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Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington’s disease

Abstract

Huntington’s disease (HD) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene, encoding a homopolymeric polyglutamine (polyQ) tract. Although mutant HTT (mHTT) protein is known to aggregate, the links between aggregation and neurotoxicity remain unclear. Here we show that both translation and aggregation of wild-type HTT and mHTT are regulated by a stress-responsive upstream open reading frame and that polyQ expansions cause abortive translation termination and release of truncated, aggregation-prone mHTT fragments. Notably, we find that mHTT depletes translation elongation factor eIF5A in brains of symptomatic HD mice and cultured HD cells, leading to pervasive ribosome pausing and collisions. Loss of eIF5A disrupts homeostatic controls and impairs recovery from acute stress. Importantly, drugs that inhibit translation initiation reduce premature termination and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

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Fig. 1: uORF modulates translation of HTT under normal and stress conditions.
Fig. 2: uORF reduces truncation and aggregation of mHTT protein.
Fig. 3: Ribosome collisions cause abortive translation termination of mHTT.
Fig. 4: polyQ expansions deplete eIF5A.
Fig. 5: Loss of eIF5A disrupts global translation elongation dynamics.
Fig. 6: Loss of eIF5A disrupts recovery from acute stress.
Fig. 7: mHtt expression alters striatal cell sensitivity to ribotoxicy.

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Data availability

Previously published ribosome profiling analyses were from GSE87328 (Fig. 1c), GSE72064 (Fig. 1d,e), SRA160745, GSE74279 and GSE94460 (Extended Data Fig. 1a), GSE66715, GSE81283 and GSE36892 (Extended Data Fig. 1b) and GSE53743 and GSE119615 (Extended Data Fig. 1c). For analysis of previously published proteomic data, processed MS intensity tables were downloaded from the supplementary information section of the following published articles: Hosp et al.6, Supplementary Tables 1 and 3 (label-free quantification, LFQ); Sap et al.50, Supplementary Tables 1 and 3 (LFQ); Newcombe et al.43, Supplementary Table 2 (LFQ); and Sui et al.45, Supplementary Table 1 (raw intensities). No additional normalization of the data was performed. Statistical tests were done using two-sided Student’s t-test. Sequencing data were deposited in the Sequence Read Archive (SRA) database under BioProject number PRJNA730032. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE72 partner repository with the dataset identifier PXD026012. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank I. Munoz-Sanjuan and D. Marchionini, as well as members of the Frydman lab, for their helpful comments. This work was supported by NIH grants GM05643321, NS092525 to J.F. and AI36178, AI40085 and AI091575 to R. Andino and Cure Huntington’s Disease Initiative (CHDI) grant A-13887 to J.F.

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R. Aviner and J.F. designed the study. R. Aviner and T.-T.L. carried out experiments, analysed data and performed statistical analyses. K.H.L. performed LC–MS/MS data acquisition. V.B.M. performed cloning. R. Aviner, R. Andino and J.F. wrote the manuscript, and all authors approved the manuscript.

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Correspondence to Judith Frydman.

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Extended data

Extended Data Fig. 1 HTT translation initiation is dynamically regulated by its uORF.

(a) Ribo-seq traces of initiating ribosomes (A-sites, top) and elongating ribosomes (footprints, bottom) on human HTT uORF and start of main ORF. (b) Top, ribo-seq traces of elongating ribosomes on Htt uORF and start of main ORF in different organisms and tissues. Bottom, Htt protein level (low, moderate, high) in the indicated tissues, from ProteinAtlas. (c) Ribo-seq traces of elongating ribosomes on Htt and Atf4 mRNA in mouse embryonic fibroblasts before and after thapsigargin (Tg) treatment (left, n = 2) and SH-SY5Y neuroblasts before and after differentiation with retinoic acid (right, n = 2).

Source data

Extended Data Fig. 2 HTT uORF reduces truncation and aggregation of mHTT.

(a) 293 T cells were transfected with constructs expressing wild-type polyQ(25) or mutant polyQ(97) HTT-ex1 with a C-terminal fluorescent GFP. Shown are live cell images at the indicated times post-transfection. Images were obtained using 20x magnification. Representative images of 2 independent replicates. (b-c) Immunoblot analyses of 293 T (b) and mouse striatal cells (c) transfected as above and separated into soluble and insoluble fractions (n = 2). WB, western blot. (d) Flow cytometry analysis of transfected 293 T cells. (e) Effect of 4EGI-1 on global translation. 293 T cells were treated with increasing concentrations of 4EGI-1 for 24 h, followed by puromycin for 15 min (n = 2). Immunoblot analysis was performed on whole cell lysates using a puromycin-specific antibody. (f) 293 T cells were transfected with mHTT-ex1-GFP and increasing concentrations of 4EGI-1 were added 6 h post-transfection. Immunoblot analysis of the insoluble fraction was performed at 24 h post-transfection (n = 3). Means +/- s.d. P, p-value of a two-tailed Student t-test comparing 50 and 0 µM 4EGI-1. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Translation of mHTT from poliovirus genome is associated with ribosome collisions.

