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  • Letter
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Polyglutamine tracts regulate beclin 1-dependent autophagy

Nature volume 545, pages 108–111 (2017)Cite this article

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Abstract

Nine neurodegenerative diseases are caused by expanded polyglutamine (polyQ) tracts in different proteins, such as huntingtin in Huntington’s disease and ataxin 3 in spinocerebellar ataxia type 3 (SCA3)1,2. Age at onset of disease decreases with increasing polyglutamine length in these proteins and the normal length also varies3. PolyQ expansions drive pathogenesis in these diseases, as isolated polyQ tracts are toxic, and an N-terminal huntingtin fragment comprising exon 1, which occurs in vivo as a result of alternative splicing4, causes toxicity. Although such mutant proteins are prone to aggregation5, toxicity is also associated with soluble forms of the proteins6. The function of the polyQ tracts in many normal cytoplasmic proteins is unclear. One such protein is the deubiquitinating enzyme ataxin 3 (refs 7, 8), which is widely expressed in the brain9,10. Here we show that the polyQ domain enables wild-type ataxin 3 to interact with beclin 1, a key initiator of autophagy11. This interaction allows the deubiquitinase activity of ataxin 3 to protect beclin 1 from proteasome-mediated degradation and thereby enables autophagy. Starvation-induced autophagy, which is regulated by beclin 1, was particularly inhibited in ataxin-3-depleted human cell lines and mouse primary neurons, and in vivo in mice. This activity of ataxin 3 and its polyQ-mediated interaction with beclin 1 was competed for by other soluble proteins with polyQ tracts in a length-dependent fashion. This competition resulted in impairment of starvation-induced autophagy in cells expressing mutant huntingtin exon 1, and this impairment was recapitulated in the brains of a mouse model of Huntington’s disease and in cells from patients. A similar phenomenon was also seen with other polyQ disease proteins, including mutant ataxin 3 itself. Our data thus describe a specific function for a wild-type polyQ tract that is abrogated by a competing longer polyQ mutation in a disease protein, and identify a deleterious function of such mutations distinct from their propensity to aggregate.

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Figure 1: Ataxin 3 contributes to autophagosome formation by regulating the levels of beclin 1.
Figure 2: Deubiquitination of beclin 1 by ataxin 3.
Figure 3: The ataxin 3 polyQ domain contributes to the interaction between ataxin 3 and beclin 1.
Figure 4: Expanded polyQ tracts inhibit ataxin 3–beclin 1 interaction, decrease beclin 1 levels and impair starvation-induced autophagy.

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Acknowledgements

We thank the Wellcome Trust (Principal Research Fellowship to D.C.R. (095317/Z/11/Z), Wellcome Trust Strategic Grant to Cambridge Institute for Medical Research (100140/Z/12/Z)), National Institute for Health Research Biomedical Research Centre at Addenbrooke’s Hospital, and Addenbrooke’s Charitable Trust and Federation of European Biochemical Societies (FEBS Long-Term Fellowship to A.A.) for funding; R. Antrobus for mass spectrometry analysis; S. Luo for truncated HTT constructs; M. Jimenez-Sanchez and C. Karabiyik for assistance with the primary mouse cell cultures; and J. Lim and Z. Ignatova for reagents.

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Authors and Affiliations

  1. Department of Medical Genetics, Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge, UK

    Avraham Ashkenazi, Carla F. Bento, Thomas Ricketts, Mariella Vicinanza, Farah Siddiqi, Mariana Pavel, Maarten C. Hardenberg, Sara Imarisio, Fiona M. Menzies & David C. Rubinsztein

  2. IRCCS Casa Sollievo della Sofferenza, Huntington and Rare Diseases Unit, San Giovanni Rotondo, Italy

    Ferdinando Squitieri

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Contributions

A.A., C.F.B. and D.C.R. developed the study rationale. A.A. and D.C.R. wrote the manuscript, which was commented on by all authors. A.A. designed and performed most of the experiments. C.F.B. analysed some patient cell lines and structural models. T.R. and F.Si. performed mouse experiments. M.V. and M.C.H. performed and analysed confocal experiments. M.P. assisted with data analysis. S.I. contributed preliminary data. F.Sq. provided some control and patient samples. F.M.M. generated stable cell lines and constructs. D.C.R. supervised the study.

