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. 2019 Feb 15;363(6428):eaav2606.
doi: 10.1126/science.aav2606.

Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity

Yong-Jie Zhang  1   2 , Lin Guo  3 , Patrick K Gonzales  4 , Tania F Gendron  1   2 , Yanwei Wu  1 , Karen Jansen-West  1 , Aliesha D O'Raw  1 , Sarah R Pickles  1 , Mercedes Prudencio  1   2 , Yari Carlomagno  1 , Mariam A Gachechiladze  5 , Connor Ludwig  6 , Ruilin Tian  6 , Jeannie Chew  1   2 , Michael DeTure  1   2 , Wen-Lang Lin  1 , Jimei Tong  1 , Lillian M Daughrity  1 , Mei Yue  1 , Yuping Song  1 , Jonathan W Andersen  1 , Monica Castanedes-Casey  1 , Aishe Kurti  1 , Abhishek Datta  7 , Giovanna Antognetti  7 , Alexander McCampbell  8 , Rosa Rademakers  1   2 , Björn Oskarsson  9 , Dennis W Dickson  1   2 , Martin Kampmann  6 , Michael E Ward  5 , John D Fryer  1   2 , Christopher D Link  4 , James Shorter  3 , Leonard Petrucelli  10   2
Affiliations

Heterochromatin anomalies and double-stranded RNA accumulation underlie C9orf72 poly(PR) toxicity

Yong-Jie Zhang et al. Science. .

Abstract

How hexanucleotide GGGGCC (G4C2) repeat expansions in C9orf72 cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is not understood. We developed a mouse model engineered to express poly(PR), a proline-arginine (PR) dipeptide repeat protein synthesized from expanded G4C2 repeats. The expression of green fluorescent protein-conjugated (PR)50 (a 50-repeat PR protein) throughout the mouse brain yielded progressive brain atrophy, neuron loss, loss of poly(PR)-positive cells, and gliosis, culminating in motor and memory impairments. We found that poly(PR) bound DNA, localized to heterochromatin, and caused heterochromatin protein 1α (HP1α) liquid-phase disruptions, decreases in HP1α expression, abnormal histone methylation, and nuclear lamina invaginations. These aberrations of histone methylation, lamins, and HP1α, which regulate heterochromatin structure and gene expression, were accompanied by repetitive element expression and double-stranded RNA accumulation. Thus, we uncovered mechanisms by which poly(PR) may contribute to the pathogenesis of C9orf72-associated FTD and ALS.

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Conflict of interest statement

Competing interests: B.O. served as a paid consultant for Flex Pharma, Mitsubishi Tanabe, and Biogen Idec. G.A. and A.M. are full-time employees of and own equity in Biogen Idec. M.K. serves on the scientific advisory board of Engine Biosciences and is a consultant for Maze Therapeutics. M.K. has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449). Other authors declare no competing financial interests.

