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. 2017 Apr 18;5(1):29.
doi: 10.1186/s40478-017-0432-x.

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration

Sarah Mizielinska  1   2 , Charlotte E Ridler  1 , Rubika Balendra  1   3 , Annora Thoeng  1 , Nathan S Woodling  3 , Friedrich A Grässer  4 , Vincent Plagnol  5 , Tammaryn Lashley  6 , Linda Partridge  3   7 , Adrian M Isaacs  8
Affiliations

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration

Sarah Mizielinska et al. Acta Neuropathol Commun. .

Abstract

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

Keywords: C9orf72; Dipeptide repeat proteins; FTLD; Nucleolar stress; Poly(GR); RNA foci.

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Figures

Fig. 1
Fig. 1
Decreased nucleolar volume in neurons from C9FTLD patient brain compared with neurons from neurologically-normal control brain. a Representative images of frontal cortex from neurologically-normal controls and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD cases immunostained for the nucleolar protein nucleophosmin (NPM, green), the neuronal marker (NeuN, magenta) with DAPI nuclear stain (blue). Scale bar represents 2 μm. b, c Quantification of neuronal nucleolar volume determined by nucleophosmin immunoreactivity. Frequency distribution of pooled control and C9FTLD (heterozygous cases only) nucleolar volumes show a shift to reduced volumes in C9FTLD cases (b). Median nucleolar volume was significantly decreased in C9FTLD cases compared with controls (c). Each dot represents an individual case with the homozygous C9FTLD case shown in red, and the average and SEM of heterozygous cases shown as long and short horizontal bars, respectively. Significance was determined by unpaired t test: *p < 0.05
Fig. 2
Fig. 2
Increased nucleolar volume in poly(GR) inclusion-bearing neurons in C9FTLD patient brain. a Representative images 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 typical poly(GR) inclusion is arrowed. Scale bar represents 2 μm. 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 without inclusions (b). Median nucleolar volume in C9FTLD cases was significantly larger in neurons with poly(GR) inclusions (GR+) compared with neurons without inclusions (GR-) (c). Each dot represents an individual case with the homozygous C9FTLD case shown in red, grey lines link medians from the same cases in neurons with or without poly(GR) inclusions, and the average and SEM of heterozygous cases are shown as long and short horizontal bars, respectively. Significance was determined by paired regression analysis: ****p < 0.0001
Fig. 3
Fig. 3
Expression of GR100, and to a lesser extent GA100, in Drosophila adult neurons is sufficient to increase nucleolar volume in inclusion-bearing neurons. a Representative images of Drosophila brain expressing GR100 or GA100 in adult neurons using the elav-GeneSwitch (elavGS) driver immunostained for the nucleolar protein fibrillarin (FIB, red), the dipeptide repeat (DPR) protein poly(GR) or poly(GA) (green), with DAPI nuclear stain (blue); typical poly(GR) and poly(GA) inclusions are arrowed. Scale bar represents 2 μm. b, c Quantification of neuronal nucleolar volume determined by fibrillarin immunoreactivity. Median nucleolar volume in Drosophila adult neurons expressing GR100 was significantly larger in neurons bearing poly(GR) inclusions (GR+) than in neurons without inclusions (GR-) (b). Using the same scale as in b, the median nucleolar volume increase in Drosophila adult neurons expressing GA100 is not apparent, highlighting the difference in magnitude of the changes; magnifying the scale (inset), reveals nucleolar volume was significantly larger in neurons bearing poly(GA) inclusions (GA+) than in neurons without inclusions (GA-) (c). Controls in b and c are Drosophila transgenic for DPR proteins that were not induced for gene expression. Each dot represents an individual fly, grey lines link medians from the same individual fly in neurons with or without DPR protein inclusions, and average and SEM are shown as long and short horizontal bars, respectively. Significance was determined by paired regression analysis: ***p < 0.001. Genotypes were: w; UAS-GR100/+; elavGS/+ (elavGS > GR100), w; UAS-GA100/+; elavGS/+ (elavGS > GA100)
Fig. 4
Fig. 4
Nucleolar volume is increased in RNA foci-bearing neurons in C9FTLD patient brain. a Representative images of frontal cortex from a heterozygous C9FTLD case immunostained for the nucleolar protein nucleophosmin (NPM, green), with RNA fluorescent in situ hybridisation for sense RNA foci (red) and DAPI nuclear stain (blue); typical RNA foci-bearing (Foci+) and non foci-bearing (Foci-) neurons are highlighted with dotted boxes. Scale bar represents 2 μm. b, c Quantification of neuronal nucleolar volume determined by nucleophosmin immunoreactivity. Frequency distribution of pooled C9FTLD (heterozygous cases) nucleolar volumes show a slight shift to increased volumes in neurons bearing RNA foci compared with neurons without foci (b). Median nucleolar volume in C9FTLD cases was significantly larger in neurons with RNA foci compared with neurons without foci inclusions (c). Each dot represents an individual heterozygous C9FTLD case, grey lines link medians from the same cases in neurons with or without RNA foci, and the average and SEM are shown as long and short horizontal bars, respectively. Significance was determined by paired regression analysis: *p < 0.05
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References

    1. Ash PE, Bieniek KF, Gendron TF, Caulfield T, Lin WL, Dejesus-Hernandez M, van Blitterswijk MM, Jansen-West K, Paul JW, III, Rademakers R, Boylan KB, Dickson DW, Petrucelli L. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron. 2013;77:639–646. doi: 10.1016/j.neuron.2013.02.004. - DOI - PMC - PubMed
    1. Beck J, Poulter M, Hensman D, Rohrer JD, Mahoney CJ, Adamson G, Campbell T, Uphill J, Borg A, Fratta P, Orrell RW, Malaspina A, Rowe J, Brown J, Hodges J, et al. Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population. Am J Hum Genet. 2013;92:345–353. doi: 10.1016/j.ajhg.2013.01.011. - DOI - PMC - PubMed
    1. Boeynaems S, Bogaert E, Kovacs D, Konijnenberg A, Timmerman E, Volkov A, Guharoy M, De Decker M, Jaspers T, Ryan VH, Janke AM, Baatsen P, Vercruysse T, Kolaitis RM, Daelemans D, et al. Phase separation of C9orf72 dipeptide repeats perturbs stress granule dynamics. Mol Cell. 2017;65:1044–1055. doi: 10.1016/j.molcel.2017.02.013. - DOI - PMC - PubMed
    1. Burguete AS, Almeida S, Gao FB, Kalb R, Akins MR, Bonini NM. GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function. Elife. 2015;4:e08881. - PMC - PubMed
    1. Cooper-Knock J, Walsh MJ, Higginbottom A, Robin HJ, Dickman MJ, Edbauer D, Ince PG, Wharton SB, Wilson SA, Kirby J, Hautbergue GM, Shaw PJ. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain. 2014;137:2040–2051. doi: 10.1093/brain/awu120. - DOI - PMC - PubMed

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