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Correspondence to David C. Rubinsztein.

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F.M.M. is currently an employee of Eli Lilly & Co. Ltd.

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Reviewer Information Nature thanks I. Dikic, J. Yuan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Ataxin 3 contributes to autophagosome formation.

a, Primary cultures of mouse cortical neurons were transduced with control or Atxn3 lentiviral shRNAs and analysed for the levels of LC3-I under starvation condition (HBSS, 4 h) or with BafA1 (400 nM, 4 h). Results are normalized to control cells (HBSS + BafA1). Mean ± s.e.m., n = 5 replicates from two independent cultures. Two-way ANOVA (NS, not significant). b, HeLa cells were transfected with different Atxn3 siRNAs or scrambled siRNA as a control. Ataxin 3 knockdown (KD) efficiency is presented as well as basal LC3-II levels. LC3-II levels in ataxin-3-depleted HeLa cells were normalized to control cells; n = 4 independent experiments. One-way ANOVA with post hoc Tukey’s test. c, p62 levels in HeLa cells depleted of ataxin 3 by siRNA. p62 levels were normalized to control cells (n = 4 independent experiments, one-tailed paired t-test). d, HeLa cells stably expressing mTagRFP–mWasabi–LC3 reporter were transfected with either scrambled or Atxn3 siRNA and were analysed with the ThermoFisher cellomics system to assess the number of autophagosomes and autolysosomes in the cells. Results are mean number of autophagosomes or autolysosomes per cell ± s.e.m. in eight fields from a representative experiment out of three independent experiments (one-tailed unpaired t-test). Representative images of the cells were taken by confocal microscopy (total 800 cells). Scale bars, 10 μm. e, Control and ataxin 3 KD HeLa cells were starved (HBSS, 4 h) or kept in full medium. The number of PtdIns3P (PI3P) phospholipid dots was analysed by staining with anti-PtdIns3P antibody. Results are mean dots per cell ± s.d. from a representative experiment out of three independent experiments as well as representative confocal images of PtdIns3P dots (red) for each condition (n = 20 cells). Scale bars, 10 μm. Two-way ANOVA (column factor siRNA P < 0.001, row factor starvation P < 0.01, interaction P < 0.05) with Bonferroni’s post-test. *P < 0.05, **P < 0.01, NS, not significant.

Extended Data Figure 2 Ataxin 3 regulates starvation-induced autophagy.

a, b, HeLa cells stably expressing GFP–LC3 were treated with control or Atxn3 siRNA and incubated for 1 h with carrier alone or carrier with 1 μM PtdIns3P phospholipid. Then, the control cells and ataxin 3 KD cells with the different treatments were shifted to starvation conditions (HBSS, 4 h) or kept in full medium. a, The number of LC3 dots was analysed for each of the conditions and presented as mean LC3 dots per cell ± s.e.m, determined from n = 5 fields from a single representative experiment out of three independent experiments. Two-way ANOVA (column factor siRNA P < 0.001, row factor starvation P < 0.05, interaction P value not significant (NS)) with Bonferroni’s post-test: for basal condition, NS; for starvation, P < 0.001. b, Representative confocal images of LC3 dots (green) from the different treatments are presented for the starvation condition. For a and b, the total number of cells analysed in basal control, n = 25; basal KD, basal KD carrier, basal KD carrier PtdIns3P, n = 30; HBSS control, n = 34; HBSS KD, HBSS KD carrier PtdIns3P, n = 37; HBSS KD carrier, n = 32. Scale bars, 10 μm. c, Number of dots of the endogenous PtdIns3P effector, WIPI2, in HeLa cells that were transfected with Flag–ataxin 3 or empty vector and starved (EBSS, 1 h) with or without the PtdIns3P inhibitor Wortmannin (Wm, 20 nM). Data are presented as means ± s.e.m. of the number of WIPI2 dots per cell determined from the total number of cells analysed using software described in Methods from a representative experiment out of two independent experiments. Confocal images of WIPI2 dots (green) from the different treatments are shown. Number of cells analysed and used for the s.e.m. in empty Flag, n = 47; Flag–ataxin 3, n = 45; Flag–ataxin 3/Wm, n = 37. Scale bars, 10 μm. One-way ANOVA with post hoc Tukey’s test. d–f, Mice were depleted of ataxin 3 in the liver by injection of Atxn3 siRNA or control/scrambled siRNA formulations into the lateral caudal vein. The knockdown was left for 5 days with fasting on the fourth day. Livers from these mice were dissected and homogenized and proteins were resolved by SDS–PAGE. d, Representative blots and in vivo ataxin 3 knockdown efficiency. For quantification of beclin 1 and LC3-II, see Fig. 1d, e. e, Quantification of p62 levels; f, quantification of LC3-I levels in each group of mice (control fed, n = 9; ataxin 3 KD fed, n = 6; control fasted, n = 8, ataxin 3 KD fasted, n = 6; a.u, arbitrary units). e, Two-way ANOVA (column factor siRNA P < 0.05, row factor fasting P < 0.05, interaction P < 0.05) with Bonferroni’s post-test. For f, two-way ANOVA (column factor siRNA NS, row factor starvation P < 0.05, interaction NS) with Bonferroni’s post-test (NS). This suggests no obvious difference in LC3-I levels between the control and ataxin-3 KD groups. **P < 0.01, ***P < 0.001.

