Age five ofFig. 1 Decreased nucleolar volume in neurons from C9FTLD patient brain compared with neurons from neurologically-normal control brain. a Representative photos of frontal cortex from neurologically-normal controls and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD instances immunostained for the nucleolar protein nucleophosmin (NPM, green), the TIGIT Protein HEK 293 neuronal marker (NeuN, magenta) with DAPI nuclear stain (blue). Scale bar represents two m. b, c Quantification of neuronal nucleolar volume determined by nucleophosmin immunoreactivity. Frequency distribution of pooled control and C9FTLD (heterozygous instances only) nucleolar volumes show a shift to lowered volumes in C9FTLD situations (b). Median nucleolar volume was substantially decreased in C9FTLD instances compared with controls (c). Each dot represents an individual case with the homozygous C9FTLD case shown in red, and also the average and SEM of heterozygous situations shown as long and brief horizontal bars, respectively. Significance was determined by unpaired t test: *p 0.(Fig. 3c). These findings show that the arginine-rich DPR protein poly(GR) can exert nucleolar stress to a far higher extent than poly(GA) in vivo.Nucleolar volume is increased in RNA foci-bearing neurons in C9FTLD patient brainIn addition to reports of association in between the arginine-rich DPR proteins and also the nucleolus, C9orf72 sense repeat RNA has also been shown to bind nucleolar proteins in vitro [5, 10]. To investigate whether any adjustments in nucleolar volume were connected with RNA foci in patient tissue, we performed fluorescent in situ hybridisation for C9orf72 sense RNA combined with immunofluorescence for the nucleolar marker nucleophosmin on C9FTLD patient frontal cortex (Fig. 4a). Tissue was also stained for poly(GR) protein, andneurons containing poly(GR) inclusions had been excluded from the analysis (Further file 1: Figure S6a ). RNA foci were present in an average of 35.0 4.three of neurons (Extra file 1: Figure S6b). C9FTLD frontal cortical neurons bearing sense RNA foci showed no difference in the quantity of nucleophosmin-positive nucleoli per cell (Extra file 1: Figure S6d), but a smaller shift towards enhanced nucleophosmin volume compared with neurons with out foci (Fig. 4b). Evaluation of median nucleophosmin volume per case showed that RNA foci-bearing neurons had on average 24 larger nucleoli than neurons without foci (ten.0 1.1 versus 8.1 0.9 m3, p 0.05, Fig. 4c). We were not able to decide nuclear size as DAPI staining was not robust RSPO3 Protein C-Fc-6His sufficient to allow volume analysis immediately after our combined FISH and immunostaining protocol (FISH at 80Mizielinska et al. Acta Neuropathologica Communications (2017) five:Web page 6 ofFig. 2 Elevated nucleolar volume in poly(GR) inclusion-bearing neurons in C9FTLD patient brain. a Representative photos of frontal cortex from a heterozygous C9FTLD case immunostained for the nucleolar protein nucleophosmin (NPM, green), poly(GR) protein (red), the neuronal marker (NeuN, magenta) with DAPI nuclear stain (blue); a standard poly(GR) inclusion is arrowed. Scale bar represents two m. b, c Quantification of neuronal nucleolar volume determined by nucleophosmin immunoreactivity. Frequency distribution of pooled C9FTLD (heterozygous cases only) nucleolar volumes show a shift to increased volumes in neurons bearing poly(GR) inclusions compared with neurons with no inclusions (b). Median nucleolar volume in C9FTLD situations was considerably bigger in neurons with poly(GR) inclusions (GR) compared wi.