(a) Poliovirus replicates more slowly when engineered to express mutant versus wild-type HTT-ex1. Wild-type polyQ(8) and mutant polyQ(73) human HTT-ex1 were cloned into poliovirus genome. Infectious viruses were generated by transfection of in vitro transcribed RNA. Plaque assays were performed to determine virus titers (n = 3). Means +/- s.d. (left) and representative images of viral plaques (right). (b-c) Huh7 cells (b) and SH-SY5Y neuroblasts (c) were infected with engineered viruses. At the indicated times, puromycin was added to tissue culture media to label nascent polypeptide chains and lysates were analyzed by immunoblotting. Shown is a representative of 2 independent repeats. Purple boxes and arrowheads indicate virus-Htt polyproteins prior to cleavage by the viral protease. WB, western blot. (d) Quantification of blots from Fig. 3c (n = 3). Means +/- s.d. (e) Huh7 cells were infected with HTT-ex1-expressing viruses for 3.5 h, and ribosomes were isolated by ultracentrifugation on a sucrose cushion, followed by immunoblot analysis using antibodies specific to viral epitopes upstream and downstream of HTT-ex1 insert (n = 2). Unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 HD mouse brains show age-dependent changes in levels of soluble translation and RQC factors.

(a) eIF5A, but not other elongation factors, is inversely correlated with mHTT levels in mouse brains at 12 weeks of age. Pearson’s correlation coefficients for wild-type or mutant Htt and the indicated translation elongation factors. Reanalysis of published MS analysis of soluble brain proteomes from 12-week-old R6/2 and control mice (n = 16/12). (b-c) Levels of RQC factors increase, while those of elongation factors decrease, in the soluble brain proteome of presymptomatic R6/2 HD mice. Shown are levels of the indicated proteins in wild-type and R6/2 HD cells at 5, 8 and 12 weeks of age (b) and their Student’s t-test differences between wild-type and R6/2 at 8 weeks. n = 16, 12 and 12 for WT or n = 16, 12 and 16 for R6/2 at weeks 5, 8 and 12, respectively. Means +/-s.d. (c). Histogram shows the distribution of t-test differences of the entire soluble proteome at 8 weeks.

Source data

Extended Data Fig. 5 Disruption of proteostasis machines in R6/2 mice.

(a) Proteins encoded by pause-containing transcripts detected in striatal cells expressing polyQ(111) mHtt are enriched in the insoluble brain proteome of aged 12-week-old R6/2 mice (n = 12/16 for WT and R6/2, respectively). (b) Kurtosis is a measure of tailedness or outliers relative to a normal distribution, and can be used to estimate stoichiometry of large molecular complexes. (c-d) Stoichiometry of ribosomal proteins (c) and proteasomal subunits (d) is disrupted in R6/2 brains starting at 8 weeks of age. Shown are kurtosis scores for all core ribosomal proteins or proteasomal subunits as measured by MS. n = 16, 12 and 12 for WT or n = 16, 12 and 16 for R6/2 at weeks 5, 8 and 12, respectively. (e) Ribosomal proteins are enriched in the insoluble brain proteome of symptomatic 12-week-old but not asymptomatic 8-week-old R6/2 mice. n = 16, 12 and 12 for WT or n = 16, 12 and 16 for R6/2 at weeks 5, 8 and 12, respectively. Proteasomal subunits were not detected in the insoluble brain proteome of either wild-type or R6/2 mice at any age. For a, c-e, center lines show medians; box limits—upper and lower quartiles; and whiskers extend 1.5 times the interquartile range.

Source data

Extended Data Fig. 6 Global and Atf4-specific translation levels in mouse striatal cells.

(a) Global translation is lower in striatal cells expressing polyQ(111) mHtt. Translation rates were monitored by puromycin labeling and quantified by densitometry (left, n = 3, Means +/-s.d.) and polysome profiles on 10-50% sucrose gradients (right, n = 2, representative traces). WB, western blot. (b) Translation efficiency of Atf4 is lower in polyQ(111) cells at steady-state. Means of values from RNA- and ribo-seq analyses (n = 2 each). (c) Ribo-seq traces of elongating ribosomes on Atf4 mRNA (n = 2). Unprocessed blots are available in source data.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1-6

MS data, ribosome stalling analyses and plasmid/oligonucleotide information.

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Aviner, R., Lee, TT., Masto, V.B. et al. Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington’s disease. Nat Cell Biol 26, 892–902 (2024). https://doi.org/10.1038/s41556-024-01414-x

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