Source data

Extended Data Figure 3 Ataxin 3 regulates beclin 1 stability and ubiquitination.

a, Beclin 1 levels in control siRNA-treated HeLa cells and ataxin 3 KD cells (t = 0) and after cycloheximide (CHX, 50 μg ml−1) treatment (t = 8 h). The percentage of beclin 1 degradation in control or ataxin 3 KD cells was determined by comparing to cells without CHX treatment (n = 3 independent experiments, one-tailed paired t-test). b, Beclin 1 levels in control siRNA-treated HeLa cells and ataxin 3 KD cells that were treated for the last 6 h with proteasome inhibitor (MG132, 5 μM). n = 3 independent experiments, one-way ANOVA (P < 0.01) with post hoc Tukey’s test. c, Beclin 1 levels in ataxin-3-depleted HeLa cells that were transfected with wild-type (WT) Flag–ataxin 3 or protease-dead mutant FLAG–ataxin 3 C14A for 48 h. Results normalized to control siRNA, n = 3 independent experiments, one-way ANOVA (P < 0.01) with post hoc Tukey’s test. d, Control siRNA-treated and Atxn3 siRNA-treated HeLa cells were transfected with the indicated vectors for 24 h and treated for the last 6 h with MG132 (10 μM). Endogenous beclin 1 was immunoprecipitated to detect beclin 1 ubiquitination. e, VPS34 levels in ataxin-3-depleted HeLa cells normalized to control siRNA, n = 3 independent experiments. One-tailed paired t-test. f, HeLa cells were transfected with the indicated vectors for 24 h and treated for the last 6 h with MG132 (10 μM). VPS34 was immunoprecipitated to detect VPS34 ubiquitination. The levels of the VPS34 components are co-ordinately regulated, and decreased beclin 1 levels in ataxin-3-depleted cells were accompanied by decreased levels of VPS34. Still, no obvious change in VPS34 ubiquitination was observed in ataxin-3-overexpressing cells, supporting a selective effect on beclin 1. g, Flag–ataxin 3 WT and Flag–ataxin 3 C14A were co-expressed with GFP–huntingtin (HTT) exon 1 Q74 in HeLa cells for 48 h. The number of aggregates was analysed by monitoring GFP fluorescence in 400 cells. n = 4 independent experiments. Results normalized to control (empty vector). One-way ANOVA (P < 0.01) with post hoc Tukey’s test. *P < 0.05, **P < 0.01, NS, not significant.

Extended Data Figure 4 Analysis of beclin 1 lysine 402 modification.

