Enhanced detection of expanded repeat mRNA foci with hybridization chain reaction

Abstract

Transcribed nucleotide repeat expansions form detectable RNA foci in patient cells that contribute to disease pathogenesis. The most widely used method for detecting RNA foci, fluorescence in situ hybridization (FISH), is powerful but can suffer from issues related to signal above background. Here we developed a repeat-specific form of hybridization chain reaction (R-HCR) as an alternative method for detection of repeat RNA foci in two neurodegenerative disorders: C9orf72 associated ALS and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome. R-HCR to both G₄C₂ and CGG repeats exhibited comparable specificity but > 40 × sensitivity compared to FISH, with better detection of both nuclear and cytoplasmic foci in human C9 ALS/FTD fibroblasts, patient iPSC derived neurons, and patient brain samples. Using R-HCR, we observed that integrated stress response (ISR) activation significantly increased the number of endogenous G₄C₂ repeat RNA foci and triggered their selective nuclear accumulation without evidence of stress granule co-localization in patient fibroblasts and patient derived neurons. These data suggest that R-HCR can be a useful tool for tracking the behavior of repeat expansion mRNA in C9 ALS/FTD and other repeat expansion disorders.

Keywords: Amyotrophic lateral sclerosis; C9orf72; FXTAS; Fragile X; G3BP; RNA Gelation; RNA foci; Repeat-associated non-AUG (RAN) translation; Stress granules.

Fig. 1

Hybridization chain reaction increases detection of GC rich repeats over FISH. a FISH probe with a 5′ conjugated fluorophore. Signal strength is dependent on number of RNA molecules present. b In R-HCR, two probe types are used: the initiator probe which binds directly to the RNA of interest, and 5' fluorophore conjugated hairpin probes (H1 and H2) complementary to 3′ and 5′ extensions on the initiator probe. Upon binding, H1 and H2 unfold to reveal new binding sites for the other hairpin probe. In this way, signal from one RNA molecule is amplified > 100 fold, dramatically enhancing detection. c, d MEFs transfected with (G₄C₂)₇₀-NL-3xFlag (c) or CGG₁₀₀-3xFlag (d) vectors and probed with indicated concentrations of either FISH or R-HCR probe. e MEFs transfected with (G₄C₂)₇₀-NL-3xFlag expressing vector (top) or CGG₁₀₀-3xFlag vector (bottom) and treated with DNase (left) or RNase (right) prior to R-HCR. f ICC-R-HCR of MEFs transfected with antisense (G₂C₄)₄₇-NL-3xFlag (top) or (CCG)₆₀-NL-3xFlag (bottom) expressing plasmids. Top: N = 434; Bottom: N = 38.Error bars indicate 95% confidence intervals (CI). Scale bar = 20 μm in c, d, e; 50 μm in f

Fig. 2

R-HCR probe signal intensity is repeat length dependent for G₄C₂ and CGG repeats. a R-HCR on MEFs transfected with G₄C₂ repeat constructs with increasing repeat sizes. b Quantification of cells with detectable G₄C₂ repeat RNA foci in total cells. (G₄C₂)₃: N = 313; (G₄C₂)₃₅ N = 575; (G₄C₂)₇₀ N = 514. c Quantification of R-HCR signal intensity in MEFs transfected with (G₄C₂)_n-NL-3xF reporters with indicated repeat length. (G₄C₂)₃₅: N = 175; (G₄C₂)₇₀: N = 110. d R-HCR on MEFs transfected with CGG repeat constructs with increasing repeat sizes. e Quantification of Flag + and CGG RNA + cells expressed as proportion of total transfected cells. f Quantification of R-HCR signal intensity in MEFs transfected with 2(CGG)n-NL-3xF reporters with indicated repeat length. e-fN ≥ 35/condition. Error bar indicates 95% CI. Statistics: Chi-square test for b, c, e and f. Fisher’s exact test for b, **p < 0.01, ****p < 0.0001, ns: not significant. Scale bar = 50 μm in a and d

Fig. 3

R-HCR is more sensitive than FISH at detecting endogenous G₄C₂ repeats in C9orf72 ALS-FTD patient fibroblasts. a–c FISH (a) and R-HCR (b) in three (C1–C3) C9orf72 ALS-FTD patient and control fibroblast lines, c treated with DNase and RNase prior to R-HCR. White arrows were used indicate RNA foci positive cells in some images. Higher magnification images of the boxed areas are shown on the right. d Quantification of a and b. e–f Quantification of RNA foci number per RNA + cell and the distribution for RNA foci. Error bars = 95% CI (d and f ) and ± SEM (e). N > 150 for each line. Statistics: Fisher’s exact test (d), unpaired t test (e) and Chi-square test (f). ***p < 0.001, ****p < 0.0001, ns = not significant. Scale bar = 50 μm in left for a and b; 20 μm in right for a and b, and c

