TSC1 loss increases risk for tauopathy by inducing tau acetylation and preventing tau clearance via chaperone-mediated autophagy

Abstract

Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions are known as tauopathies. We previously linked a loss-of-function mutation in the TSC1 gene to tau accumulation and frontotemporal lobar degeneration. Now, we have identified genetic variants in TSC1 that decrease TSC1/hamartin levels and predispose to tauopathies such as Alzheimer’s disease and progressive supranuclear palsy. Cellular and murine models of TSC1 haploinsufficiency, as well as human brains carrying a TSC1 risk variant, accumulated tau protein that exhibited aberrant acetylation. This acetylation hindered tau degradation via chaperone-mediated autophagy, thereby leading to its accumulation. Aberrant tau acetylation in TSC1 haploinsufficiency resulted from the dysregulation of both p300 acetyltransferase and SIRT1 deacetylase. Pharmacological modulation of either enzyme restored tau levels. This study substantiates TSC1 as a novel tauopathy risk gene and includes TSC1 haploinsufficiency as a genetic model for tauopathies. In addition, these findings promote tau acetylation as a rational target for tauopathy therapeutics and diagnostic.

Fig. 1.. Genetic variants in TSC1 are overrepresented in tauopathy patients.

(A) TSC1 genetic variants found in PSP subjects, compared to healthy control individuals from the ADSP database. (B) Representation of TSC1/hamartin protein with the PEST motif indicated in blue and the disease-associated variants in red. (C) Immunoblots showing TSC1-FLAG levels after the CHX time-course treatment. The plot and the table represent TSC1/hamartin half-life. (D) Immunoblots showing TSC1-FLAG levels at 8 and 24 hours after CHX and MG132 treatments. (E) Quantification of the effect of proteasome in the clearance of WT and mutant TSC1. (F) Immunoblot showing decreased TSC1/hamartin levels in the brain of PSP subjects carrying the G1035S variant in TSC1 gene. (G and H) TSC1/hamartin and tau levels in iNeurons derived from a family carrying a TSC1 LOF mutation (p.Arg22CysfsTer5) (G) and isogenic TSC1^+/− induced pluripotent stem cells (iPSCs) generated in the background of the PGP1 line (H). (G) Immunoblots show two representative individuals (MHF130 WT and MHF104 mutant). The quantification is the mean ± SEM of four independent experiments including two WT (MHF130 and F11350) and three mutant lines (MH104, MH128, and MHF129). (H) Plot shows the mean ± SEM of four independent experiments. Statistical significance was assessed by Student’s t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 2.. TSC1/hamartin haploinsufficiency induces tau accumulation independent of macroautophagy impairment and mTORC1 pathway activation.

(A) Plot showing the number of LC3-II puncta per cell in TSC1^+/+ and TSC1^+/− fibroblasts. Total autophagic vesicles consist of autophagosomes and autolysosomes. n.s., not significant. (B) Anti-LC3 immunoblot showing LC3-I and -II levels before and after 6 hours of treatment with 100 nM bafilomycin 1 (Baf1) in control or TSC1^+/−-differentiated SH-SY5Y cells. (C) Immunoblots showing tau levels before and after 72 hours of treatment with rapamycin (Rap, 25 nM) or Torin-1 (50 nM) in WT and TSC1^+/−-differentiated SH-SY5Y cells. All plots show the means ± SEM from at least three independent experiments. Two-way analysis of variance (ANOVA) tests followed by Bonferroni’s comparison was used to assess statistical significance. Unless otherwise indicated, P values indicate comparison to the first, leftmost column in the graph.

Fig. 3.. Loss of TSC1/hamartin impairs tau degradation in the lysosomes by CMA.

(A) Tau mRNA levels in WT and TSC1^+/−-differentiated SH-SY5Y cells measured by qPCR. (B and C) Control and TSC1^+/−-differentiated SH-SY5Y cells (B) and iNeurons (C) immunostained with antibodies against tau (green) and the lysosomal marker Lamp2A (purple). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The imaging experiments were performed in triplicates. Plots show the average of Pearson’s R correlation value for both channels and the two tau antibodies. The statistical significance was assessed by Student’s t test. (D) CMA active (+) and CMA inactive (−) lysosomal fractions were isolated from control and TSC1^+/−-differentiated SH-SY5Y cells. Representative immunoblots showing the distribution of tau protein in the lysosomal compartments are shown. Lamp-1 and Hsc70 were used as markers for CMA⁻ lysosomes and CMA⁺ lysosomes, respectively. Plot shows the means ± SEM of tau levels from two independent experiments. (E) Tau protein was immunoprecipitated from TSC1^+/+ and TSC1^+/− cells using a monoclonal anti-tau antibody (HT7). The plot shows the quantification of Hsc70/tau coimmunoprecipitation. The experiment was performed three times. Student’s t test was used to assess statistical significance.

Fig. 4.. TSC1-mediated tau acetylation alters the association of tau with CMA lysosomes.

