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Cited by 105Alzheimer's disease research and treatmentsHistone Deacetylase Inhibitors ResearchDementia and Cognitive Impairment Research

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An HDAC6-dependent surveillance mechanism suppresses tau-mediated neurodegeneration and cognitive decline

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Trzeciakiewicz, Hanna, Ajit, Deepa, Tseng, Jui-Heng, Chen, Youjun, Ajit, Aditi, Tabassum, Zarin, Lobrovich, Rebecca, Peterson, Claire, Riddick, Natallia V., Itano, Michelle S., Tripathy, Ashutosh, Moy, Sheryl S., Lee, Virginia M. Y., Trojanowski, John Q., Irwin, David J., Cohen, Todd J.·2020·Nature Communications
DOI 10.1038/s41467-020-19317-4PMC PMC7606452
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Abstract

Tauopathies including Alzheimer’s disease (AD) are marked by the accumulation of aberrantly modified tau proteins. Acetylated tau, in particular, has recently been implicated in neurodegeneration and cognitive decline. HDAC6 reversibly regulates tau acetylation, but its role in tauopathy progression remains unclear. Here, we identified an HDAC6-chaperone complex that targets aberrantly modified tau. HDAC6 not only deacetylates tau but also suppresses tau hyperphosphorylation within the microtubule-binding region. In neurons and human AD brain, HDAC6 becomes co-aggregated within focal tau swellings and human AD neuritic plaques. Using mass spectrometry, we identify a novel HDAC6-regulated tau acetylation site as a disease specific marker for 3R/4R and 3R tauopathies, supporting uniquely modified tau species in different neurodegenerative disorders. Tau transgenic mice lacking HDAC6 show reduced survival characterized by accelerated tau pathology and cognitive decline. We propose that a HDAC6-dependent surveillance mechanism suppresses toxic tau accumulation, which may protect against the progression of AD and related tauopathies.

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Figures

Fig. 1 An HDAC6-chaperone complex targets aberrant tau species. a Co-IP assay evaluating the binding of a panel of human deacetylases to tau in HEK-293A cells by FLAG (FL) pull-down followed by immunoblotting with a total tau (K9JA) antibody. WT (wild-type) tau preferentially interacts with HDAC3, HDAC6, and SIRT1 as indicated. b Co-IP assay to determine HDAC6 interacting domains. WT HDAC6, H216/611A (catalytically dead HDAC6 harboring both H216/H611A mutations), 1–840 (lacking 841–1215), ΔBUZ (lacking 1045–1215), and ΔSE14 (lacking 884–1022) were evaluated. C-terminal mutants (1–840 and ΔSE14) exhibited decreased binding to tau, as quantified in c. Values are means ± SEM of the ratio of IP total tau to input total tau (background subtracted and normalized to WT) with unpaired t-test statistical analysis for each HDAC6 mutant compared to WT HDAC6 (CD, *p = 0.0107; 1–840, **p = 0.0013; ΔBUZ, ns; ΔSE14, **p = 0.0048). d Co-IP assay using tau mutants lacking individual MTBR repeats or the entire MTBR (R1, Q244-K274; R2, V275-S305; R3, V306-Q336; R4, V337-E372; R1-4, Q244-E372). Removal of R2, R3, or complete removal of the MTBR (R1–4) reduced HDAC6 binding. The binding of MTBR mutants to HDAC6 were quantified in e (ΔR1, *p = 0.0498; ΔR2, *p = 0.0125; ΔR3, ***p = 0.0002; ΔR4, ns; ΔR1–4, ****p = <0.0001). f Co-IP assay assessing HDAC6 binding to tau isoforms varying in the number of N-terminal inserts (0N, 1N, or 2N) or MTBR domains (3R or 4R). 3R-tau (0N3R, 1N3R, and 2N3R) and 4R-tau (0N4R, 1N4R, and 2N4R) binding was quantified in g (2N4R vs. 2N3R, *p = 0.0125; 1N4R vs. 1N3R, *p = 0.0435; 0N4R vs. 0N3R, *p = 0.0499). h Co-IP assay assessing HDAC6 binding to FTD disease-causing tau mutations was quantified in i (A125T, ns; G272V, ns; ΔK280, **p = 0.0069; P301L, ****p < 0.0001; L315R, **p = 0.0029; S320F, **p = 0.0013; V337M, ns; K369I, *p = 0.0384). j HDAC6 activity assays were used to evaluate the ability of full-length recombinant WT, P301L, L315R, S320F, and ΔR1–4 tau proteins to inhibit HDAC6 activity, which were normalized to the TSA control. Values are means ± SEM. k Co-IP assay to evaluate tau binding to ectopically over-expressed heat shock proteins (Hsps) including Hsp27, Hsc70, Hsp70, and Hsp90. Only Hsc70 and Hsp70 bound robustly to tau. l Co-IP assay to evaluate HDAC6 binding to WT and P301L (PL) compared to their Hsc/Hsp70 binding-deficient mutants (4Δ and PL4Δ; ΔI277/I278/V308/V309). HDAC6 binding was decreased in the presence of chaperone binding-deficient tau mutations, as quantified in m. n Schematic of the HDAC6-chaperone complex in association with tau. Statistical tests: p value determined by two-sided unpaired t-test (c, e, g, i, j, m) from n = 3 (e, g, i,), n = 4 (j), and n = 5 (m) biologically independent experiments. Representative co-IP assays (a, k) are shown from n = 3 biologically independent experiments. Error bars represent means ± SEM (c, e, g, i, j, m). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.