Figures

Fig. 1.
Fig. 1.. GFP-(PR)50 mice exhibited neurodegeneration and behavioral deficits.
Immunohistochemical (A) and quantitative (B) analyses of anti-PR immunoreactivity in the cortices of GFP-(PR)50 mice at 1 month (1M) (n = 8 mice) and 3 months (3M) (n = 10 mice) of age. Scale bars, 100 μm. (C) An immunoassay was used to compare poly(PR) levels in cortex and hippocampus lysates of GFP-(PR)50 mice at 1 (n = 8 mice) and 3 (n = 9 mice) months of age. (D) Representative images of NeuN-labeled cells in the cortices of 3-month-old GFP or GFP-(PR)50 mice. Scale bars, 100 μm. (E) Quantification of NeuN-labeled cells in the cortices of GFP mice at 1 (n = 8 mice) and 3 (n = 10 mice) months of age or GFP-(PR)50 mice at 1 (n = 8 mice) and 3 (n = 10 mice) months of age. (F) Results from a 4-day rotarod test used to assess motor deficits of 3-month-old mice expressing GFP (n = 12 mice) or GFP-(PR)50 (n = 11 mice) by evaluating their latency to fall from a rotating rod. (G) Results from the fear-conditioning test used to assess the associative learning and memory of 3-month-old mice expressing GFP (n = 12 mice) or GFP-(PR)50 (n = 11 mice) by evaluating the percentage of time frozen in response to a conditioned (cued) or unconditioned (context) stimulus. Data are presented as the mean ± SEM. Male mice are represented by solid symbols, whereas female mice are represented by empty symbols. In (B) and (C), ****P < 0.0001, **P = 0.0072, two-tailed unpaired t test. In (E), ****P < 0.0001; NS (not significant), P = 0.1130; two-way ANOVA and Tukey’s post hoc analysis. In (F), NS, P = 0.1269; **P = 0.0096; ##P = 0.0015; ***P = 0.0005; two-way ANOVA and Tukey’s post hoc analysis. In (G), ****P 0.0001; NS, P = 0.6007; two-tailed unpaired t test.
Fig. 2.
Fig. 2.. Poly(PR) proteins localized to heterochromatin in GFP-(PR)50 mice and c9FTD/ALS patients.
(A) Double immunofluorescence staining for GFP-(PR)50 and nucleolar markers (NPM1 and fibrillarin) in the cortices of 3-month-old GFP-(PR)50 mice (n = 5). Scale bars, 5 μm. (B) Immunoelectron microscopy using an anti-PR antibody labeled with gold particles in the cortices of 3-month-old GFP-(PR)50 mice. The selected region in the low-magnification image (left) is shown at high magnification (right). * indicates the nucleolus. Arrows indicate gold particles. Scale bars, 0.5 μm (left) and 0.2 μm (right). (C) Immunoelectron microscopy analysis of purified mouse genomic DNA incubated with (PR)8 peptide by using an anti-PR antibody labeled with gold particles. Arrows indicate gold particles. Scale bars, 50 nm. (D) Electrophoretic mobility shift assays using single- and double-stranded DNA co-incubated (+) or not (–) with (PR)8 or (PA)8 peptides. AT, AT-rich oligonucleotides; GC, GC-rich oligonucleotides.(E) Double immunofluorescence staining for GFP-(PR)50 and heterochromatin (H3K9me3 and H3K27me3) or euchromatin (H3K4me3) markers in the cortices of 3-month-old GFP-(PR)50 mice (n = 7). Scale bars, 5 μm. (F) Double immunofluorescence staining for poly(PR) and H3K27me3 or H3K4me3 in the cortices of c9FTD/ALS patients. All nuclear poly(PR) inclusions colocalized with H3K27me3 (n = 7 cases) and with H3K4me3 (n = 4 cases). Representative images from case 4 are shown. See also fig. S5F and table S1. Scale bars, 5 μm.
Fig. 3.
Fig. 3.. Poly(PR) proteins caused lamin invaginations, reduced HP1α levels, and disrupted HP1α liquid droplets.
(A) Double immunofluorescence staining for GFP-(PR)50 and lamin A/C or lamin B in the cortices of 3-month-old GFP (n = 11) or GFP-(PR)50 (n = 7) mice. Scale bars, 10 μm. (B) Double immunofluorescence staining for GFP-(PR)50 and HP1α in the cortices of 3-month-old GFP-(PR)50 mice (n = 7). Scale bars, 5 μm. (C) Differential interference contrast (DIC) microscopy images of HP1α droplets at a concentration of 90 μM HP1α before the addition of (PR)8. The arrow indicates the fusing of two liquid droplets. By contrast, (PR)8 peptides at a concentration of 245 μM did not form droplets under these conditions. (D) DIC microscopy images of HP1α droplets at 90 μM HP1α (top, low magnification; bottom, high magnification) after the addition of 245 μM (PR)8. Black arrows indicate the bursting of a preformed HP1α droplet after the addition of (PR)8. Small blue arrows indicate the solid components within HP1α droplets. (E) DIC microscopy images of HP1α droplets at 90 μM HP1α (top, low magnification; bottom, high magnification) after the addition of 245 μM (PA)8 peptides. In (D) and (E), turquoise arrows between images indicate progression over time.
Fig. 4.
Fig. 4.. Transcriptome alterations were identified in the brains of mice expressing GFP-(PR)50.
(A) Hierarchical clustering of the 1000 most variable genes between 3-month-old GFP mice (n = 4) and GFP-(PR)50 mice (n = 7). (B) MA plots of up- and down-regulated genes (FDR < 0.01) in the cortices and hippocampi of 3-month-old mice expressing GFP-(PR)50 (n = 7) compared with those of GFP controls (n = 4). The MA plot is based on the Bland-Altman plot, where M represents the log2 fold change (y axis) and A represents the log of mean gene expression (x axis). (C) Gene modules identified in brains of 3-month-old mice expressing GFP-(PR)50 (n = 7 mice) through weighted gene coexpression correlation network analyses using differentially expressed genes.
Fig. 5.
Fig. 5.. Poly(PR) caused abnormal expression of REs and dsRNA accumulation.
(A) MA plots of RNA-seq data show up- and down-regulated REs in the cortices and hippocampi of 3-month-old GFP-(PR)50 mice (n = 7) compared with GFP mice (n = 4). Red dots represent the REs with a significant change (FDR < 0.10). (B) Validation of REs identified by RNA-seq in the cortices and hippocampi of 3-month-old GFP (n = 10) or GFP-(PR)50 (n = 9) mice by qPCR. (C) Double immunofluorescence staining for GFP-(PR)50 and dsRNA in the cortices of 3-month-old GFP mice (n = 3) or GFP-(PR)50 mice (n = 7). Scale bars, 5 μm. (D) Double immunofluorescence staining for HP1α and dsRNA in human dCas9-iPSC-differentiated neurons stably expressing HP1α sgRNA 1. Scale bars, 5 μm. Ctrl, control. (E) Double immunofluorescence staining for active caspase-3 and dsRNA in human dCas9-iPSC-differentiated neurons stably expressing HP1α sgRNA 1. Scale bars, 5 μm. Data are presented as the mean ± SEM. Male mice are represented by solid symbols, whereas female mice are represented by empty symbols. In (B), = 0.0015; ##P = 0.0065; = 0.0004; and &&P = 0.0036; two-tailed unpaired t test.
Fig. 6.
Fig. 6.. Expression of GFP-(PR)50 in mice caused abnormalities of RanGAP1 and NPC proteins but did not lead to TDP-43 pathology.
(A) Double immunofluorescence staining for GFP-(PR)50 and RanGAP1 in the cortices of 3-month-old GFP (n = 11) or GFP-(PR)50 (n = 7) mice. Scale bars, 5 μm. (B) Double immunofluorescence staining for GFP-(PR)50 and NPC proteins in the cortices of 3-month-old GFP or GFP-(PR)50 mice (n = 4 mice per group). Scale bars, 5 μm. Insets in (A) and (B) show boxed areas at higher magnification. (C) Double immunofluorescence staining for GFP-(PR)50 and TDP-43 in the cortices of 3-month-old GFP or GFP-(PR)50 mice (n = 3 mice per group). Scale bars, 5 μm.
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