Abstract
A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly-PR inclusions were rare throughout the brain but significantly more abundant in the CA3/4 region of FTLD cases than in MND cases. Thus, although DPR distribution is not correlated with neurodegeneration spatially, it correlates with neuropathological subtypes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1450-z) contains supplementary material, which is available to authorized users.
Highlights
About 10 % of all patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) or mixed presentation of both diseases (ALS/FTD) are caused by a massive expansion of a GGGGCC repeat upstream of the C9orf72-coding region [11, 18, 43]
To compare dipeptide repeat (DPR) expressed from synthetic genes and DPR inclusions in C9orf72 mutation patients under optimal conditions, we raised novel monoclonal antibodies
Since all RNA dyes cross-react with DNA to some extent, we focused on the specific enrichment of heterochromatin DNA in paranucleolar DPR inclusions
Summary
About 10 % of all patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) or mixed presentation of both diseases (ALS/FTD) are caused by a massive expansion of a GGGGCC repeat upstream of the C9orf72-coding region [11, 18, 43]. Inclusions of poly-(glycine– alanine) (poly-GA), poly-(glycine–arginine) (poly-GR) and poly-(glycine–proline) (poly-GP) proteins encoded by the sense strand are far more abundant than poly(proline–alanine) (poly-PA) and poly-(proline–arginine) (poly-PR) proteins encoded by the antisense strand [17, 36]. None of these mechanisms, has so far explained the origin of neuronal and glial TDP-43 inclusions found in almost all cases with C9orf mutation, and the variable expression of dementia and motor symptoms even within the same family [16, 33]. The first clinical symptoms and neurodegeneration seem to arise prior to the onset of TDP-43 pathology when DPR inclusion pathology is already widespread [2, 36, 38, 42]
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