Fig. 1 An HDAC6-chaperone complex targets aberrant tau species. a Co-IP assay evaluating the binding of a panel of human deacetylases to tau in HEK-293A cells by FLAG (FL) pull-down followed by immunoblotting with a total tau (K9JA) antibody. WT (wild-type) tau preferentially interacts with HDAC3, HDAC6, and SIRT1 as indicated. b Co-IP assay to determine HDAC6 interacting domains. WT HDAC6, H216/611A (catalytically dead HDAC6 harboring both H216/H611A mutations), 1–840 (lacking 841–1215), ΔBUZ (lacking 1045–1215), and ΔSE14 (lacking 884–1022) were evaluated. C-terminal mutants (1–840 and ΔSE14) exhibited decreased binding to tau, as quantified in c. Values are means ± SEM of the ratio of IP total tau to input total tau (background subtracted and normalized to WT) with unpaired t-test statistical analysis for each HDAC6 mutant compared to WT HDAC6 (CD, *p = 0.0107; 1–840, **p = 0.0013; ΔBUZ, ns; ΔSE14, **p = 0.0048). d Co-IP assay using tau mutants lacking individual MTBR repeats or the entire MTBR (R1, Q244-K274; R2, V275-S305; R3, V306-Q336; R4, V337-E372; R1-4, Q244-E372). Removal of R2, R3, or complete removal of the MTBR (R1–4) reduced HDAC6 binding. The binding of MTBR mutants to HDAC6 were quantified in e (ΔR1, *p = 0.0498; ΔR2, *p = 0.0125; ΔR3, ***p = 0.0002; ΔR4, ns; ΔR1–4, ****p = <0.0001). f Co-IP assay assessing HDAC6 binding to tau isoforms varying in the number of N-terminal inserts (0N, 1N, or 2N) or MTBR domains (3R or 4R). 3R-tau (0N3R, 1N3R, and 2N3R) and 4R-tau (0N4R, 1N4R, and 2N4R) binding was quantified in g (2N4R vs. 2N3R, *p = 0.0125; 1N4R vs. 1N3R, *p = 0.0435; 0N4R vs. 0N3R, *p = 0.0499). h Co-IP assay assessing HDAC6 binding to FTD disease-causing tau mutations was quantified in i (A125T, ns; G272V, ns; ΔK280, **p = 0.0069; P301L, ****p < 0.0001; L315R, **p = 0.0029; S320F, **p = 0.0013; V337M, ns; K369I, *p = 0.0384). j HDAC6 activity assays were used to evaluate the ability of full-length recombinant WT, P301L, L315R, S320F, and ΔR1–4 tau proteins to inhibit HDAC6 activity, which were normalized to the TSA control. Values are means ± SEM. k Co-IP assay to evaluate tau binding to ectopically over-expressed heat shock proteins (Hsps) including Hsp27, Hsc70, Hsp70, and Hsp90. Only Hsc70 and Hsp70 bound robustly to tau. l Co-IP assay to evaluate HDAC6 binding to WT and P301L (PL) compared to their Hsc/Hsp70 binding-deficient mutants (4Δ and PL4Δ; ΔI277/I278/V308/V309). HDAC6 binding was decreased in the presence of chaperone binding-deficient tau mutations, as quantified in m. n Schematic of the HDAC6-chaperone complex in association with tau. Statistical tests: p value determined by two-sided unpaired t-test (c, e, g, i, j, m) from n = 3 (e, g, i,), n = 4 (j), and n = 5 (m) biologically independent experiments. Representative co-IP assays (a, k) are shown from n = 3 biologically independent experiments. Error bars represent means ± SEM (c, e, g, i, j, m). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 HDAC6 co-aggregates with tau and accumulates in pathological tau lesions. a Primary cortical neurons from mouse embryos (E16) of tau knockout (KO) mice were cultured for 10 days in vitro (10 DIV) and transfected with P301L Tau-GFP (green) and HDAC6-FLAG (FL) (red) expression constructs followed by imaging. Arrows indicate regions of extensive co-localization along processes. Scale bar = 50 μm. b Primary cortical neurons (E16) from WT (top) and PS19 tau transgenic (bottom) mice were analyzed by immunofluorescence (IF), detecting co-localized focal swellings of tau (green), endogenous HDAC6 (red), and the nuclei marked by DAPI (blue). The white rectangle highlights a magnified insert of a neuronal process harboring tau-positive foci. Scale bar = 50 μm. c Biochemical fractionation of soluble high-salt fractions and insoluble urea fractions were extracted from frontal cortex of control and AD brains followed by immunoblotting to detect HDAC6, tau, Hsc70, and acetylated tubulin. The arrows highlight acetylated tubulin fragments ~37 kDa and higher molecular weight species ~150 kDa. d, e