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, 21 (8), 1038-1048

Pathogenic Tau-Induced piRNA Depletion Promotes Neuronal Death Through Transposable Element Dysregulation in Neurodegenerative Tauopathies

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Pathogenic Tau-Induced piRNA Depletion Promotes Neuronal Death Through Transposable Element Dysregulation in Neurodegenerative Tauopathies

Wenyan Sun et al. Nat Neurosci.

Abstract

Transposable elements, known colloquially as 'jumping genes', constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. Here we utilize Drosophila melanogaster and postmortem human brain samples to identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, we find that heterochromatin decondensation and reduction of piwi and piRNAs drive transposable element dysregulation in tauopathy. We further report a significant increase in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer's disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, our data identify heterochromatin decondensation, piwi and piRNA depletion and consequent transposable element dysregulation as a pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy.

Conflict of interest statement

Competing Financial Interests Statement. The authors declare no competing financial interests.

Figures

Figure 1 |
Figure 1 |. Transposable element transcription in tauR406W transgenic Drosophila.
a, Transposable element transcripts that are differentially expressed in tauR406W transgenic Drosophila heads versus control based on RNA-seq (two-sided Wald test, FDR, P<0.01, n=3 biologically independent replicates, each consisting of RNA pooled from six heads). b, Pie charts depicting all classes of transposable elements in Drosophila, and classes of transposable elements that are increased or decreased in tauR406W transgenic Drosophila. NanoString-based validation of transposable element transcripts that are increased in tauopathy based on RNA-seq (c), and transposable elements transcripts that are decreased in tauR406W transgenic Drosophila based on RNA-seq (d), n=6 biologically independent replicates each consisting of RNA pooled from 6 heads, values are relative to control, which was set to 1. Unpaired, two-tailed Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Values are mean ± s.e.m. All flies are 10 days old. Full genotypes are listed in Supplementary Table 1. Transposable elements recognized by “generic” probes are listed in Supplementary Table 4.
Figure 2 |
Figure 2 |. Loss of function mutations in the flamenco locus enhance tauR406W-induced neurotoxicity.
Compared to tauR406W expressed alone, tauR406W transgenic Drosophila harboring loss of function mutations in the flamenco locus have a, increased neuronal death, based on TUNEL (one-way ANOVA with Tukey’s multiple comparison test) and b, reduced locomotor activity (one-way ANOVA with Tukey’s multiple comparison test), and c, increased activation of the cell cycle based on PCNA staining (one-way ANOVA with Tukey’s multiple comparison test). n=20 animals per genotype, per assay. All flies are 10 days old. Values are mean ± s.e.m. n=20 animals per genotype, per assay, **P=0.005, ***P<0.001, ****P<0.0001. Full genotypes are listed in Supplementary Table 1.
Figure 3 |
Figure 3 |. Decreased expression of piwi and piRNAs mediate pathogenic tauR406W-induced increase in transposable element transcripts and drive neuronal death.
a, Heatmap reflecting fold change of piRNAs that are differentially expressed in tauR406W transgenic Drosophila heads versus controls based on small RNA-seq (two-sided Wald test, FDR, p<0.01, n=4 biologically independent replicates, each consisting of RNA pooled from 6 heads). b, Decreased levels of piwi protein (red) in cortex of the tauR406W transgenic Drosophila brain based on immunostaining and c, western blotting (unpaired, two-tailed Student’s t-test, **P=0.005, n=6 animals per genotype). In b, piwi immunostaining was repeated in 6 animals of each genotype with similar results. Western blot is cropped in c, full blot presented in Supplementary Figure 10. d, NanoString analysis of transposable element expression in response to RNAi-mediated knockdown of piwi versus control (unpaired, two-tailed Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=6 biologically independent replicates each consisting of RNA pooled from 6 heads, values are relative to control, which was set to 1). e, Neuronal death assayed by TUNEL in Drosophila caused by pan-neuronal RNAi-mediated knockdown of piwi (one-way ANOVA with Tukey’s multiple comparison test, ****P<0.0001, n=20 animals per genotype). f, NanoString analysis of transposable element expression in response to pan-neuronal piwi overexpression in tauR406W transgenic Drosophila versus tau expressed alone (unpaired, two-tailed Student’s t-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=6 biologically independent replicates each consisting of RNA pooled from 6 heads, values are relative to tau expressed alone, which was set to 1). g, Neuronal death assayed by TUNEL in tauR406W transgenic Drosophila with pan-neuronal piwi overexpression (one-way ANOVA with Tukey’s multiple comparison test, ****P<0.0001, n=20 animals per genotype). All flies are 10 days old. Values are mean ± s.e.m. Full genotypes are listed in Supplementary Table 1. Transposable elements recognized by “generic” probes are listed in Supplementary Table 4.
