Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease
2022; Springer Nature; Volume: 14; Issue: 7 Linguagem: Inglês
10.15252/emmm.202215851
ISSN1757-4684
AutoresChunyue Liu, Zixing Fu, Shanshan Wu, Xiaosong Wang, Shengrong Zhang, Chu Chu, Yuan Hong, Wenbo Wu, Shengqi Chen, Yueqing Jiang, Yang Wu, Yongbo Song, Yan Liu, Xing Guo,
Tópico(s)Spaceflight effects on biology
ResumoArticle7 June 2022Open Access Source DataTransparent process Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease Chunyue Liu Chunyue Liu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Zixing Fu Zixing Fu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shanshan Wu Shanshan Wu State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Methodology Search for more papers by this author Xiaosong Wang Xiaosong Wang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shengrong Zhang Shengrong Zhang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Chu Chu Chu Chu State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Yuan Hong Yuan Hong State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Wenbo Wu Wenbo Wu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shengqi Chen Shengqi Chen State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Yueqing Jiang Yueqing Jiang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Resources, Methodology, Project administration Search for more papers by this author Yang Wu Yang Wu orcid.org/0000-0002-6069-3553 State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, China Contribution: Investigation, Methodology Search for more papers by this author Yongbo Song Yongbo Song Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China Contribution: Methodology, Project administration Search for more papers by this author Yan Liu Corresponding Author Yan Liu [email protected] orcid.org/0000-0003-2918-5398 State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Xing Guo Corresponding Author Xing Guo [email protected] orcid.org/0000-0002-0216-0310 State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Department of Endocrinology, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu, China Contribution: Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Chunyue Liu Chunyue Liu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Zixing Fu Zixing Fu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shanshan Wu Shanshan Wu State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Methodology Search for more papers by this author Xiaosong Wang Xiaosong Wang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shengrong Zhang Shengrong Zhang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Chu Chu Chu Chu State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Yuan Hong Yuan Hong State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Wenbo Wu Wenbo Wu State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Shengqi Chen Shengqi Chen State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Investigation Search for more papers by this author Yueqing Jiang Yueqing Jiang State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Contribution: Resources, Methodology, Project administration Search for more papers by this author Yang Wu Yang Wu orcid.org/0000-0002-6069-3553 State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, China Contribution: Investigation, Methodology Search for more papers by this author Yongbo Song Yongbo Song Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China Contribution: Methodology, Project administration Search for more papers by this author Yan Liu Corresponding Author Yan Liu [email protected] orcid.org/0000-0003-2918-5398 State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Xing Guo Corresponding Author Xing Guo [email protected] orcid.org/0000-0002-0216-0310 State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China Department of Endocrinology, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu, China Contribution: Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Chunyue Liu1,2,†, Zixing Fu1,†, Shanshan Wu2,†, Xiaosong Wang1,†, Shengrong Zhang1,†, Chu Chu2,†, Yuan Hong2, Wenbo Wu1, Shengqi Chen1, Yueqing Jiang1, Yang Wu3, Yongbo Song4, Yan Liu *,2 and Xing Guo *,1,5 1State Key Laboratory of Reproductive Medicine, Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Neurobiology, Interdisciplinary InnoCenter for Organoids, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China 2State Key Laboratory of Reproductive Medicine, Interdisciplinary InnoCenter for Organoids, Institute for Stem Cell and Neural Regeneration, School of Pharmacy, Nanjing Medical University, Nanjing, China 3State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan, China 4Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China 5Department of Endocrinology, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu, China † These authors contributed equally to this work *Corresponding author. Tel: +86 25 86868478; E-mail: [email protected] *Corresponding author. Tel: +86 25 86869345; E-mail: [email protected] EMBO Mol Med (2022)14:e15851https://doi.org/10.15252/emmm.202215851 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Aberrant