Immunoblotting results were quantified by densitometry. f AD cortical sections were analyzed by double-labeling IF using either conformational tau (MC1) or total tau (TAU-5) antibodies (blue), combined with Thioflavin S (ThS) (green), and HDAC6 (red) to evaluate localization in proximity to AD neuritic plaques. White arrows highlight regions of tau and HDAC6 immunoreactivity that are associated with ThS positive plaques. Scale bar = 50 μm. Statistical tests: p value determined by two-sided unpaired t-test with Welch’s correction from n = 5 biologically independent samples (d, e). Representative neurons are shown from n = 3 biologically independent experiments (a, b) and n = 3 independent AD brains (f). Error bars represent means ± SEM and are plotted relative to control (d) or relative to AD (e). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.
Fig. 2 HDAC6 co-aggregates with tau and accumulates in pathological tau lesions. a Primary cortical neurons from mouse embryos (E16) of tau knockout (KO) mice were cultured for 10 days in vitro (10 DIV) and transfected with P301L Tau-GFP (green) and HDAC6-FLAG (FL) (red) expression constructs followed by imaging. Arrows indicate regions of extensive co-localization along processes. Scale bar = 50 μm. b Primary cortical neurons (E16) from WT (top) and PS19 tau transgenic (bottom) mice were analyzed by immunofluorescence (IF), detecting co-localized focal swellings of tau (green), endogenous HDAC6 (red), and the nuclei marked by DAPI (blue). The white rectangle highlights a magnified insert of a neuronal process harboring tau-positive foci. Scale bar = 50 μm. c Biochemical fractionation of soluble high-salt fractions and insoluble urea fractions were extracted from frontal cortex of control and AD brains followed by immunoblotting to detect HDAC6, tau, Hsc70, and acetylated tubulin. The arrows highlight acetylated tubulin fragments ~37 kDa and higher molecular weight species ~150 kDa. d, e Immunoblotting results were quantified by densitometry. f AD cortical sections were analyzed by double-labeling IF using either conformational tau (MC1) or total tau (TAU-5) antibodies (blue), combined with Thioflavin S (ThS) (green), and HDAC6 (red) to evaluate localization in proximity to AD neuritic plaques. White arrows highlight regions of tau and HDAC6 immunoreactivity that are associated with ThS positive plaques. Scale bar = 50 μm. Statistical tests: p value determined by two-sided unpaired t-test with Welch’s correction from n = 5 biologically independent samples (d, e). Representative neurons are shown from n = 3 biologically independent experiments (a, b) and n = 3 independent AD brains (f). Error bars represent means ± SEM and are plotted relative to control (d) or relative to AD (e). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 AD and related tauopathies present with distinct tau acetylation profiles. a Immunoblotting of control and AD brains homogenates using antibodies to detect ac-K311, total tau (TAU-5), and GAPDH. b Immunoblots from a were quantified and normalized to control (soluble) or AD (insoluble) samples. c Immunohistochemistry (IHC) analysis using ac-K311 (#5089) in a panel of 3R and 4R tauopathies: Alzheimer’s disease (AD), Pick’s disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). Only 3R-tauopathies (AD and PiD) demonstrated ac-K311-immunoreactive pathological lesions. Scale bar = 50 μm. d Double-labeling IF to mark ac-K311 (red) in combination with tau-isoform specific antibodies. The green channel represents 4R-tau (RD4) in AD, CBD, and PSP or 3R-tau (RD3) in AD and PiD. Ac-K311 colocalized strongly to 3R-tau pathology in AD and PiD. Scale bar = 50 μm. e Immunoblotting of isolated PHF-tau from the indicated tauopathies probed with ac-K311, 3R-tau (RD3), 4R-tau (RD4), and total tau (K9JA) antibodies. The arrows highlighting tau bands 1–5 represent distinctly migrating tau bands that are resolved by immunoblotting. Statistical tests: p value determined by unpaired t-test with Welch’s correction from n = 5 independent samples (b). Representative images are shown from n = 10 (c, d) and n = 2 (e) independent samples. Error bars represent means ± SEM. *p < 0.05 and **p < 0.01. Source data are provided as a Source Data file.