a, b, HeLa cells were transfected with Flag–beclin 1 and HA–Ub for 24 h and treated for the last 6 h with MG132 (10 μM). Flag–beclin 1 was immunoprecipitated for mass spectrometry analysis. Tryptic digests of ubiquitin-conjugated beclin 1 resulted in peptides that contain a ubiquitin remnant derived from the ubiquitin C terminus (‘GG’ motif). a, Identification of a putative site of ubiquitination in beclin 1. Top, MS–MS spectrum of the unmodified beclin 1 peptide spanning residues 401–416. Amino acids with corresponding y ions are shown in blue. Bottom, MS–MS spectrum of an ion with a mass 114 Da greater than the unmodified peptide. The matching y ions and presence of a modified b2 ion indicate –GG modification of lysine 402. b, MS–MS spectra filtered to high confidence covering 100% of the ubiquitin sequence. Tryptic peptide spanning residues 43–54 including lysine 48 was identified as the sole high-confidence peptide with a modification corresponding to a –GG motif and the MS–MS spectra of the peptide demonstrate fragments corresponding to a –GG modified lysine 48. c, Levels of Flag–beclin 1 and Flag–beclin 1 K402R in HeLa cells that were treated for the last 6 h with MG132 (10 μM). Results normalized to control (Flag–beclin 1 WT). n = 3 replicates from two independent experiments. Two-way ANOVA (column factor K402R NS, row factor MG132 P < 0.001, interaction P < 0.05) with Bonferroni’s post-test. d, HeLa cells were transfected with the indicated vectors for 24 h and shifted in the last 4 h to HBSS. Beclin 1 and LC3-II levels were analysed and results normalized to control (Flag–beclin 1 WT). For LC3-II levels, n = 3 independent experiments, two-way ANOVA (column factor ataxin 3 P < 0.05, row factor K402R mutation P < 0.01, interaction P < 0.01) with Bonferroni’s post-test. For beclin 1 levels, n = 4 independent experiments, two-way ANOVA (column factor ataxin 3 P < 0.05, row factor K402R mutation P < 0.001, interaction NS) with Bonferroni’s post-test. *P < 0.05, ***P < 0.001, NS, not significant.

Extended Data Figure 5 Analysis of the interaction of the polyQ domain with beclin 1.

a, Flag–beclin 1 ECD alone, Flag–beclin 1 ΔECD, Flag–beclin 1 full length and GFP–Q35 were transfected into HeLa cells for 24 h and the cell lysates were immunoprecipitated with anti-Flag antibody. Immunocomplexes were analysed using anti-GFP antibody. b, Superimposition of human beclin 1 ECD (pdb 4DDP) and Vps30 (pdb 5DFZ), the yeast orthologue of beclin 1. The N-terminal helix (dark blue helix) of the human structure is displaced, probably owing to protein truncation for crystallographic purposes. The yeast structure suggests that this helix is part of the coiled-coil CC2 of beclin 1 instead of the ECD. c, Two binding sites in human beclin 1 ECD reveal high docking scores for polyQ7 (the docking scores for site 1 and site 2 are −10.394 and −10.721, respectively). Sites comprising the region adjacent to the N-terminal helix (dark blue) were not considered for the docking. d, e, Surface charge illustrations of human beclin 1 ECD with the two sites of polyQ interaction. Site 2 is close to a protruding hydrophobic loop (aromatic finger) comprising Phe359, Phe360 and Trp361 (top right e, cartoon view), which are thought to be implicated in anchorage of beclin 1 to lipid membranes.

Extended Data Figure 6 Expression of polyQ tracts impairs beclin 1-dependent starvation-induced autophagy.

a, HeLa cells were transfected with empty GFP or GFP–Q35 with or without Flag–ataxin 3 Q22 for 24 h and were shifted to HBSS for the last 4 h. LC3-II and beclin 1 levels were analysed from the cell lysates. Results are mean ± s.e.m. normalized to control (empty GFP), n = 5 independent experiments, one-way ANOVA (for LC3-II P < 0.01, for beclin 1 P < 0.05) with post hoc Tukey’s test. b, HeLa cells were transfected with empty GFP or GFP–Q35 for 24 h and were shifted to HBSS for the last 4 h. p62 levels were than analysed from the lysates. Results normalized to control (empty GFP), n = 3 independent experiments, one-tailed paired t-test. c, HeLa cells were treated with 20 nM Becn1 siRNA or scrambled siRNA (control) for 3 days. Beclin 1 KD efficiency is presented. d, e, Control and beclin 1 KD HeLa cells were transfected with empty GFP or GFP–Q35 for 24 h and were shifted to HBSS for the last 4 h. The number of endogenous LC3 dots (red) was analysed in the GFP-expressing cells (green). Results are mean number of LC3 dots per cell in four fields from a representative experiment out of three independent experiments, as well as confocal images for each condition (number of cells analysed in control GFP n = 32, control GFP–Q35 n = 27, beclin 1 KD GFP n = 25, beclin 1 KD GFP–Q35 n = 23). Scale bars, 10 μm. Two-way ANOVA (column factor GFP Q35 P < 0.01, row factor beclin 1 KD P < 0.001, interaction P < 0.01) with Bonferroni’s post-test. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