localization of proteins to mitochondria disturbs mitochondrial function and contributes to the pathogenesis of Huntington’s disease (HD). However, the crucial factors and the molecular mechanisms remain elusive. Here, we found that heat shock transcription factor 1 (HSF1) accumulates in the mitochondria of HD cell models, a YAC128 mouse model, and human striatal organoids derived from HD induced pluripotent stem cells (iPSCs). Overexpression of mitochondria-targeting HSF1 (mtHSF1) in the striatum causes neurodegeneration and HD-like behavior in mice. Mechanistically, mtHSF1 facilitates mitochondrial fission by activating dynamin-related protein 1 (Drp1) phosphorylation at S616. Moreover, mtHSF1 suppresses single-stranded DNA-binding protein 1 (SSBP1) oligomer formation, which results in mitochondrial DNA (mtDNA) deletion. The suppression of HSF1 mitochondrial localization by DH1, a unique peptide inhibitor, abolishes HSF1-induced mitochondrial abnormalities and ameliorates deficits in an HD animal model and human striatal organoids. Altogether, our findings describe an unsuspected role of HSF1 in contributing to mitochondrial dysfunction, which may provide a promising therapeutic target for HD. Synopsis Inhibition of mitochondrial HSF1(mtHSF1) restores mitochondrial function and slows neurodegeneration in Huntington's Disease (HD) mice, suggesting that mtHSF1 might be a therapeutic target for treating HD. HSF1 mislocalizes to mitochondria in various HD models. mtHSF1 induces mitochondrial dysfunction by disrupting their dynamic balance and mtDNA replication. mtHSF1 causes neurodegeneration and HD-like behavior in mice. HD-associated neuropathology and behavioral deficits are improved by peptide inhibitor DH1. The paper explained Problem Huntington’s disease (HD) is a neurodegenerative disease caused by genetic mutation of huntingtin (htt), which disrupts protein distribution and causes striatal medium spiny neural degeneration. So far, there is no effective treatment of HD. We previously found the deficits in mitochondrial quality control caused by mislocalized proteins are closely associated with HD. Heat shock transcription factor (HSF) 1 is a predominantly cytosolic transcription factor, which contributes to the pathogenesis of HD. However, it remains elusive whether HSF1 directly regulates mitochondrial function independent of its role in regulating nuclear gene expression. Moreover, whether HSF1 could be a potential therapeutic target for the treatment of HD is also unclear. Results We first found HSF1 accumulates in the mitochondria of various HD models. Overexpression of mitochondria-targeting HSF1 (mtHSF1) in the striatum causes neurodegeneration and HD-like behavior. Mechanistically, mtHSF1 facilitates mitochondrial fission by activating dynamin-related protein 1 (Drp1) phosphorylation at S616. Moreover, mtHSF1 suppresses single-stranded DNA-binding protein 1 (SSBP1) oligomer formation, which results in mitochondrial DNA (mtDNA) deletion. Furthermore, to block Drp1/HSF1 binding that may reduce HSF1 accumulation in mitochondria, we next design and synthesize a unique peptide inhibitor, DH1, according to the region of homology between Drp1 and HSF1. DH1 remarkably abolishes HSF1-induced mitochondrial abnormalities and ameliorates deficits in an HD animal model and human striatal organoids. Impact We remarkably show that HSF1 accumulates on mitochondria and causes neurodegeneration and movement deficits. Notably, treatment with DH1 improved neurotoxicity and animal behavior, indicating that blocking HSF1 translocation to mitochondria may slow the pathogenesis of HD. Indeed, our results reveal an unsuspected role of HSF1 in mitochondria, which may provide a therapeutic target for HD. Introduction Huntington's disease (HD) is a neurodegenerative disease caused by genetic mutation of huntingtin (htt) (Tabrizi et al, 2020). Mutant huntingtin (mtHtt) disrupts protein distribution, resulting in the dysfunction of physiological processes and subsequent neurodegeneration. Valosin-containing protein (VCP) recruitment to mitochondria triggers excessive mitophagy and neurodegeneration in HD (Guo et al, 2016). The tumor suppressor P53 shuttles from the nucleus to the mitochondria and activates mitochondrial fission (Guo et al, 2014). These findings indicate that defects in mitochondrial quality control caused by mislocalized proteins are closely associated with HD. Therefore, identifying proteins that are mislocalized to mitochondria and revealing the underlying mechanisms may be useful for HD treatment. Heat shock transcription factor (HSF) 1 is a predominantly cytosolic transcription factor that is inhibited by Hsp40, Hsp70, Hsp90, and TRiC under physiological conditions (Neef et al, 2014; Gomez-Pastor et al, 2018). Upon activation, phosphorylated HSF1 assembles into a homotrimer and accumulates in the nucleus to regulate gene expression (Morimoto, 1998; Akerfelt et al, 2010). Previous studies have revealed that HSF1 deficiency contributes to the pathogenesis of HD. The transcriptional activity of HSF1, a master regulator of heat shock protein (HSP), is suppressed by mtHtt