Fig. 4

R-HCR is more sensitive than FISH at detecting endogenous G₄C₂ repeats in C9orf72 ALS-FTD patient brains. a–e FISH (a, c) and R-HCR (b, d, e) cerebellum (a, b, e) and frontal cortex (c, d) from three C9orf72 ALS-FTD patients and controls. e C9 cases treated with DNase and RNase prior to R-HCR. White arrows indicate RNA foci positive cells. Higher magnification images of the boxed areas are shown on the right. f Quantification of the percentage of cerebellar granule cells with detectable G₄C₂ repeat RNA foci in 3 cases. g Quantification of the percentage of cortical neurons and glia with detectable G₄C₂ repeat RNA foci in three C9orf72 cases ± 95%CI. N > 150 for each sample. Statistics: Fisher’s exact test for f and g. ***p < 0.001, ****p < 0.0001. Scale bar = 50 μm in left for a–d; 20 μm in right for a–e. cb = cerebellum, ctx = frontal cortex

Fig. 5

G₄C₂ repeat RNAs redistribute in the nucleus during stress in C9orf72 ALS-FTD patient fibroblasts. a R-HCR of C9orf72 ALS-FTD patient fibroblasts treated with H₂O (vehicle) or sodium arsenite (SA) for the indicated times. b) Quantification of cells with detectable G₄C₂ repeat RNA foci in total cells. The dot shapes represent different C9orf72 patient fibroblast lines. N > 150 for each line for vehicle, SA 1.0 h, and SA 2.0 h. c Distribution of G₄C₂ repeat RNA foci with or without SA treatment ± 95%CI. N = 400 for vehicle, 679 for SA 1.0 h, and 775 for SA 2.0 h. Statistics: matched one-way ANOVA and paired t test (b), and Chi-square test (c). *p < 0.05, ***p < 0.001, ****p < 0.0001. ns not significant. Scale bar = 10 μm in a

Fig. 6

G₄C₂ repeat RNAs redistribute in the nucleus during stress in C9orf72 ALS-FTD patient neurons. a R-HCR-ICC of C9orf72 ALS-FTD patient derived neurons treated with H₂O or SA. G3BP1 = stress granule marker. A higher magnification of the boxed areas are shown on the right. White arrows indicate rare colocalization events between cytoplasmic G₄C₂ repeats and G3BP1. b Quantification of (a). Bars represent proportion of neurons with detectable G₄C₂ repeat RNA among total neurons ± 95%CI. N = 156 for vehicle and 535 for SA 1.0 h. c Quantification of the distribution of repeat RNA in all repeat positive neurons ± 95% CI. N = 104 for vehicle and 233 for SA 1.0 h. Statistics: Fisher’s exact test (b) and Chi-square test (c). ****p < 0.0001. Scale bar = 10 μm in (a) except for the zoomed in images (2.5 μm)

Fig. 7

G₄C₂ repeat RNAs rarely co-localize with cytoplasmic G3BP1 or nuclear TDP-43 during stress. a R-HCR-ICC of C9orf72 patient derived fibroblasts treated 1 h with H₂O or SA. G3BP1 = stress granule marker. Higher magnification images of the boxed areas are shown on the right. White arrows indicate co-localization of G₄C₂ repeat and G3BP1. b Quantification of cytoplasmic G₄C₂ repeat foci co-localization with G3BP1 as a fraction of all cytoplasmic G₄C₂ repeats. c R-HCR-ICC of C9orf72 patient derived fibroblasts treated with H₂O or SA (1 h, 2 h). TDP-43 = nuclear body marker. White arrows indicate co-localization of G₄C₂ repeat and TDP-43. d Quantification of nuclear G₄C₂ repeat foci colocalization with TDP-43 as a fraction of all observed nuclear G₄C₂ repeat foci. % shown as proportion of co-localization for each condition ± SEM. Statistics: Fisher’s exact test (b) and Chi-square test (d). **p < 0.01, ***p < 0.001. ns: not significant. Scale bar = 10 μm in (a) and (c) except for zoomed images (2.5 μm)