A growing body of evidence suggests that a loss of chromosome 9 open reading frame 72 (C9ORF72) expression, formation of
dipeptide-repeat
proteins, and generation of
RNA foci contribute to disease pathogenesis in
amyotrophic lateral sclerosis and
frontotemporal dementia. Although the levels of C9ORF72 transcripts and
dipeptide-repeat
proteins have already been examined thoroughly, much remains unknown about the role of
RNA foci in C9ORF72-linked diseases. As such, we performed a comprehensive
RNA foci study in an extensive pathological cohort of C9ORF72 expansion carriers (n = 63). We evaluated two brain regions using a newly developed computer-automated pipeline allowing recognition of cell nuclei and
RNA foci (sense and antisense) supplemented by manual counting. In the frontal cortex, the percentage of cells with sense or
antisense RNA foci was 26 or 12%, respectively. In the cerebellum, 23% of granule cells contained sense
RNA foci and 1%
antisense RNA foci. Interestingly, the highest percentage of cells with
RNA foci was observed in cerebellar Purkinje cells (~70%). In general, more cells contained sense
RNA foci than
antisense RNA foci; however, when
antisense RNA foci were present, they were usually more abundant. We also observed that an increase in the percentage of cells with
antisense RNA foci was associated with a delayed age at onset in the frontal cortex (r = 0.43, p = 0.003), whereas no other associations with clinico-pathological features were seen. Importantly, our large-scale study is the first to provide conclusive evidence that
RNA foci are not the determining factor of the clinico-pathological variability observed in C9ORF72 expansion carriers and it emphasizes that the distribution of
RNA foci does not follow the pattern of neurodegeneration, stressing the complex interplay between different aspects of C9ORF72-related diseases.