in R6/2 mice (Labbadia et al, 2011). Hyperactivation of the E3 ligase Fbxw7 promotes HSF1 polyubiquitination and proteasome-mediated degradation (Kourtis et al, 2015). In addition, recent studies have demonstrated that deletion of HSF1 elicits mitochondrial dysfunction by inducing oxidative stress, attenuating respiration activity, and inhibiting mitochondrial biogenesis (Homma et al, 2007; Ma et al, 2015; Qiao et al, 2017; Intihar et al, 2019). However, it remains elusive whether HSF1 directly regulates mitochondrial function independent of its role in regulating nuclear gene expression. Here, we show that HSF1 accumulates on mitochondria and causes neurodegeneration and movement deficits. We observed that mitochondria-targeting HSF1 (mtHSF1) shortens mitochondria by evoking dynamin-related protein 1 (Drp1) phosphorylation at S616. In addition, expression of mtHSF1 results in decreased single-stranded DNA-binding protein 1 (SSBP1) oligomerization and subsequent mitochondrial DNA (mtDNA) deletion. Furthermore, blockade of HSF1 mitochondrial localization by DH1 reduces mitochondrial dysfunction and neurotoxicity in HD. Results HSF1 associates with mitochondria in models of HD We first isolated mitochondria-enriched fractions from HdhQ7 or HdhQ111 cells, which are commonly used model cell lines for HD research. Western blot (WB) analysis showed that endogenous HSF1 levels in mitochondria were dramatically higher in HdhQ111 cells than in HdhQ7 cells (Fig 1A). However, other members of the HSF family, including HSF2 and HSF4, were not affected by the presence of mtHtt (Fig EV1A). Wild-type (WT) striatal cells exposed to 3-nitropropionic acid (3-NP), an inducer of HD-like symptoms, strongly promoted HSF1 association with mitochondria (Fig 1B). Consistently, we observed higher HSF1 levels in mitochondria in YAC128 HD transgenic mice than in their age-matched littermates (Fig 1C). Moreover, elevated binding of HSF1 with mitochondria was confirmed in three lines of HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals (Figs 1D and EV1B). Induced pluripotent stem cells (iPSCs) derived from a HD patient carrying 75 CAG repeats were confirmed by WB and pluripotency staining (Fig EV1C and D). There were significantly higher HSF1 levels in mitochondria in HD iPSCs (HDUE003) than in control iPSCs (IMR90-4) (Fig 1E). Overexpression of exon 1 of htt with 73 polyglutamine repeats sharply increased HSF1 translocation to mitochondria (Fig EV1E). Notably, the total protein levels of HSF1 were not different between the HD models and their controls (Fig EV1F and G). To further confirm the results in human tissues, we established a method to generate human striatal organoids for research on the etiology of HD (Fig EV2A). The striatal organoids expressed markers for medium spiny neurons (MSNs, DARPP32+) and lateral ganglionic eminences (LGEs, GSH2+), as well as telencephalic markers (Figs 1F and, EV2A–C), indicating a striatal fate. We evaluated the characteristics of the striatal organoids by performing 10x Genomics single-cell RNA sequencing (scRNA-seq) on day 30 and day 60 (Fig 1G). Uniform Manifold Approximation and Projection (UMAP) visualization showed that the number of cells in the subcluster of MSN and LGE progenitors was significantly increased in day (D) 60 organoids (Figs 1G and EV2D–F). Moreover, comparison of our scRNA-seq data with the BrainSpan database showed a positive correlation between D60 striatal organoids and the fetal striatum at 12 to 19 weeks post-conception (Fig EV2G). Furthermore, differentially expressed gene (DEG) analysis revealed upregulation of striatal genes but downregulation of neural progenitor genes in the D60 striatal organoids (Fig EV2H). Overall, we generated human striatal organoids confirmed by immunohistochemistry and scRNA-seq. HD striatal organoids exhibited increased colocalization of HSF1 and mitochondria compared with WT striatal organoids (Fig 1H). Treatment with proteinase K resulted in the digestion of the outer mitochondrial membrane (OMM) protein Mcl1 and approximately 60% of mtHSF1, suggesting that part of HSF1 localizes to the OMM (Fig EV2I). Immunogold electron microscopy further determined that HSF1 localizes to the OMM, inner membrane space, and matrix (Fig 1I). Deletion of mtHtt in HdhQ111 cells with shRNA against Htt reduced the association between HSF1 and mitochondria (Figs 1J and EV2J). Taken together, these data suggest that HSF1 accumulates on mitochondria in the models of HD. Figure 1. HSF1 associates with mitochondria in models of HD Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control (n = 6 biological replicates). HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control (n = 5 biological replicates). HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates (n = 5 biological replicates). mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group). Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs (n = 3 biological replicates). Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO, Hoechst (blue). The scale bar represents 100 µm. Left, schematic of the procedure by which the striatal organoids were dissociated for scRNA-seq. Right, UMAP visualization of single-cell RNA expression in striatal organoids at D30 (n = 5,939 cells) and D60 (n = 5,188 cells). Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n = 15 organoids; HD: n = 14 organoids. Two-tailed unpaired t-test; means ± SEMs. Images were taken using structured illumination microscopy (SIM). The scale bar represents 5 μm. The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm. HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions (n = 3 biological replicates). Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student’s t-test was used in (A–E) and (H); one-way ANOVA followed by Tukey’s multiple comparison test was used in (J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 1 [emmm202215851-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. HSF1 associates with mitochondria A. Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. HSF2 and HSF4 protein levels in the mitochondria were detected by WB analysis (n = 3 biological replicates). B, C. Total lysates were harvested from control iPSCs, HD iPSCs, control fibroblasts, and HD patient fibroblasts. Immunoblot analysis of htt was performed with 1C2 and MAB2166 (n = 3 biological replicates). D. Characterization of hiPSCs by immunostaining for the pluripotency markers SOX2 and NANOG and staining for alkaline phosphatase (AP) (n = 2 biological replicates). The scale bar represents 100 μm. E. Flag-HSF1 was transfected with Myc-Q23 or Myc-Q73 into HEK293 cells. Mitochondria-localized HSF1 was examined by WB analysis (n = 3 biological replicates). The data are the means ± SEMs; unpaired Student’s t-test was used. ***P < 0.001. F. Total HSF1 levels in fibroblasts were measured by WB analysis (n = 2 biological replicates). G. Total HSF1 protein levels were tested in the indicated samples by immunoblotting (n = 3 biological replicates). Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Construction of mtHSF1 and the procedure for human striatal organoid differentiation Schematic of the protocol for differentiating human striatum-like organoids from hPSCs and immunostaining for the LGE progenitor marker GSH2 and the MSN marker DARPP32 at D30, D45, and D60. The scale bar represents 100 μm. Identification of telencephalon and striatum markers by immunostaining. The scale bar represents 100 μm. Confirmation of maturation of striatal organoids by robust MAP2 staining and sparse GSH2 and KI67 staining. The scale bar represents 100 μm. UMAP visualization of single-cell RNA expression in striatal organoids (n = 11,127 cells). Cell-type compositions of human striatal organoids at D30 and D60. UMAP plots showing the gene expression patterns of representative marker genes for each cell type. The relative expression level is indicated by the color from gray to red. Correlation with BrainSpan dataset of the developing human brain (PCW 8–19). Volcano and violin plots for cell type-specific genes differentially expressed in neuronal (left) cells and neural progenitor cells (right) from striatal organoids at D30 and D60. Mitochondria-enriched fractions isolated from HdhQ111 cells were exposed to proteinase K for 20 min at 3 µg/ml. Protein levels were detected with the indicated antibodies. The htt gene expression was measured by real-time PCR (n = 3 biological replicates). Data information: The data are the means ± SEMs; unpaired Student’s t-test was used. *P < 0.05 and ****P < 0.0001. Source data are available online for this figure. Download figure Download PowerPoint mtHSF1 disturbs dynamic balance by promoting Drp1 phosphorylation To determine the effect of mtHSF1 on mitochondrial morphology, we constructed Flag-tagged mtHSF1 (Flag-mtHSF1) by fusing a mitochondrial targeting sequence (MTS) to the N-terminus of HSF1 to mimic the accumulation of HSF1 on mitochondria in HD (Figs 2A and EV3A). We observed shortened and swollen mitochondria in striatal cells expressing Flag-mtHSF1, suggesting that the mitochondria were fragmented (Fig 2B). To understand the molecular basis of the observed mitochondrial fragmentation, we examined the protein levels of mitochondrial dynamics-related proteins. The presence of Flag-mtHSF1 had no effect on the total protein levels of Drp1, optic atrophy 1 (OPA1), mitofusin 2 (MFN2), or mitochondria-localized Drp1 (Figs 2C and EV3B). Figure 2. mtHSF1 disturbs the dynamic balance by promoting Drp1 phosphorylation Sketch of mitochondrial targeting by HSF1. HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar shows the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm (n = 3 biological replicates; at least 100 cells per group were counted). The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1, and actin (loading control) were determined by WB analysis (n = 3 biological replicates). Timeline overview of mitochondrial analysis in mice. AAV-Con and AAV-mtHSF1 were injected into the striata of WT mice at 8 weeks of age. Mitochondrial morphology was analyzed after 3 weeks. Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green), and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar represents 2 mm,
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