The microtubule-associated protein tau is involved in a number of neurodegenerative disorders, including Alzheimer's disease. Previous studies have linked oxidative stress and subsequent DNA damage to neuronal death in Alzheimer's disease and related tauopathies. Given that DNA damage can substantially alter chromatin structure, we examined epigenetic changes in tau-induced neurodegeneration. We found widespread loss of heterochromatin in tau transgenic Drosophila and mice and in human Alzheimer's disease. Notably, genetic rescue of tau-induced heterochromatin loss substantially reduced neurodegeneration in Drosophila. We identified oxidative stress and subsequent DNA damage as a mechanistic link between transgenic tau expression and heterochromatin relaxation, and found that heterochromatin loss permitted aberrant gene expression in tauopathies. Furthermore, large-scale analyses from the brains of individuals with Alzheimer's disease revealed a widespread transcriptional increase in genes that were heterochromatically silenced in controls. Our results establish heterochromatin loss as a toxic effector of tau-induced neurodegeneration and identify chromatin structure as a potential therapeutic target in Alzheimer's disease.
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A. Alekseyenko, N. Riddle and A. Minoda provided critical advice for the H3K9me2 ChIP experiments. J. Eissenberg (Saint Louis University School of Medicine), K. Maggert (Texas A&M University), J. Brennecke (Austrian Academy of Science) and F. Missirlis (National Polytechnic Institute) provided Drosophila stocks. We thank C. Lemere (Brigham and Women's Hospital), D. Borchelt and G. Xu (University of Florida) for generously providing important reagents. We performed confocal imaging at the Harvard NeuroDiscovery Center Enhanced Neuroimaging Core Facility. The TRiP at Harvard Medical School (NIH/HIGMS R01-GM084947) and the Vienna Drosophila RNAi Center provided transgenic RNAi fly stocks. Antibodies obtained from the Developmental Studies Hybridoma Bank were developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa. Antibodies obtained from the University of California Davis/US National Institutes of Health NeuroMab facility are supported by NIH grant U24NS050606 and maintained by the University of California Davis. This work was supported by a Ruth L. Kirschstein National Research Service Award F32AG039193 (B.F.), R01AG33518, a Senior Scholar Award from the Ellison Medical Foundation and a grant from the American Health Assistance Foundation/BrightFocus (M.B.F.), and US National Institutes of Health (1R21NS070250) and National Science Foundation grants (0954570) in support of M.H. Support was provided to J.L. through the University of Florida Department of Neuroscience.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Heterochromatin in wild-type tau or pseudohyperphosphorylated tau transgenic Drosophila, Drosophila harboring mutations in or RNAi-mediated reduction of chromatin modifying genes, and tau phosphorylation in the context of chromatin modifiers.
(a) Su(var)3-9 and Su(var)205 mRNA levels in tau transgenic flies, n = 3 trials, 10 heads per trial. (b) H3K9me2 and HP1α levels in homogenates from fly heads transgenic for human wild type tau (tauWT) or pseudohyperphosphorylated tau (tauE14). Full-length blots are shown in Supplementary Fig. 6a. (c) Quantification of b, n = 7 heads (for H3K9me2, *p = 0.04, F = 4.39, for HP1α, **p = 0.001, F = 12.26, six degrees of freedom, one-way ANOVA). (d) H3K9me2- and HP1α-staining in control and tau transgenic brains based on DAB staining, n = 3 brains. Arrows indicate a nucleus with typical focal chromocenter labeling; arrowheads indicate a nucleus lacking focal chromocenter labeling. The region presented is cortex. Scale bar is 10 μm. (e) Quantification of β-galactosidase reporter activation in control, tauWT and tauE14 transgenic fly brains. Control is elav-GAL4/BL2, n = 6 brains, p < 0.001, F = 198.54 for five degrees of freedom, one-way ANOVA. (f) H3K9me2 and HP1α levels in homogenates from heads of control flies heterozygous for a loss-of-function mutation in ash1 or expressing an RNAi transgene targeted to NURF301. Full-length blots are shown in Supplementary Fig. 6b. (g) Quantification of f, n = 3 heads. (h) mRNA levels of ash1, NURF38, and NURF301 in tau transgenic Drosophila, compared to control, n = 3 trials, 10 heads per trial. (i) RT-PCR of ash1, NURF38, and NURF301 in flies harboring RNAi transgenes targeted to ash1, NURF38, and NURF301, n = 3, 10 heads per trial, *p = 0.02, F = 5.86 for two degrees of freedom, one-way ANOVA. H3K9me2 and HP1α levels in homogenates from heads of control flies heterozygous for loss-of-function mutations in Su(var)205 or Su(var)3-9 (j, quantified in k, n = 3 heads). Full-length blots are shown in Supplementary Fig. 6c. (l) Total tau levels and tau phosphorylation in homogenates from heads of tau transgenic flies with the indicated transgenes at 1 day, n = 3 heads. Full-length blots are shown in Supplementary Fig. 6d. Flies are 10 days old unless otherwise specified. Controls are elav-GAL4/+. Data are mean ± s.e.m.