Figure 4 |
Figure 4 |. Active mobilization of transposable elements in neurons of tau transgenic Drosophila.
a, Gypsy-TRAP (GFP, green), a reporter of transposable element mobilization, is activated in retinal neurons of 10- and 40-day old tauV337M transgenic Drosophila compared to control. Brains were stained with the cTau antibody (red) to recognize transgenic tau (unpaired, two-tailed Student’s t-test, n=6 animals per genotype, per age, n.s.=not significant, **P=0.004, ***P=0.0001). b, Quantification of gypsy-TRAP activation based on western blotting with an antibody recognizing GFP (unpaired, two-tailed Student’s t-test, n.s.=not significant, **P=0.003, ****P<0.0001). Western blot is cropped, full blot presented in Supplementary Figure 10. n=6 animals per genotype, per age. Values are mean ± s.e.m. Full genotypes are listed in Supplementary Table 1.
Figure 5 |
Figure 5 |. Dietary restriction significantly suppresses tau-induced transposable element mobilization and tau-induced neurotoxicity in Drosophila.
a, 66% dietary restriction reduces gypsy-TRAP reporter activation in retinal neurons of tauV337M transgenic Drosophila based on GFP fluorescence (one-way ANOVA with Tukey’s multiple comparison test, *P=0.03, **P=0.002, n=6 animals per genotype, per treatment) and b, western blotting (unpaired, two-tailed Student’s t-test, n.s.=not significant, *P=0.04, n=6 animals per genotype, per treatment). Western blot is cropped in b, full blot presented in Supplementary Figure 10. c, 66% dietary restriction significantly reduces tauR406W-induced neuronal death based on TUNEL (one-way ANOVA with Tukey’s multiple comparison test, ****P<0.0001, n=20 animals per genotype, per treatment). All flies are 10 days old. Values are mean ± s.e.m. Full genotypes are listed in Supplementary Table 1.
Figure 6 |
Figure 6 |. 3TC (Lamivudine), an FDA-approved nucleoside analog reverse transcriptase inhibitor, suppresses tau-induced transposable element mobilization and tau-induced neurotoxicity in Drosophila.
a, 10 mM 3TC reduces gypsy-TRAP reporter activation in retinal neurons of tauV337M transgenic Drosophila based on GFP fluorescence (one-way ANOVA with Tukey’s multiple comparison test, *P=0.01, **P<0.01, n=6 animals per genotype, per treatment) and b, western blotting (unpaired, two-tailed Student’s t-test, n.s.=not significant, **P=0.003, n=6 animals per genotype, per drug treatment). Western blot is cropped in b, full blot presented in Supplementary Figure 10. c, 10 mM 3TC significantly reduces tauR406W-induced neuronal death based on TUNEL (one-way ANOVA with Tukey’s multiple comparison test, ****P<0.0001, n=20 animals per genotype, per treatment). d, 10 mM 3TC significantly alleviates tauR406W-induced deficits in locomotor activity (one-way ANOVA with Tukey’s multiple comparison test, ***P=0.0008, ****P<0.0001, n=20 animals per genotype, per treatment). All flies are 10 days old. Values are mean ± s.e.m. Full genotypes are listed in Supplementary Table 1.
Figure 7 |
Figure 7 |. Transposable element expression in cortex of human tauopathy.
Heatmaps reflecting fold change of differentially expressed transposable elements in human cortex in a, control versus Alzheimer’s disease (AD), and b, control versus progressive supranuclear palsy (PSP), based on RNA-seq. (two-sided Wald test, FDR, P<0.01). c, Differentially expressed transposable elements in postmortem Alzheimer’s disease and progressive supranuclear palsy cortex compared to controls. HERVs are significantly over-represented among transposable element transcripts that are increased in tauopathy (hypergeometric test, adjusted P=0.004). Non-LTR are significantly over-represented among transposable elements that are decreased in tauopathy (hypergeometric test, adjusted P=4*10−8). d, Principal component analyses of differentially expressed transposable elements in control, Alzheimer’s disease, and progressive supranuclear palsy cortex (two-sided Kolmogorov-Smirnov test, P<10−13). e, Based on principal component analysis of transposable element expression in control versus tauopathy, violin plots show that control samples are relatively farther from the center of the cluster, as defined by the median of Alzheimer’s disease and progressive supranuclear palsy samples. Euclidian distance was computed using the first principal components of those transposable elements that are differentially expressed among the three conditions. For control, AD and PSP respectively, minima=0.8, 0.4, 0.3, maxima=12.3, 11.1, 10.1, median=3.2, 2.2, 2.2, mean=3.8, 2.8, 2.6, 1st quantile=2.5, 1.5, 1.3, 3rd quantile=4.2, 3.4, 3.2. Human control n=21, Alzheimer’s disease n=80, progressive supranuclear palsy n=82 biologically independent replicates in a-e.

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