Fig. 3 AD and related tauopathies present with distinct tau acetylation profiles. a Immunoblotting of control and AD brains homogenates using antibodies to detect ac-K311, total tau (TAU-5), and GAPDH. b Immunoblots from a were quantified and normalized to control (soluble) or AD (insoluble) samples. c Immunohistochemistry (IHC) analysis using ac-K311 (#5089) in a panel of 3R and 4R tauopathies: Alzheimer’s disease (AD), Pick’s disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). Only 3R-tauopathies (AD and PiD) demonstrated ac-K311-immunoreactive pathological lesions. Scale bar = 50 μm. d Double-labeling IF to mark ac-K311 (red) in combination with tau-isoform specific antibodies. The green channel represents 4R-tau (RD4) in AD, CBD, and PSP or 3R-tau (RD3) in AD and PiD. Ac-K311 colocalized strongly to 3R-tau pathology in AD and PiD. Scale bar = 50 μm. e Immunoblotting of isolated PHF-tau from the indicated tauopathies probed with ac-K311, 3R-tau (RD3), 4R-tau (RD4), and total tau (K9JA) antibodies. The arrows highlighting tau bands 1–5 represent distinctly migrating tau bands that are resolved by immunoblotting. Statistical tests: p value determined by unpaired t-test with Welch’s correction from n = 5 independent samples (b). Representative images are shown from n = 10 (c, d) and n = 2 (e) independent samples. Error bars represent means ± SEM. *p < 0.05 and **p < 0.01. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 The targeting of tau by HDAC6 is regulated by tau kinases. a Immunoblotting of HEK-293A cells cotransfected for 48 h with WT tau, the acetyltransferase CBP, and WT HDAC6 (where indicated) using acetylation or phosphorylation specific tau antibodies. b–d The relative levels of modified tau within the MTBR (ac-280, ac-K311, p-S262, and p-S356) or outside the MTBR (AT8, p-S202, p-T205, and p-S396) were quantified by densitometry of tau PTMs normalized to total tau (Tau 12). e Cells expressing the indicated tau constructs (WT, 4Δ, PL, and PL4Δ) were treated with tubastatin A (TBST) to inhibit HDAC6 or cotransfected with WT HDAC6 to suppress tau acetylation followed by immunoblotting with acetylation or phosphorylation specific tau antibodies as in a. f–l Primary cortical neurons (E16) from PS19 derived tau-transgenic mice cultured for ten DIV were treated overnight with TBST at a concentration of 1–5 μM and analyzed by immunoblotting followed by quantification by densitometry of tau PTMs normalized to human, mouse, or total tau (K9JA). The arrows in f highlight overexpressed human tau migrating at ~65 kDa compared to mouse tau at ~50 kDa (see also Supplementary Fig. 6c to distinguish between human and mouse tau). m Co-IP assay evaluating HDAC6 binding to WT tau, tau-P301L, and tau-P301L/4SE mutant containing S → E substitutions at all four MARK2-targeted phosphorylation sites (S262/S290/S324/S356E). Despite P301L tau showing greater binding to HDAC6, the addition of the MARK2 phosphorylation-mimic mutations reduced HDAC6 binding. n Co-IP assay evaluating tau-P301L mutant tau binding to HDAC6 in the presence of MARK2 or GSK-3β kinases. o HEK-293A cells co-transfected with WT tau, MARK2, or GSK-3β (where indicated) were analyzed by immunoblotting using acetylation or phosphorylation specific tau antibodies followed by quantification of ac-K311 and ac-Tubulin levels normalized to GAPDH levels in p. q HEK-293A cells co-transfected with WT and MARK2 were treated with a panel of HDAC inhibitors including tubastatin A (TBST, HDAC6 inhibitor, 5 μM), trichostatin A (TSA, class I/II HDAC inhibitor, 1 μM) or nicotinamide (NCA, class III HDAC inhibitor, 5 mM) and analyzed as described in o. Statistical tests: p value determined by two-sided unpaired t-test from n = 6 (b–d), n = 3 (g–l), and n = 4 (p) biologically independent experiments. Error bars represent means ± SEM (b–d, g–l, p). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.