Supplementary Figure 2 Efficiency of RNAi-mediated knockdown of gene expression, and tau phosphorylation in the context of RNAi-mediated reduction of Ago3 in Drosophila.
(a) mRNA levels of Su(var)205 and Su(var)3-9 in flies with Su(var)205 and Su(var)3-9 RNAi transgenes, n = 3 trials, 10 heads per trial, **p < 0.001, F = 56.07 for two degrees of freedom, one-way ANOVA. (b) Tau levels and phosphorylation in homogenates from heads of tau transgenic flies with RNAi mediated reduction of Ago3, n = 3 heads. Full blots are shown in Supplementary Fig. 6e. (c) Ago3 mRNA levels in 1 day old flies harboring RNAi transgenes targeted to Ago3, n = 3 trials, 10 heads per trial, **p = 0.002, F = 21.53 for two degrees of freedom, one-way ANOVA. Control is elav-GAL4/+. Flies are 1 day old. Data are mean ± s.e.m.
(a) HP1α and AT8 immunostaining in human AD hippocampal neurons. Arrowheads indicate AT8 negative neurons and arrows indicate AT8 positive neurons. Upper boxes are higher magnifications of HP1α staining in AT8 negative or AT8 positive neurons, n = 6 brains. (b) Publicly available histone modification data used to define chromatin states. Average mRNA levels of heterochromatic (c) and euchromatic (d) genes from liver biopsies of HIV positive patients with and without GB virus C infection. Gray lines indicate 50% gene expression change threshold. (e) Principal component analysis of AD (n = 10) and control (n = 13) laser captured hippocampal neurons, hippocampi from control (n = 9) and three stages of AD (incipient (n = 7), moderate (n = 8), and severe (n = 7)), and fetal brain (n = 2), yellow box indicates principal component 4, which groups AD hippocampal neurons, severe AD hippocampi, and fetal brain. Average mRNA levels of heterochromatic (f) and euchromatic (g) genes in peripheral blood mononuclear cells (BMC) from control and AD (n = 14) patients. (h) Bar plot of genes from f and g with expression changes greater than 50%. For increased expression of genes in AD that are classified as heterochromatic and expressed at low levels in control, p < 0.0002, chi-square. Error bars in h reflect standard deviation from 1,000 bootstraps.
In neurons affected by tauopathies, tau aggregation and/or aberrant phosphorylation causes oxidative DNA damage, which is one mechanism whereby tau induces relaxation of heterochromatin. Heterochromatin loss permits aberrant expression of genes, particularly those genes associated with development, which induces dedifferentiation of neurons toward a more stem cell-like state. Neuronal dedifferentiation then triggers aberrant cell cycle activation and subsequent apoptosis.
Supplementary Figures 1–6 and Supplementary Tables 1–4 (PDF 1902 kb)
Chromatin states within the human hippocampus. (PDF 123562 kb)
List of genes that are heterochromatically silenced in control human hippocampal neurons and are expressed at least 50% higher in AD hippocampal neurons. (PDF 925 kb)
List of genes that are heterochromatically silenced in control peripheral blood mononuclear cells and expressed at least 50% higher in AD peripheral blood mononuclear cells. (PDF 380 kb)
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Frost, B., Hemberg, M., Lewis, J. et al. Tau promotes neurodegeneration through global chromatin relaxation. Nat Neurosci 17, 357–366 (2014). https://doi.org/10.1038/nn.3639
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