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. 2021 Feb 15;9(1):26.
doi: 10.1186/s40478-021-01125-6.

Nucleolar stress in C9orf72 and sporadic ALS spinal motor neurons precedes TDP-43 mislocalization

Olubankole Aladesuyi Arogundade  1 , Sandra Nguyen  1 , Ringo Leung  1 , Danielle Wainio  1 , Maria Rodriguez  1 , John Ravits  2
Affiliations

Nucleolar stress in C9orf72 and sporadic ALS spinal motor neurons precedes TDP-43 mislocalization

Olubankole Aladesuyi Arogundade et al. Acta Neuropathol Commun. .

Abstract

Nucleolar stress has been implicated in the pathology and disease pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) from repeat expansions of GGGGCC in C9orf72 (C9-ALS/FTLD) but not in sporadic ALS (SALS). Previously we reported that antisense RNA transcripts are unique in C9-ALS because of their nucleolar localization in spinal motor neurons and correlation with TDP-43 mislocalization, the hallmark proteinopathy of ALS and FTLD. Here we report our further studies of 11 SALS, 11 C9-ALS and 11 control spinal cords. We find that nucleolar stress manifests specifically as shrinkage in nucleoli of C9-ALS spinal motor neurons. Nucleolar size reduction is greatest in similarly sized alpha motor neurons from C9-ALS cases and results are not skewed by the number of surviving neurons from each ALS spinal cord. Surprisingly, nucleolar shrinkage occurs before main pathological hallmarks-TDP-43 mislocalization or antisense RNA foci-appear and this suggest that nucleolar stress can precede pathology in C9-ALS, findings previously identified in C9-FTLD using sense RNA foci and dipeptide repeat proteins as pathological markers. Importantly, these observations are also seen in SALS motor neurons and thus nucleolar stress appears to be a significant and probably upstream problem in sporadic disease.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Nucleolar shrinkage occurs in C9-ALS and SALS spinal motor neurons. a Nucleolar area is identified by fibrillarin staining (green), nuclear area is identified by DAPI and cytoplasmic area is identified by autofluorescence in control, C9-ALS and SALS spinal motor neurons. b–g Average nucleolar (b, c), nuclear (d, e) and cytoplasmic area (f, g) were decreased in C9-ALS and SALS cases compared to controls
Fig. 2
Fig. 2
Gamma motor neurons are identified by ESRRG immunohistochemistry and immunofluorescence. a Immunohistochemistry of ESRRG in control, C9-ALS and SALS lumbar spinal cord with cresyl violet counterstain. Alpha motor neurons (third column) lack nuclear ESRRG immunofluorescence. Gamma motor neurons (fourth column) have nuclear ESRRG immunofluorescence. Magnification scales are consistent for each column. b Distribution of cytoplasmic area for gamma and alpha motor neurons in control, C9-ALS and SALS lumbar spinal cord (dashed line: threshold for alpha motor neurons). c–f Stratification of immunofluorescence data. c Distribution of cytoplasmic area from un-stratified immunofluorescence data. d Comparison of similar distribution of cytoplasmic area in sub-sample of control, C9-ALS and SALS alpha motor neurons (cytoplasmic area thresholds at 2000 µm2 and 3000 µm2). e Nucleolar area is decreased in sub-sample of C9-ALS neurons compared to control neurons with similar cytoplasmic size. f Nucleolar area in subsamples based on cytoplasmic size. g Distribution of cytoplasmic area in control, C9-ALS and SALS gamma motor neurons. h TDP-43 mislocalization occurs in alpha motor neurons (white dashed outline) and in gamma motor neurons (yellow dashed outline) identified by nuclear ESRRG immunofluorescence (arrowheads)
Fig. 3
Fig. 3
Nucleolar size reduction occurs in neurons with and without antisense RNA foci in C9-ALS. a Nucleolar area is identified by fibrillarin staining (green), nuclear area is identified by DAPI and cytoplasmic area is identified by autofluorescence in control, C9-ALS and SALS spinal motor neurons. C-terminal TDP-43 immunofluorescence (cyan) was always nuclear in control neurons and was either nuclear or mislocalized to the cytoplasm in ALS neurons. Antisense RNA foci are identified by FISH staining (red) (nucleolar antisense RNA foci are show in the insert, arrowheads). b–g Nucleolar area (b, c), nuclear area (d, e) and cytoplasmic area (f, g) are decreased in C9-ALS and SALS neurons with or without nucleolar antisense RNA foci compared to controls
Fig. 4
Fig. 4
Nucleolar size reduction occurs in neurons with and without TDP-43 mislocalization. a–f Nucleolar area (a, b), nuclear area (c, d) and cytoplasmic area (e, f) are decreased in C9-ALS with or without TDP-43 mislocalization and in SALS neurons compared to controls. g–i Scatterplots of group average (top) and individual neurons (bottom) comparing nucleolar and nuclear area (g), nucleolar and cytoplasmic area (h) and nuclear and cytoplasmic area (i) (lines indicate linear regression)
Fig. 5
Fig. 5
Ribosomal protein mRNA are upregulated in SALS spinal motor neurons. a Volcano plot of differential gene expression from laser capture microdissection of SALS spinal motor neurons compared to controls. Significance level, p = 0.05 and p = 0.01 are marked with blue and black lines, respectively. Large (dark purple) and small (light purple) ribosomal protein subunit genes are denoted (pseudogenes are included). b Fold change are shown for the top 5 differentially expressed large or small ribosomal protein subunit mRNAs. c Fold enrichment for significantly enriched gene ontology terms relating to ribosomal biogenesis and protein translation. d Schematic representation of nucleolar stress preceding antisense RNA foci and TDP-43 mislocalization in C9-ALS and SALS spinal motor neurons
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