Fig. 4 The targeting of tau by HDAC6 is regulated by tau kinases. a Immunoblotting of HEK-293A cells cotransfected for 48 h with WT tau, the acetyltransferase CBP, and WT HDAC6 (where indicated) using acetylation or phosphorylation specific tau antibodies. b–d The relative levels of modified tau within the MTBR (ac-280, ac-K311, p-S262, and p-S356) or outside the MTBR (AT8, p-S202, p-T205, and p-S396) were quantified by densitometry of tau PTMs normalized to total tau (Tau 12). e Cells expressing the indicated tau constructs (WT, 4Δ, PL, and PL4Δ) were treated with tubastatin A (TBST) to inhibit HDAC6 or cotransfected with WT HDAC6 to suppress tau acetylation followed by immunoblotting with acetylation or phosphorylation specific tau antibodies as in a. f–l Primary cortical neurons (E16) from PS19 derived tau-transgenic mice cultured for ten DIV were treated overnight with TBST at a concentration of 1–5 μM and analyzed by immunoblotting followed by quantification by densitometry of tau PTMs normalized to human, mouse, or total tau (K9JA). The arrows in f highlight overexpressed human tau migrating at ~65 kDa compared to mouse tau at ~50 kDa (see also Supplementary Fig. 6c to distinguish between human and mouse tau). m Co-IP assay evaluating HDAC6 binding to WT tau, tau-P301L, and tau-P301L/4SE mutant containing S → E substitutions at all four MARK2-targeted phosphorylation sites (S262/S290/S324/S356E). Despite P301L tau showing greater binding to HDAC6, the addition of the MARK2 phosphorylation-mimic mutations reduced HDAC6 binding. n Co-IP assay evaluating tau-P301L mutant tau binding to HDAC6 in the presence of MARK2 or GSK-3β kinases. o HEK-293A cells co-transfected with WT tau, MARK2, or GSK-3β (where indicated) were analyzed by immunoblotting using acetylation or phosphorylation specific tau antibodies followed by quantification of ac-K311 and ac-Tubulin levels normalized to GAPDH levels in p. q HEK-293A cells co-transfected with WT and MARK2 were treated with a panel of HDAC inhibitors including tubastatin A (TBST, HDAC6 inhibitor, 5 μM), trichostatin A (TSA, class I/II HDAC inhibitor, 1 μM) or nicotinamide (NCA, class III HDAC inhibitor, 5 mM) and analyzed as described in o. Statistical tests: p value determined by two-sided unpaired t-test from n = 6 (b–d), n = 3 (g–l), and n = 4 (p) biologically independent experiments. Error bars represent means ± SEM (b–d, g–l, p). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 HDAC6 depletion in tauopathy mice reduces survival and accelerates cognitive decline. a Survival analysis showed a significantly reduced survival rate in PS19 tau transgenic mice with HDAC6 deletion (PHK). Two-sided log-rank (Mantel-Cox) test with multiple comparisons was performed to assess significance among genotypes (WT, black squares, n = 22; HDAC6 KO, blue circles, n = 42; PS19, red triangles, n = 27; PHK, purple diamonds, n = 37) (*p < 0.05, ****p < 0.0001). b Decreased body weights in PS19 and PHK mice were observed at 32 weeks and 37 weeks of age (WT, n = 11; HDAC6 KO, n = 15; PS19, n = 15; PHK, n = 13). Comparisons among genotypes were as follows: *p < 0.05, **p < 0.01, ***p < 0.001, comparison to same measure in WT; ΔΔ, p < 0.01, ΔΔΔ, p < 0.001, comparison to HDAC6 KO. c No genotype differences were observed in average swimming speed across 9 days of training. d Acquisition of spatial learning using Morris water maze was evaluated for all genotypes (7–8-month old male mice). Data are means ± SEM from four trials per day. PHK mice showed impaired learning. p-values represent within-genotype repeated measures ANOVAs, effect of training day. WT, ****p < 0.0001; HDAC6 KO, ****p < 0.0001; PS19, **p = 0.0052; PHK, p = 0.4251. Asterisks represent comparisons to WT. e Quadrant preference data are means ± SEM from a 1-min probe trail without the platform following hidden platform training. Target indicates the quadrant where the platform had been placed during training, versus the opposite quadrant. Brackets signify