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Age-related Huntington’s disease progression modeled in directly reprogrammed patient-derived striatal neurons highlights impaired autophagy

Nature Neuroscience volume 25, pages 1420–1433 (2022)Cite this article

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Abstract

Huntington’s disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3′ untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs.

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Fig. 1: Differential manifestation of neurodegeneration between MSNs derived from healthy control individuals and patients with HD.
Fig. 2: Identification of gene module associated with autophagy dysfunction in HD-MSNs by WGCNA.
Fig. 3: Autophagy inhibitor induces neurodegeneration of pre-HD-MSNs.
Fig. 4: Autophagy activator rescues HD-MSNs from degeneration.
Fig. 5: Comparative analysis of chromatin accessibility between pre-HD-MSNs and HD-MSNs.
Fig. 6: Inhibition of miR-29b-3p enhances autophagy and rescues HD-MSNs from degeneration.
Fig. 7: Inhibition of miR-29b-3p–STAT3 axis enhances autophagy and rescues HD-MSNs from degeneration.

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Data availability

All the deep-sequencing data (RNA-seq and ATAC-seq) have been uploaded to the Gene Expression Omnibus repository: GSE194243. Source data that support all findings of the study are available as supplementary data. The data that support this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

No new code was used in this study.

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Acknowledgements

We thank the Genome Technology Access Center at Washington University for deep-sequencing experiments. HD and control brain tissues were received through the NIH NeuroBioBank. Images were created with BioRender. We thank D. H. Geschwind and R. Kawaguchi (UCLA) for their suggestions on statistics of miRNAs on gene networks. This study was supported by the following programs, grants and fellowships: Cellular and Molecular Biology Training Program (T32 GM007067) (K.C.), Cure Alzheimer’s Fund, CHDI Fund, Hereditary Disease Foundation Grant, RF1AG056296 (National Institute on Aging), R01NS107488 (National Institute of Neurological Disorders and Stroke), Farrell Foundation Fund and Mallinckrodt Scholar Award (A.S.Y.).

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Author notes

  1. These authors contributed equally: Young Mi Oh, Seong Won Lee.

Authors and Affiliations

  1. Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA

    Young Mi Oh, Seong Won Lee, Woo Kyung Kim, Shawei Chen, Victoria A. Church, Kitra Cates, Tiandao Li, Bo Zhang & Andrew S. Yoo

  2. Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Tiandao Li, Bo Zhang & Andrew S. Yoo

  3. Department of Biochemistry, Washington University School of Medicine, St. Louis, MO, USA

    Roland E. Dolle

  4. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Sonika Dahiya

  5. Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA

    Stephen C. Pak, Gary A. Silverman & David H. Perlmutter

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Contributions

Y.M.O. and A.S.Y. conceived and developed the idea, designed the experiments and analyzed data. Y.M.O. performed all experiments and associated assays and analyses. Y.M.O. and S.W.L. performed neuronal reprogramming throughout figures shown in the study. S.W.L. performed western blot and immunostaining for p62. Y.M.O. and W.K.K. performed ATAC-seq analysis. Y.M.O. and S.C. performed SYTOX assay of G2 analog. T.L. and B.Z. performed WGCNA. V.A.C and S.D. provided the RNAs of human brain samples. K.C. performed LGE analysis. R.E.D., S.C.P., G.A.S. and D.H.P. developed the G2 analog. Y.M.O. and A.S.Y. wrote the manuscript. A.S.Y. supervised the overall project.

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Correspondence to Andrew S. Yoo.

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Nature Neuroscience thanks Johan Jakobsson and Neelroop Parikshak for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Healthy control and HD patient fibroblasts can be directly reprogrammed into MSNs.

a. Reprogrammed cells by transduction of miR-9/9*-124+CDM immunostained for neuronal markers, TUBB3, and MSN marker, DARPP32 at PID30. Scale bars 20 μm. b. Expression of each CDM factor in reprogramming cells by immunostaining for CTIP2, DLX1, DLX2, MYT1L, and TUBB3. Non-transduced fibroblasts were used as a negative control for immunostaining. Scale bars 20 μm. c. The neuronal morphology across all samples we used in the current study stained positive for TUBB3 successfully undergo direct conversion by miR-9/9*-124+CDM. Scale bars 20 μm. d. Left, representative images of healthy control Young, Old, Pre-HD, and HD MSNs marked by TUBB3. Images processed by CellProfiler to identify neurites and associated cell soma. Scale bars, 100 μm. Right, the measurement of mean neurite length and mean number of neurite branches in reprogrammed Healthy control Young, Old, Pre-HD, and HD-MSNs at PID21 and PID35 (n = 24 individual’s reprogrammed MSNs). Statistical significance was determined using one-way ANOVA, ****p < 0.0001, ***p < 0.001 (Old vs. HD in neurite length p = 0.0007, Old vs. HD in neurite branches p = 0.0001, Pre-HD vs. HD in neurite branches p = 0.0002), **p < 0.01 (Young vs. HD in neurite length p = 0.0012, Pre-HD vs. HD in neurite length p = 0.0026), ns, not significant. Mean±s.e.m.

