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. 2016 Aug 12;353(6300):708-12.
doi: 10.1126/science.aaf7791.

Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts

Nicholas J Kramer  1 , Yari Carlomagno  2 , Yong-Jie Zhang  2 , Sandra Almeida  3 , Casey N Cook  2 , Tania F Gendron  2 , Mercedes Prudencio  2 , Marka Van Blitterswijk  2 , Veronique Belzil  2 , Julien Couthouis  4 , Joseph West Paul 3rd  4 , Lindsey D Goodman  5 , Lillian Daughrity  2 , Jeannie Chew  2 , Aliesha Garrett  2 , Luc Pregent  2 , Karen Jansen-West  2 , Lilia J Tabassian  2 , Rosa Rademakers  2 , Kevin Boylan  6 , Neill R Graff-Radford  6 , Keith A Josephs  7 , Joseph E Parisi  7 , David S Knopman  7 , Ronald C Petersen  7 , Bradley F Boeve  7 , Ning Deng  4 , Yanan Feng  4 , Tzu-Hao Cheng  8 , Dennis W Dickson  2 , Stanley N Cohen  4 , Nancy M Bonini  5 , Christopher D Link  9 , Fen-Biao Gao  3 , Leonard Petrucelli  10 , Aaron D Gitler  11
Affiliations

Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts

Nicholas J Kramer et al. Science. .

Abstract

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.

