate the potential loss-of-function effect of C9ORF72 gene product in the regulation of endosomal trafficking and in the pathogenesis of TDP-43 proteinopathy, which are common neuropathological features in these cases (Farg et al, 2014; Gomez-Deza et al, 2015). …”

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

“…The lower limb onset of ALS symptoms may mask or make it difficult to elicit spinocerebellar features in these cases. Importantly, lower limb onset of itself in other ALS cases (sporadic and C9ORF72) does not predispose Purkinje cells to retrograde degeneration, and neither does having another type of repeat expansion (despite increased cerebellar pathology with C9ORF72 expansions; present study and previous works 5,23 ). Interestingly, both ATXN2 and C9ORF72 expansion-specific proteins do not accumulate in motor neurons (present study and previous works 5,23 ) with abnormal ATXN2 accumulation also not observed in Purkinje cells in any of the ALS cases with the expansion (present study and see Table 5).…”

RNA-binding proteins in neurodegeneration: mechanisms in aggregate

“…While toxicity of DPRs has been shown in model organisms and cell culture (Wen et al 2014;Lopez-Gonzalez et al 2016), this could reflect nonphysiological effects resulting from overexpression. Understanding how DPRs are made (Green et al 2016) and how abundant they truly are in degenerating regions (Gomez-Deza et al 2015) will be important in order to gauge how critical they are to the pathology of the ALS/FTD spectrum.…”