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, 17 (3), 357-66

Tau Promotes Neurodegeneration Through Global Chromatin Relaxation

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Tau Promotes Neurodegeneration Through Global Chromatin Relaxation

Bess Frost et al. Nat Neurosci.

Abstract

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.

Figures

Figure 1
Figure 1
Tau transgenic Drosophila have widespread alterations in chromatin structure. (a) H3K9me2 and HP1α levels in control and tau transgenic fly head homogenates. The full-length blot is shown in Supplementary Figure 5a (b) Quantification of a, n = 5 flies, (for H3K9me2, *p = 0.04, t = 3.14 for HP1α, *p = 0.04, t = 3.08 for four degrees of freedom, unpaired t-test). (c) H3K9me2 and HP1α immunostaining of tau transgenic fly brains. Arrows indicate chromocenters. The region presented is cortex. Scale bar is 3 μm. (d) Quantification of experiments in c, n = 3 brains (for H3K9me2, **p = 9e-05, t = 105.36 for two degrees of freedom, for HP1α, **p = 4.7e-05, t = 145.24 for two degrees of freedom, unpaired t-test). Control is elav-GAL4/+ in a-d. (e) Quantification BL2 (β-galactosidase) and Y10C (GFP) chromatin reporter activation in control and tau transgenic flies. Controls are elav-GAL4/BL2 and elav-GAL4/Y10C, n = 6 brains (for BL2, **p = 5.12e-09, t = 81.94 for five degrees of freedom, for HP1α, **p = 1.13e-05, t = 17.47 for five degrees of freedom, unpaired t-test). β-galactosidase and AT8 (f) cleaved PARP (cPARP) (g) or PCNA (h) immunostaining in flies transgenic for the BL2 chromatin reporter, n = 3 brains. Control is elav-GAL4/BL2 in f and h, and elav-GAL4/BL2;UAS-PARP/+ in g. Scale bars are 10 μm in f-h. Data are mean ± s.e.m., all flies are 10 days old.
Figure 2
Figure 2
Genetic manipulation of chromatin structure modifies tau-induced toxicity in Drosophila. (a) H3K9me2 and HP1α levels in tau transgenic 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 Figure 5b. (b) Quantification of a, n = 3 heads, (for H3K9me2, *p = 0.04, F = 4.38 for two degrees of freedom, for HP1α, *p = 0.02, F = 5.6 for two degrees of freedom, one-way ANOVA). (c) Quantification of H3K9me2- and HP1α-positive chromocenters in tau transgenic flies heterozygous for a loss-of-function mutation in ash1 or expressing an RNAi transgene targeted to NURF301, n = 3 brains (for H3K9me2, **p = 0.01, F = 11.64 for two degrees of freedom, for HP1α, **p < 0.001, F = 46.2 for two degrees of freedom, one-way ANOVA). (d) Quantification of BL2 reporter activation in control and tau transgenic flies heterozygous for a loss-of-function mutation in ash1 or expressing an RNAi transgene targeted to NURF301 (**p = .001, F = 8.85 for two degrees of freedom, one-way ANOVA). Neuronal degeneration assayed by TUNEL staining (e) and cell cycle activation assayed by PCNA staining (f) in brains of control and tau transgenic flies heterozygous for a loss-of-function mutation in ash1 or expressing an RNAi transgene targeted to ash1, NURF301, or NURF38 (for TUNEL, **p < 0.001, F = 22.1 for two degrees of freedom, for PCNA, **p < 0.001, F = 14.569 for two degrees of freedom, one-way ANOVA). (g) Locomotor activity in control and tau transgenic flies expressing an RNAi transgene targeted to ash1 or NURF301, n = 18 flies, **p < 0.001, F = 11.66 with two degrees of freedom, one-way ANOVA. (h) H3K9me2 and HP1α levels in homogenates from heads of tau transgenic flies heterozygous for loss-of-function mutations in Su(var)205 or Su(var)3-9. Full-length blots are shown in Supplementary Figure 5c. (i) Quantification of h, n = 3 heads (for H3K9me2, **p = 0.0002, F = 1,080.2 for two degrees of freedom, for HP1α, **p = 0.01, F = 16.83 for two degrees of freedom, one-way ANOVA). (j) Quantification of H3K9me2- and HP1α-positive chromocenters in tau transgenic flies heterozygous for a loss-of-function mutation in Su(var)205 or Su(var)3-9 based on immunofluorescence, n = 3 brains (For H3K9me2, **p = 0.01, F = 13.16 for two degrees of freedom, for HP1α, *p = 0.02, F = 8.964 for two degrees of freedom, one-way ANOVA). (k) Quantification of BL2 reporter activation in control and tau transgenic flies heterozygous for loss-of-function mutations in Su(var)205 or Su(var)3-9, **p < 0.001, F = 21.2 for two degrees of freedom, one-way ANOVA. Neuronal apoptosis (l) and PCNA positive foci (m) in control and tau transgenic flies heterozygous for loss-of-function mutations in Su(var)205 or Su(var)3-9 (for TUNEL, **p < 0.001, F = 10.26 for four degrees of freedom, for PCNA, **p < 0.001, F = 34.48 for four degrees of freedom, one-way ANOVA). (n) Locomotor activity in control and tau transgenic flies heterozygous for a loss-of-function mutation in Su(var)205 or Su(var)3-9, n = 18 flies, **p < 0.001, F = 12.84 for two degrees of freedom, one-way ANOVA. Flies are 10 days old and n = 6 unless otherwise specified. Controls are elav-GAL4/+. Data are mean ± s.e.m.
Figure 3
Figure 3
Oxidative stress causes DNA damage and heterochromatin loss in Drosophila. pH2Av and elav staining in neurons of control and Trxr-1481 hemizygous (a) or Sod2Δ02 heterozygous (b) mutant flies, n = 3 brains. Arrows indicate pH2Av positive nuclei. Scale bar is 10 μm. Quantification of comet tails in Trxr-1481 hemizygous (c) and Sod2Δ02 heterozygous (d) mutant flies compared to control, n = 6 brains (for c, **p = 0.01, t = 9.2 for two degrees of freedom, for d, **p = 0.01, t = 10.84 for two degrees of freedom, unpaired t-test). (e) H3K9me2 and HP1α levels in homogenates from heads of flies harboring loss-of-function mutations in Trxr-1 or Sod2. Full-length blots are shown in Supplementary Figure 5d. (f) Quantification of experiments in e, n = 3 heads (for H3K9me2, *p = 0.02, F = 7.98 for two degrees of freedom, for HP1α, *p = 0.03, F = 6.5 for two degrees of freedom, one-way ANOVA). Quantification of BL2 reporter activation in brains of Trxr-1481 (g) and Sod2Δ02 (h) flies; control is hemizygous for the BL2 reporter. n = 6 brains (for g, **p < 0.001, t = 20.19 for five degrees of freedom, for h, **p < 0.001, t = 26.56 for five degrees of freedom, unpaired t-test). (i) H3K9me2 and HP1α levels in homogenates from heads of flies with neuronal overexpression of CycA or Rheb. Full-length blots are shown in Supplementary Figure 5e. (j) Quantification of experiments in i, n = 3 heads (for H3K9me2, p = 0.92, F = 0.08 for two degrees of freedom, for HP1α, p = 0.54, F = 0.71 for two degrees of freedom, one-way ANOVA). (k) Cell cycle activation assayed by PCNA staining in brains of flies with RNAi-mediated knockdown of Su(var)205 or Su(var)3-9. n = 6 brains, **p < 0.001, F = 23.52 for two degrees of freedom, one-way ANOVA. Control is w1118 in a-h, and elav-GAL4/+ in i-k. Trxr-1481 hemizygous flies and respective controls are 2 days old, all other flies are 10 days old. Data are mean ± s.e.m., except in c, d, g and h in which data are represented as box and whisker plots comprised of minimum, lower quartile, median, upper quartile and maximum values.
Figure 4
Figure 4
H3K9me2 loss and increased gene expression in tau transgenic Drosophila. (a) H3K9me2 distribution in genes with significant H3K9me2 loss in tau transgenic flies based on H3K9me2 ChIP-seq, n = 2 rounds of ChIP-seq on biological replicates, (FDR < 0.01). Arrows indicate the locations of primers used for qPCR. Rectangles indicate exons, dashed lines indicate introns. (b) qPCR of each gene identified by ChIP-seq as H3K9me2 depleted in tau transgenic flies, n = 3 trials, 10 heads per trial (for Ago3, *p = 0.02, t = 6.85, for CG15115, p = 0.004, t = 15.6, for CG15661, *p = 0.01, t = 8.25, for CG40006, *p = 0.01, t = 9.56, for Dscam, *p = 0.0003, t = 54.42, for Gprk1, *p = 0.004, t = 15.07, for IntS3, *p = 0.0003, t = 62.71, for Ir41a, *p = 0.005, t = 14.38, for nvd, *p = 0.007, t = 12.31, for Snap25, *p = 0.004, t = 15.75, for uif, *p = 0.003, t = 19.15, for