within-genotype comparisons, effect of quadrant (#p < 0.05; ###p < 0.001; ns not significant). Comparisons among genotypes were as follows: **p < 0.01, ***p < 0.001, comparison to same measure in WT; ΔΔΔ, p < 0.001, comparison to HDAC6 KO; ϕϕ, p < 0.01 comparison to PS19. f Swim path crosses during a 1 min probe trial without the platform following hidden platform training. Target indicates the location where the platform had been placed during training, versus the corresponding location in the opposite quadrant. Within-genotype comparisons were used to determine effect of quadrant (##p < 0.01; ns not significant). Comparisons among genotypes were as follows: *p < 0.05, **p < 0.01, comparison to same measure in WT; Δ, p < 0.05, comparison to HDAC6 KO. Statistical tests: p value determined by repeated measure ANOVAs followed by Fisher’s protected least-significant difference (PLSD) posthoc test from WT (n = 11), HDAC6 KO (n = 16), PS19 (n = 15), and PHK (n = 13) mice (c–f). All error bars throughout this figure represent means ± SEM (b–f). Source data are provided as a Source Data file.
Fig. 5 HDAC6 depletion in tauopathy mice reduces survival and accelerates cognitive decline. a Survival analysis showed a significantly reduced survival rate in PS19 tau transgenic mice with HDAC6 deletion (PHK). Two-sided log-rank (Mantel-Cox) test with multiple comparisons was performed to assess significance among genotypes (WT, black squares, n = 22; HDAC6 KO, blue circles, n = 42; PS19, red triangles, n = 27; PHK, purple diamonds, n = 37) (*p < 0.05, ****p < 0.0001). b Decreased body weights in PS19 and PHK mice were observed at 32 weeks and 37 weeks of age (WT, n = 11; HDAC6 KO, n = 15; PS19, n = 15; PHK, n = 13). Comparisons among genotypes were as follows: *p < 0.05, **p < 0.01, ***p < 0.001, comparison to same measure in WT; ΔΔ, p < 0.01, ΔΔΔ, p < 0.001, comparison to HDAC6 KO. c No genotype differences were observed in average swimming speed across 9 days of training. d Acquisition of spatial learning using Morris water maze was evaluated for all genotypes (7–8-month old male mice). Data are means ± SEM from four trials per day. PHK mice showed impaired learning. p-values represent within-genotype repeated measures ANOVAs, effect of training day. WT, ****p < 0.0001; HDAC6 KO, ****p < 0.0001; PS19, **p = 0.0052; PHK, p = 0.4251. Asterisks represent comparisons to WT. e Quadrant preference data are means ± SEM from a 1-min probe trail without the platform following hidden platform training. Target indicates the quadrant where the platform had been placed during training, versus the opposite quadrant. Brackets signify within-genotype comparisons, effect of quadrant (#p < 0.05; ###p < 0.001; ns not significant). Comparisons among genotypes were as follows: **p < 0.01, ***p < 0.001, comparison to same measure in WT; ΔΔΔ, p < 0.001, comparison to HDAC6 KO; ϕϕ, p < 0.01 comparison to PS19. f Swim path crosses during a 1 min probe trial without the platform following hidden platform training. Target indicates the location where the platform had been placed during training, versus the corresponding location in the opposite quadrant. Within-genotype comparisons were used to determine effect of quadrant (##p < 0.01; ns not significant). Comparisons among genotypes were as follows: *p < 0.05, **p < 0.01, comparison to same measure in WT; Δ, p < 0.05, comparison to HDAC6 KO. Statistical tests: p value determined by repeated measure ANOVAs followed by Fisher’s protected least-significant difference (PLSD) posthoc test from WT (n = 11), HDAC6 KO (n = 16), PS19 (n = 15), and PHK (n = 13) mice (c–f). All error bars throughout this figure represent means ± SEM (b–f). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 6 HDAC6 depletion accelerates tau pathology and neurodegeneration. Representative IHC images of pathological tau detected with the MC1 (a), phosphorylated tau (AT8) (b), and total tau (Tau 12) (c) antibodies in the cortex (top panels) and hippocampus (bottom panels) of 12-month old WT, PS19, and PHK mice. Representative images are shown from n = 3 mice per genotype. Scale bar = 50 μm (a–c, scale bar is identical for both hippocampus and cortex). d ThS-positive tau pathology (green) is double-labeled with total tau (TAUY9, red) and DAPI (blue) to highlight cortical amyloid burden. The high magnification inset depicts a neuron with robust tau pathology. e Quantification of MC1 and AT8 immunoreactivity in the hippocampus and ThS-positive inclusions in the cortex (n = 3 mice per genotype). Error bars represent means ± SEM. Statistical analysis was performed using two-sided unpaired t-test and normalized to PHK. f Quantification of hippocampal area using total hippocampus, dentate gyrus, and CA brain area from 12-month-old mice (n = 4 mice per genotype). Measurements were performed in an unbiased manner using ImageJ software. Statistical tests: one-way ANOVAs were performed with Tukey’s test for multiple comparisons among groups. Data shown represent means ± SEM normalized to WT. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.
Fig. 6 HDAC6 depletion accelerates tau pathology and neurodegeneration. Representative IHC images of pathological tau detected with the MC1 (a), phosphorylated tau (AT8) (b), and total tau (Tau 12) (c) antibodies in the cortex (top panels) and hippocampus (bottom panels) of 12-month old WT, PS19, and PHK mice. Representative images are shown from n = 3 mice per genotype. Scale bar = 50 μm (a–c, scale bar is identical for both hippocampus and cortex). d ThS-positive tau pathology (green) is double-labeled with total tau (TAUY9, red) and DAPI (blue) to highlight cortical amyloid burden. The high magnification inset depicts a neuron with robust tau pathology. e Quantification of MC1 and AT8 immunoreactivity in the hippocampus and ThS-positive inclusions in the cortex (n = 3 mice per genotype). Error bars represent means ± SEM. Statistical analysis was performed using two-sided unpaired t-test and normalized to PHK. f Quantification of hippocampal area using total hippocampus, dentate gyrus, and CA brain area from 12-month-old mice (n = 4 mice per genotype). Measurements were performed in an unbiased manner using ImageJ software. Statistical tests: one-way ANOVAs were performed with Tukey’s test for multiple comparisons among groups. Data shown represent means ± SEM normalized to WT. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 HDAC6 depletion accelerates tau pathology and causes synaptic dysfunction. a Immunoblotting of soluble high salt fractions from 12-month-old WT, PS19, and PHK cortex to evaluate the following: acetylated tau (ac-K280 and ac-K311), phosphorylated tau in the MTBR (p-S262 and p-S356), nonMTBR phosphorylated tau (AT8, p-S202, and p-S396), cleaved tau (TauC3), endogenous mouse tau (TAU-5, ~50 kDa), human tau (TAU-5 and Tau 12, ~65 kDa), HDAC6, and the HDAC6 substrate ac-tubulin. b Similar immunoblotting as in a was performed using insoluble SDS fractions. Acetylated tau (ac-K280 and ac-K311), phosphorylated tau in the MTBR (p-S262) and total tau (TAU-5) were detected in the insoluble fractions. c, d Dot blot analysis was used to detect oligomeric tau in soluble (c) and insoluble (d) fractions with the TOC1 antibody. e Synaptic markers including postsynaptic (PSD-95), NMDA receptors (p-NR2B and total NR2B), and presynaptic (Synaptophysin) were evaluated by immunoblotting. f–i Quantification of acetylated tau (f), conformational tau (g), phosphorylated tau (h), and synaptic markers (i) were determined by densitometry and values were normalized relative to PHK mice (f–h) or WT mice (i). Statistical tests: one-way ANOVAs were performed (p-values are listed above graphs) and Tukey’s multiple comparison tests were used to compare among groups. Representative immunoblotting data are shown from n = 3 biologically independent samples (a–e). Error bars represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Source data are provided as a Source Data file.
