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.