(A) List of the acetylated lysine (K) residues found by mass spectrometry in TSC1^+/− but not WT SH-SY5Y cells. The expectation value (e-value), a measure of the statistical significance of a peptide assignment, is also shown. (B) Diagram of tau protein annotated with the functional domains, KFERQ motifs, and the acetyl-lysine residues. (C) SH-SY5Y cells expressing FLAG-tagged WT 2N4R tau, acetyl-mimetic tau (2N4R 6K➔Q tau), and non-acetylatalable tau (2N4R 6K➔R tau) were stained with anti-FLAG and anti-Lamp2A antibodies that recognized overexpressed tau (green) and endogenous Lamp2A (dark blue), respectively. Nuclei were stained with DAPI (cyan). Plot shows the average of Pearson’s R correlation value. Statistical significance was assessed using one-way ANOVA. (D) Acetyl-mimetic and non-acetylatable tau was immunoprecipitated using anti-FLAG magnetic beads. The immunoblots show the levels of Hsc70 and tau proteins in the whole lysate (input) and after the immunoprecipitation. Immunoglobulin (Ig) heavy chain and tubulin were used as loading controls for the immunoprecipitation and input, respectively. The plot shows the quantification of Hsc70/tau coimmunoprecipitation. The experiment was performed three times. Student’s t test was used to assess statistical significance.

Fig. 5.. TSC1/hamartin insufficiency regulates p300 HAT activity and SIRT1 HDAC levels in SH-SY5Y cells and iNeurons.

(A) p300 HAT activity in control and TSC1^+/−-differentiated SH-SY5Y cells indirectly assessed by the levels of histone 3 (H3) acetylated at lysine 18 (H3^acK18). (B) Enzymatic activity of HAT in differentiated SH-SY5Y cells before and after the treatment with a specific p300 inhibitor. (C and D) SIRT1 protein (C) and mRNA levels (D) measured in control and TSC1^+/−-differentiated SH-SY5Y cells. (E) Immunoblot showing tau acetylation in TSC1^+/+ and TSC1^+/− iPSC-derived iNeurons. (F and G) Immunoblots showing the p300 activity, assessed by the levels of H3^acK18, and SIRT1 levels in TSC1^+/+ and TSC1^+/− iPSC-derived iNeurons. All experiments were performed at least in triplicate. Plots show the means ± SEM for each experiment. Statistical significance was assessed using Student’s t test or two-way ANOVA followed by Bonferroni’s comparison.

Fig. 6.. Blocking tau acetylation restores tau levels in TSC1^+/− cells.

(A) Control and TSC1^+/− differentiated SH-SY5Y cells were treated for 72 hours with a specific p300 inhibitor, A485 (15 μM), or with the SIRT1 activator, resveratrol (RSV) (25 μM). Immunoblots show the effect of these treatments on tau acetylation, phosphorylation, and total tau levels. DMSO, dimethyl sulfoxide. (B) Effect of p300 inhibition on SIRT1 levels in control and TSC1^+/−-differentiated SH-SY5Y cells. (D) Control and TSC1^+/− cells were treated for 72 hours with AZD1080 (0.5 μM), a specific phospho-Tau inhibitor. Blots represent the effect of AZD1080 in the phosphorylation status and levels of tau protein. The plot below shows the quantification and statistics of five independent experiments. (C) Control and TSC1^+/− cells were treated with Torin-1. Seventy-two hours later, cells were lysed and the tau acetylation status was assessed by immunoblot. Experiments were performed at least in triplicate. Plots show the means ± SEM for each experiment. Statistical significance was assessed using two-way ANOVA followed by Bonferroni’s correction.

Fig. 7.. TSC1/hamartin insufficiency promotes tau acetylation and neurodegeneration in mice.

(A) PHF1 tau immunostaining performed in the RSCx of Tsc1^Syn1CKO and control mice, at 6 and 15 months old. Plots represent the percentage of positive tau staining. (B and C) Auditory conditioning test (B) and nesting behavior score (C) performed in 15-month-old mice. (D) Immunoblots showing tau levels, total and acetylated at lysine 343, in the RSCx of 15-month-old mice. The banding pattern was compared to that of the tau ladder, which contains all six recombinant tau isoforms. The HT7 antibody recognized the six tau isoforms whereas the AcK343 antibody only recognized 1N3R and 0N4R isoforms. (E) Representative images of the RSCx of 15-month-old mice stained with the AcK343 antibody. The plot represents the means ± SEM of the percentage of the area stained. (F) p300 HAT activity, assessed by the acetylation of H3 at K18, and SIRT1 levels were measured in the RSCx of 15-month-old mice. Plots show the means ± SEM of the protein levels. Ten control and 16 Tsc1^Syn1CKO mice were used for immunostainings/immunoblots. Behavioral tests were performed in 8 control and 11 Tsc1^Syn1CKO mice. Student’s t test or two-way ANOVA followed by Bonferroni’s correction were used to assess statistical significance.

Fig. 8.. TSC1/hamartin insufficiency promotes p300/SIRT1 dysregulation and tau acetylation in human brains.

Protein was extracted from the inferior frontal cortex of four control subjects (C1 to C4) and four individuals with PSP (P1 to P4). The immunoblots show the protein levels of TSC1 and SIRT1 and the acetylation status of tau. The activity of p300 was measured indirectly by the acetylation status of H3 at K18. GAPDH was used as loading control. The plots show the relative levels of the proteins assessed by immunoblot. P3 and P4 individuals (red dots) exhibited lower levels of TSC1 protein that correlated with increased p300 activity, decreased SIRT1 levels, and increased tau acetylation at K343.

Fig. 9.. Proposed model for TSC1/hamartin haploinsufficiency impairing tau degradation and increasing risk for tauopathy.

Decreased TSC1/hamartin levels lead to mTORC1 overactivation, which induce the activation of p300 HAT and the decrease of SIRT1 HDAC expression. As a consequence, tau is specifically acetylated at six lysine residues. While WT tau is degraded in the lysosomes through CMA, acetylated tau fails to be degraded by this system and thus accumulates in the neurons, leading to increased risk of neurodegeneration.