Fig. 7 HDAC6 depletion accelerates tau pathology and causes synaptic dysfunction. a Immunoblotting of soluble high salt fractions from 12-month-old WT, PS19, and PHK cortex to evaluate the following: acetylated tau (ac-K280 and ac-K311), phosphorylated tau in the MTBR (p-S262 and p-S356), nonMTBR phosphorylated tau (AT8, p-S202, and p-S396), cleaved tau (TauC3), endogenous mouse tau (TAU-5, ~50 kDa), human tau (TAU-5 and Tau 12, ~65 kDa), HDAC6, and the HDAC6 substrate ac-tubulin. b Similar immunoblotting as in a was performed using insoluble SDS fractions. Acetylated tau (ac-K280 and ac-K311), phosphorylated tau in the MTBR (p-S262) and total tau (TAU-5) were detected in the insoluble fractions. c, d Dot blot analysis was used to detect oligomeric tau in soluble (c) and insoluble (d) fractions with the TOC1 antibody. e Synaptic markers including postsynaptic (PSD-95), NMDA receptors (p-NR2B and total NR2B), and presynaptic (Synaptophysin) were evaluated by immunoblotting. f–i Quantification of acetylated tau (f), conformational tau (g), phosphorylated tau (h), and synaptic markers (i) were determined by densitometry and values were normalized relative to PHK mice (f–h) or WT mice (i). Statistical tests: one-way ANOVAs were performed (p-values are listed above graphs) and Tukey’s multiple comparison tests were used to compare among groups. Representative immunoblotting data are shown from n = 3 biologically independent samples (a–e). Error bars represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Source data are provided as a Source Data file.CC BY · PubMed Central

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Authors
Trzeciakiewicz, H., Ajit, D., Tseng, J.-H., Chen, Y., Ajit, A., Tabassum, Z., Lobrovich, R., Peterson, C., Riddick, N. V., Itano, M. S., Tripathy, A., Moy, S. S., Lee, V. M. Y., Trojanowski, J. Q., Irwin, D. J., & Cohen, T. J.
Affiliations
  • University of North Carolina at Chapel Hill (US)
  • University of Pennsylvania (US)
Corresponding author
Cohen, Todd J.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-11-02
Volume
11
Issue
1
Pages
5522-5522
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-19317-4
PMC
PMC7606452
OpenAlex
W3096112623
Cited by
102
References
65
Citations by year
2026: 11 · 2025: 21 · 2024: 21 · 2023: 18 · 2022: 22 · 2021: 9
FWCI
7.67
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Carolina Digital Repository (University of North Carolina at Chapel Hill) (submittedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UNC Libraries
Funders
  • U.S. Department of Health & Human Services | NIH | National Center for Advancing Translational Sciences (UL1TR001111)
  • Alzheimer’s Association (NIRG-14-321219)
  • Association for Frontotemporal Degeneration (-)
  • U.S. Department of Health & Human Services | NIH | National Institute on Aging (R21AG058080)
  • CurePSP (656-2018-06)
  • American Federation for Aging Research (RAG15247)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Histone Deacetylase Inhibitors Research
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
HDAC6, tau acetylation, tauopathy, Alzheimer's disease, tau hyperphosphorylation, protein deacetylation, chaperone complex, tau aggregation, neuritic plaques, cognitive decline, tau transgenic mice, neurodegeneration
MeSH terms
Histone Deacetylase 6, Acetylation, Aged, Aged, 80 and over, Animals, Brain, Disease Models, Animal, Female, Humans, Male, Mice, Transgenic, Middle Aged, Phosphorylation, Protein Processing, Post-Translational, tau Proteins, Disease Progression, Tauopathies, Mice, Cognitive Dysfunction

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