Source data

Extended Data Fig. 2 Genetic networks altered in HD-MSNs by WGCNA.

a, The signed association of protein-coding genes with Age, Symptomatic onset, and Sex condition of Huntington’s disease. Modules with positive values indicate increased expression in HD-MSNs; modules with negative values indicate decreased expression in HD-MSNs. The red dotted lines indicate correlation values of 0.7 or -0.7 with p = 10-7 for age, p = 10-6 for symptomatic onset, and p = 10-6 for sex. b, An expression heatmap of our selected modules of HD samples (blue, lightcyan1, brown, and greenyellow) from WGCNA. c, Pathways enriched in downregulated (log2FC < -1) or upregulated (log2FC > 1) genes in the greenyellow, blue and lightcyan1 module of Huntington’s disease by BioPlanet analysis. d, The signed association of protein-coding genes with age and sex of healthy control. Modules with positive values indicate increased expression in Old-MSNs; modules with negative values indicate decreased expression in Old-MSNs. The red dotted lines indicate correlation values of 0.7 or -0.7 with p = 10-7 for age and p = 10-6 for sex condition. e, Pathways enriched in upregulated (log2FC > 1) genes in the honeydew1 module of healthy control by BioPlanet analysis. f, Summary preservation statistics as a function of the module size. Left: the composite preservation statistic (Zsummary), Middle: the connectivity statistics (Zconnectivity), Right: the density statistics (Zdensity). Each point represents a module, labeled by color. The dashed blue and green lines indicate the thresholds Z = 2 and Z = 10, respectively. g. Representative images of MSNs expressing the tandem monomeric mCherry-GFP-LC3 reporter immunostained for TUBB3. Autophagosome (that is, mCherry + , GFP + puncta) and autolysosome (that is, mCherry + , GFP − puncta) compartments in MSNs from HD patients and control individual. Scale bar 20 μm. h. Reprogrammed cells immunostained for p62 and TUBB3 from independent HD and healthy control lines. Scale bar 20 μm. i. Immunoblot analysis for p62 and GAPDH of three independent pre-HD- and three independent HD-MSN lines at PID26. P62 signal intensities were normalized by GAPDH signals and relative fold changes in HD-MSNs were calculated over pre-HD-MSNs (**p = 0.0019 by two-tailed unpaired t-test; Mean±s.e.m.).

Source data

Extended Data Fig. 3 The treatment of autophagy inhibitor or inducer in reprogrammed MSNs.

a, Immunoblot of p62 and GAPDH in pre-HD-MSNs treated with DMSO or 50 μM LY294002 and HD-MSNs treated with DMSO or 0.5uM G2-115 at PID26 (left). p62 Intensity values were normalized by GAPDH intensities and the relative fold change over DMSO condition was calculated from immunoblot images of three independent Pre-HD-MSNs (middle, *p = 0.0277) and HD-MSNs (*p = 0.0460). Each dot represents one individual’s reprogrammed MSN. b, left: representative images of pre-HD-MSNs treated with DMSO or 50 μM LY294002 and HD-MSNs treated with DMSO or 0.5 uM G2-115 (PID 30). MSNs express the tandem monomeric RFP-GFP-LC3 reporter. Scale bars 20 μm. right: quantification of autophagosome (that is, mCherry + , GFP + puncta) and autolysosome (that is, mCherry + , GFP − puncta) compartments from three independent Pre-HD-MSNs (***p = 0.0004) and HD-MSNs (***p = 0.0005). Measurements were performed in cells having at least 3 puncta per cell (from more than 50 cells per MSN line). Each dot represents one individual’s reprogrammed MSNs. c. Synthetic route for the preparation of G2-115. d, Measurement of neuronal cell death of HD.40-MSNs with the treatment of DMSO or three different concentrations of G2 compound at PID 30 by Caspase-3/7 reagents (left, *p = 0.0199, ****p < 0.0001) or Annexin V reagents (right, *p = 0359, *p = 0.0326) (n = 4-8 cultures). For all figures shown, statistical significance was determined using two-tailed unpaired t-test (a,b) and one-way ANOVA (d); ****p < 0.0001, ***p < 0.001, *p < 0.05. Mean±s.e.m.