Extended Data Figure 7 Impaired starvation-induced autophagy and reduced beclin 1 levels in cells expressing expanded polyQ forms of huntingtin.

a, Empty Flag, Flag–huntingtin (HTT) N-terminal fragment (1–350) Q17, Flag–HTT (1–350) ΔQ and beclin 1 were transfected into HeLa cells for 24 h and cell lysates were immunoprecipitated with anti-beclin 1 antibody. Immunocomplexes were analysed using anti-Flag antibody. b, GFP–ataxin 3 Q28 and Flag–HTT Q138 (full length) were transfected into HeLa cells for 24 h and endogenous beclin 1 was immunoprecipitated. Immunocomplexes were analysed using anti-ataxin 3 antibody (detects GFP–ataxin-3) and anti-Flag antibody (detects HTT). The ratio of the bound ataxin 3 to beclin 1 is presented. c, Stable-inducible HEK293 cells were switched on for 48 h with doxycycline (Dox) to express GFP–HTT wild-type exon 1 (GFP–HTT Q23) or mutant GFP–HTT exon 1 (GFP–HTT Q74). In the last 4 h cells were starved (HBSS) and beclin 1 levels were analysed in each cell type. Results are normalized to control HTT Q23 cells with no Dox (n = 4 independent experiments). Two-way ANOVA (column factor Dox P < 0.01, row factor HEK 293 cells NS, interaction NS) with Bonferroni’s post-test. d, e, Quantification of the number of LC3 dots in the starved cells. Results are mean dots per cell in four fields of a representative experiment out of three independent experiments. Representative confocal images of endogenous LC3 dots (red) and GFP–HTT (green) in each of the conditions (number of cells analysed in GFP–HTT Q23 no Dox n = 41; GFP–HTT Q23 with Dox n = 34; GFP–HTT Q74 no Dox n = 39; GFP–HTT Q74 with Dox n = 43). Scale bars, 10 μm. Two-way ANOVA (column factor Dox P < 0.001, row factor HEK cells P < 0.05, interaction NS) with Bonferroni’s post-test. *P < 0.05, **P < 0.01, NS, not significant.

Extended Data Figure 8 Impaired starvation-induced autophagy in striatal cell lines and in brain tissue from mouse models of Huntington’s disease.

a, Striatum-derived cells from HTT (Q7/Q111) heterozygous knock-in mouse and HTT (Q7/Q7) wild-type knock-in mouse were kept in full medium or starved (EBSS, 1 h). In each experiment, cells were analysed for WIPI2 dots under different conditions. We could not detect WIPI2 dots in full medium in these cells as dots became apparent after starvation-induced autophagy. The number of WIPI2 dots per cell is normalized to control HTT (Q7/Q7) cells. n = 3 independent experiments, one-tailed paired t-test. Representative confocal images of WIPI2 (red) in each of the conditions are presented (n = 80 cells analysed). Scale bars, 10 μm. b, HTT (Q7/Q111) and HTT (Q7/Q7) striatal cells were treated with BafA1 (400 nM) in full medium or starved with HBSS together with BafA1 (400 nM) for 4 h and analysed for LC3-II levels. Results are normalized to control (HTT (Q7/Q7) in full medium). n = 3 independent experiments, two-way ANOVA (column factor mutant HTT P < 0.001, row factor starvation P < 0.01, interaction P < 0.01) with Bonferroni’s post-test. c, Beclin 1 levels in the starved HTT (Q7/Q111) and HTT (Q7/Q7) striatal cells. Results are normalized to control HTT (Q7/Q7) cells. n = 3 independent experiments. One-tailed paired t-test. d, Sections of brains from HD-N171-N82Q transgenic young and adult mice (6 and 12 weeks old, respectively) were analysed for neuronal aggregates in the motor cortex. For each brain, 400 cells were counted in at least three sections. Results are mean percentage of cells with aggregates from three brains, one-tailed unpaired t-test. e, Young wild-type (WT) mice and HD transgenic mice were fed or fasted. Brains from these mice were dissected and homogenized, and proteins were resolved by SDS–PAGE to analyse the levels of endogenous beclin 1, LC3-I, LC3-II and p62. PolyQ levels were analysed using anti-polyQ antibody showing the expression level of the polyQ HTT exon 1. Representative blots are shown that were used to generate the data in Fig. 4c, d. f, g, Quantification of p62 and LC3-I levels in each group (WT fed n = 7, HD fed n = 5, WT fasted n = 7, HD fasted n = 6). For LC3-I levels, two-way ANOVA (column factor HD P < 0.001, row factor fasting NS, interaction NS) with Bonferroni’s post-test. For p62 levels, two-way ANOVA (column factor HD P < 0.001, row factor fasting NS, interaction NS) with Bonferroni’s post-test. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