two degrees of freedom, unpaired t-test). (c) Expression levels of H3K9me2 depleted genes in tau transgenic flies versus baseline gene expression (modEncode). Transcript levels are relative to both control and the endogenous control gene RpL32. n = 3 trials, 10 heads per trial, circles are significantly different from control, which is set to 1, (for Ago3, p = 0.0003, t = 59.92, for CG15115, p = 0.05, t = 4.33, for CG15661, p = 0.04, t = 5.09, for CG40006, p = 0.35, t = 1.2, for Dscam, p = 0.48, t = 0.86, for Gprk1, p = 0.053, t = 4.15, for IntS3, *p = 0.38, t = 1.13, for Ir41a, p = 0.01, t = 9.79, for nvd, p = 0.04, t = 4.91, for Snap25, p = 0.05, t = 4.5, for uif, p = 0.02, t = 6.29, for two degrees of freedom, unpaired t-test). (d) Ago3 levels in homogenates from control and tau transgenic fly heads. Full-length blots are shown in Supplementary Figure 5f. (e) Quantification of d, n = 5 heads, **p = 3.57e-07, t = 63.98 for four degrees of freedom, unpaired t-test. Neuronal degeneration assayed by TUNEL staining (f) and cell cycle activation assayed by PCNA staining (g) in brains of control and tau transgenic flies with RNAi-mediated reduction of Ago3. n = 6 brains (for TUNEL, **p < 0.001, F = 29.92 for two degrees of freedom, for PCNA, **p < 0.001, F = 57.67 for two degrees of freedom, one-way ANOVA). (h) Locomotor activity in control and tau transgenic flies with RNAi-mediated reduction of Ago3, n = 18 flies (for Ago3RNAi-1 compared to control, **p = 4.8e-16, t = 6.56 for 17 degrees of freedom, for tau + Ago3RNAi-2 compared to tau expressed alone, **p = 0.001, t = 3.865 for 17 degrees of freedom, unpaired t-test). Control is elav-GAL4/+. Data are mean ± s.e.m., unpaired t-test or ANOVA. All flies are 10 days old.
Figure 5
Figure 5
Tauopathy model mice have heterochromatin loss and increased PIWIL1 expression. Perinucleolar chromocenter staining (arrows) of H3K9me2 (a) and HP1αD(b) in control and JNPL3 motor neurons. Upper boxes are H3K9me2 or HP1α staining in MAP2-positive neurons at higher magnification. (c) Quantification of experiments in a and b (for a, **p = 0.002, t = 6.09 for five degrees of freedom, for b, **p = 0.00002, t = 16.97 for five degrees of freedom, unpaired t-test). (d) PIWIL1 immunostaining (arrows) in control and JNPL3 motor neurons. n = 6 spinal cords per genotype. Scale bars are 10 μm. Data are mean ± s.e.m.
Figure 6
Figure 6
H3K9me2 loss and increased gene expression in human AD brains. (a) Perinucleolar chromocenter staining of H3K9me2 (arrows) in human control and AD hippocampal neurons. n = 6 brains. (b) PIWIL1 immunostaining (arrows) in control and human AD hippocampal neurons. n = 6 brains. Upper boxes are H3K9me2 or PIWIL1 staining in MAP2-positive neurons at higher magnification in a and b. (c) FACS of postmortem human cortical nuclei stained with secondary antibody AlexaFluor488, or the neuron-specific NeuN antibody and secondary antibody AlexaFluor488. (d) S100β and NeuN levels in NeuN negative (NEG) and NeuN positive (POS) nuclear populations purified via FACS. Full-length blots are shown in Supplementary Figure 5g. (e) H3K9me2 levels in FACS-purified NeuN positive neuronal nuclei from control and AD brains. Full-length blots are shown in Supplementary Figure 5h. (f) Quantification of e, n = 6 brains, data are represented as box and whisker plots comprised of minimum, lower quartile, median, upper quartile and maximum values for H3K9me2 levels, p = 0.00002, t = 15.95 for two degrees of freedom, unpaired t-test. Average mRNA levels of heterochromatic (g) and euchromatic (h) genes in laser-captured neurons from control (n = 13) and AD (n = 10) postmortem hippocampi. Gray lines indicate a 50% gene expression threshold. (i) Bar plot of genes from g and h 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 < 10−16, chi-square. Error bars in i reflect standard deviation from 1,000 bootstraps.

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