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Figures

Figure 1
Figure 1. Spt4 is required for C9orf72 mutant strand transcription, RNA foci formation, and RAN translation in Saccharomyces cerevisiae
Yeast were transformed with plasmids expressing sense (GGGGCC)n or antisense (GGCCCC)n C9orf72 repeats of varying lengths (2R or 66R) but lacking ATG translation start codons under the control of a galactose-inducible promoter. (A) qRT-PCR analysis of C9-repeat RNA levels in WT and spt4Δ yeast after six hour galactose inductions. C9-repeat RNA levels were normalized to levels of yeast actin, then quantified relative to WT yeast (two-tailed unpaired t-tests, ****p < 0.0001, *** p < 0.001, n.s. = not significant). Data expressed as relative fold changes, spt4Δ/WT (one-way ANOVA with Dunnett’s multiple comparisons test, ** p < 0.01). (B) Sense and antisense RNA foci were detected by fluorescence in situ hybridization. Percentages of yeast cells containing sense (C) and antisense (D) foci were quantified across genotypes (blue = DAPI, red = C9-repeat LNA probe; one-way ANOVA with Dunnett’s multiple comparisons test, **** p < 0.0001, * p < 0.05, n.s. = not significant, n.d. = not determined). (E) Sandwich immunoassay detection of RAN translated poly(GP) in WT and spt4Δ yeast (two-tailed unpaired t-test **** p < 0.0001).
Figure 2
Figure 2. Spt4 modulation influences pathogenicity of GGGGCC repeats in C.elegans and mitigates neurodegeneration in a Drosophila model of c9FTD/ALS
(A) RNA FISH was performed to evaluate GGGGCC RNA foci formation (red) in normal (N2) worms, (GGGGCC)66-expressing worms (C9 66R), and worms expressing both (GGGGCC)66 and human SUPT4H1 (C9 66R × SUPT4H1). The intestinal marker (green) denotes the presence of the (GGGGCC)66 transgene, and DAPI (blue) was used to label nuclei (scale bar: low magnification 50 μm, high magnification 5 μm). (B,C) Expression of GGGGCC RNA levels quantified by qPCR relative to levels in (GGGGCC)66-expressing worms (two-tailed unpaired t-test **** p < 0.0001) (B) and poly(GP) levels evaluated by immunoassay (two-tailed unpaired t-test **** p < 0.0001) (C). (D) A lifespan assay was performed to assess the impact of (GGGGCC)66 and/or SUPT4H1 overexpression on survival (N2 n=145, C9 66R n=160, C9 66R × SUPT4H1 n= 122, SUPT4H1 n=144; log-rank (Mantel-Cox) test **** p < 0.0001, n.s. = not significant. See also Fig. S3G). (E–H) qPCR was performed to measure GGGGCC (E) and Spt4 mRNA (F) levels in (GGGGCC)66-expressing worms fed control (OP50) or RNAi targeting Spt4 (two-tailed unpaired t-test **** p < 0.0001). (G) An immunoassay was utilized to evaluate the percent change in poly(GP) levels following RNAi-mediated reduction of Spt4 (two-tailed unpaired t-test **** p < 0.0001). (H) The ability of Spt4 RNAi to improve survival in (GGGGCC)66 worms was assessed by a lifespan survival assay (OP50 n=119, RNAi Spt4 n=120; log-rank (Mantel-Cox) test **** p < 0.0001. See also Fig. S3G). (I,J) External and internal retinal structures of normal flies or flies expressing (GGGGCC)n transgenes, with co-expression of (I) a control (luciferase) shRNA or (J) spt4 shRNA. A gmr-GAL4 eye specific driver was used to express UAS-transgenes. (I) Control animals with driver alone (w1118) or a short GGGGCC repeat (GGGGCC)6 have normal eye structure. Animals expressing GGGGCC29 show a mild disruption to the highly precise external ommatidial structure, and thinning and gaps in the internal retina (arrows), whereas animals expressing GGGGCC49 have severely degenerate tissue both externally and internally. (J) Reduction in spt4 has no effect on normal or GGGGCC6 expressing animals, and resulted in reduced toxicity in both the external and internal retinal structures to flies bearing 29 or 49 GGGGCC repeats. (K) Reducing the expression of spt4 in (GGGGCC)49 animals significantly increases the life expectancy for animals expressing transgenes using a ubiquitous, drug-inducible driver, da-GS. Expression was induced in adult animals to avoid effects of expressing transgenes during development. Expression of the control (luciferase) shRNA or Spt4 shRNA alone had no effect on the lifespan when expressed alone. Statistical analysis was done using a log-ranked test comparing (GGGGCC)49 animals expressing the control shRNA versus the Spt4 shRNA.
Figure 3
Figure 3. Reduction of SUPT4H1 or SUPT5H in c9ALS fibroblasts inhibits production of C9orf72 variant 3 mRNA, sense and antisense RNA foci and DPR proteins
Cultured fibroblasts from three c9ALS patients were treated with a control siRNA (siCtrl), siRNA(s) directed against SUPT4H1 and/or SUPT5H (siSUPT4H1 and siSUPT5H, respectively), or a C9orf72-repeat targeting ASO (C9-ASO) for 10 days. Post-treatment, mRNA levels of SUPT4H1 and SUPT5H were determined by qPCR (A), and SUPT4H1 and SUPT5H protein expression was examined by immunoblot followed by densitometric quantification (B). (C) The effect of SUPT4H1 and/or SUPT5H depletion on C9orf72 variant 3 mRNA expression and poly(GP) protein levels was examined by qPCR and immunoassay, respectively. (D) RNA FISH with probes for GGGGCC or GGCCCC RNA was used to detect foci containing sense or antisense repeats (red) in the nucleus of cells (blue, Hoechst 33258). The percentage of cells containing foci was then determined. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 as assessed by one way ANOVA followed by Tukey’s post-hoc analysis.
Figure 4
Figure 4. Pathological features of c9FTD/ALS correlate with SUPT4H1 and SUPT5H expression in patient brain tissue, and partial knockdown of SUPT4H1 in C9orf72 iPSC-derived cortical neurons reduces production of C9orf72 variant 3 mRNA and DPR proteins
(A,B) SUPT4H1 and SUPT5H mRNA levels in cerebellar tissue from c9FTD/ALS patients were measured by qPCR and found to associate with cerebellar levels of C9orf72 variant 3 mRNA measured by qPCR (A) and with poly(GP) proteins measured by immunoassay (B) using a Spearman’s test of correlation. (C-E) Cortical neurons differentiated from each C9orf72 iPSC line were aged for 8 weeks and then transduced with lentivirus expressing either a non-coding control shRNA or an shRNA against SUPT4H1 mRNA for 6 days. Four iPSC lines derived from 3 patients with C9orf72 repeat expansions were used. Knockdown of SUPT4H1 mRNA (C) and C9orf72 variant 3 mRNA expression levels (D) were determined by qPCR. Poly(GP) protein levels was examined by immunoassay (E). *: p < 0.05, **: p < 0.01, ****: p < 0.0001 as assessed by two-tailed unpaired t-test from three independent neuronal differentiations.
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