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Extended Data Figure 9 Expansion of the polyQ domain in ataxin 3 decreased deubiquitinase activity and increased interaction with beclin 1.

a, Beclin 1 was purified from proteasome inhibitor-treated cells that co-expressed HA–Ub and was incubated in vitro with recombinant ataxin 3 Q22 or ataxin 3 Q80 for 30 min in deubiquitination buffer. Samples were analysed for beclin 1 ubiquitination using anti-HA antibodies. b, Number of LC3 dots in ataxin 3 KD HeLa cells that were transfected with GFP–ataxin 3 Q28, GFP–ataxin 3 Q84 or GFP–ataxin 3 ΔQ and starved with HBSS for the last 4 h. Results are normalized to control siRNA-treated cells from n = 4 independent experiments. One-way ANOVA (P < 0.001) with post hoc Tukey’s test. c, Representative confocal images are presented for each of the conditions from b (LC3 dots in red and ataxin-3 staining in green, n = 20 cells analysed). Scale bar, 10 μm. d, HeLa cells were transfected with empty vector, Flag–ataxin 3 Q22, Flag–ataxin 3 Q80 and HA–Ub for 24 h and treated for the last 6 h with MG132 (10 μM). Endogenous beclin 1 was immunoprecipitated from the lysates for analysis of different polyUb linkages using K48 polyUb or K63 polyUb antibodies, and for detection of bound ataxin 3 using anti-ataxin 3 and anti-polyQ antibodies. **P < 0.01, ***P < 0.001, NS, not significant.

Extended Data Figure 10 Effect of different disease proteins with polyQ expansion on beclin 1 ubiquitination, beclin 1 levels and starvation-induced autophagy.

a, HeLa cells were transfected with empty vector, GFP–atrophin 1 (ATN-1) Q19, or GFP–ATN-1 Q71 along with HA–Ub for 24 h and treated for the last 6 h with MG132 (10 μM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound ATN-1 using anti-ATN-1 antibody. b, HeLa cells were transfected with empty vector, HA–androgen receptor (AR) Q25, or HA–AR Q120 along with HA–Ub for 24 h and treated for the last 6 h with MG132 (10 μM). Endogenous beclin 1 was immunoprecipitated from the lysates for ubiquitination analysis and for detection of bound AR using anti-AR antibody. c, Primary fibroblasts derived from healthy controls (Cont) (n = 5) and from patients with Huntington’s disease (HD) (n = 7) were starved with HBSS together with BafA1 (400 nM) for 4 h and analysed for LC3-II levels. Results are mean ± s.e.m. *P < 0.05, one-tailed Mann–Whitney test. d–f, Primary fibroblasts derived from healthy controls and from patients with different polyQ diseases (d, SCA3; e, DRPLA; f, Huntington’s disease) were kept in full medium or starved with HBSS for 4 h and analysed for LC3-II levels (LC3-II/actin ratio is presented). The BafA1 experiments for these sets of patients are presented in Fig. 4. g, Beclin 1 levels (beclin 1/actin ratio is presented) in the starved control cells compared to cells from patients with SCA3, SCA7, DRPLA or Huntington’s disease. We only had one SCA7 patient sample and thus we have not analysed it further.

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Ashkenazi, A., Bento, C., Ricketts, T. et al. Polyglutamine tracts regulate beclin 1-dependent autophagy. Nature 545, 108–111 (2017). https://doi.org/10.1038/nature22078

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