Source data

Extended Data Fig. 4 Pre-HD-MSNs and HD-MSNs display differential chromatin accessibilities.

a, A heatmaps showing gene expression levels for DEGs that positively correlated with signal intensities of ATAC-seq in their promoter regions. Signal intensity is based on normalized CPM values. Data are shown as Z-score normalized log2CPM (adjusted p < 0.05, │log2FC│>1). b, c, GO terms associated with opened and upregulated genes (b) and closed and downregulated genes (c). d, Transcription regulators predicted as upstream regulators of the brown module (Huntington’s disease) and the lavenderblush3 (healthy control) by Ingenuity Pathway Analysis (IPA). e, Mature microRNAs predicted as an upstream regulator of the brown module (Huntington’s disease) and the lavenderblush3 (healthy control) by Ingenuity Pathway Analysis (IPA). f, MicroRNAs predicted as an upstream regulator of the brown module (Huntington’s disease) by miRTarBase and TargetScan. g, Pathways enriched in target genes of miR-29b-3p in the brown module (Huntington’s disease) by BioPlanet analysis.

Extended Data Fig. 5 miR-29b-3p expression changes by miR-29-3p inhibitor or miR-29b overexpression lentivirus transduction.

a, RT-qPCR analysis of mature miR-29-3p expression levels in four HD-MSNs with control or miR-29b-3p inhibitor at PID26 (n = 4 individual’s reprogrammed HD-MSNs, ***p = 0.0002 by two-tailed unpaired t-test; Mean±s.e.m.). b, Pre-HD-MSNs expressing RFP only or RFP-miR-29b immunostained with RFP and DAPI. (Scale bars, 20 μm). c, RT-qPCR analysis of mature miR-29-3p expression levels in three independent pre-HD-MSNs expressing control or miR-29b. Each dot represents one individual’s reprogrammed MSNs. (**p = 0.0019 by two-tailed unpaired t-test; Mean±s.e.m).

Source data

Extended Data Fig. 6 The autophagy-related genes regulated by STAT3 in HD-MSNs.

a, The target genes of miR-29b-3p functionally related to autophagy in the brown module. Visualized by NetworkAnalyst. b, A heatmap of representation of ATAC-seq signal intensities for autophagy-related genes that contained STAT3 binding site in the closed DARs in HD-MSNs (n = 8-9 per group). c, Percentage of decrease of STAT3 levels in old versus young Ctrl-MSNs, and HD-MSNs versus pre-HD-MSNs, replotted from Fig. 7(f). Each dot represents one individual’s reprogrammed MSNs. (****p < 0.0001 by two-tailed unpaired t-test; Mean±s.e.m.). d, RT-qPCR analysis of STAT3 mRNA levels in three independent pre-HD-MSN lines with shControl or shSTAT3 at PID26. Each dot represents one individual’s reprogrammed Pre-HD-MSNs. (****p < 0.0001 by two-tailed unpaired t-test; Mean±s.e.m.). e, Western bot for STAT3 expression in human adult fibroblasts with shControl or shSTAT3. f, RT-qPCR analysis of STAT3 mRNA levels in three independent HD-MSN lines with Control or STAT3 overexpression at PID26. Each dot represents one individual’s reprogrammed HD-MSNs. (*p = 0.0143 by two-tailed unpaired t-test; Mean±s.e.m.).

Source data

Extended Data Fig. 7 Prediction of DAR proximal to miR-29b and HD-specific phenotype in fibroblasts.

a, Predicted transcription binding sites for the DAR proximal to miR29B1 (chr7:130,878,800-130,879,437). Image from UCSC Genome Browser on Human (GRCh38/hg38). JASPAR CORE 2022, Minimum Score: 500. b, Quantification of Sytox-positive cells, CYTO-ID signal, STAT3 expression, and miR-29b-3p expression in fibroblasts from healthy control Young/Old, Pre-HD, and HD (n = 14 or 19 individual fibroblasts, statistical significance was determined by one-way ANOVA. ns, not significant; Mean±s.e.m.).

Supplementary information

Supplementary Information

Supplementary Table 1. The list of six pre-symptomatic patients with HD, six HD patient fibroblasts and 12 healthy control fibroblasts

Reporting Summary

Supplementary Table 2

Detailed information on pathway enrichment analysis

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Oh, Y.M., Lee, S.W., Kim, W.K. et al. Age-related Huntington’s disease progression modeled in directly reprogrammed patient-derived striatal neurons highlights impaired autophagy. Nat Neurosci 25, 1420–1433 (2022). https://doi.org/10.1038/s41593-022-01185-4

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