An acetylation-phosphorylation switch that regulates tau aggregation propensity and function

Abstract

The aberrant accumulation of tau protein is a pathological hallmark of a class of neurodegenerative diseases known as tauopathies, including Alzheimer's disease and related dementias. On the basis of previous observations that tau is a direct substrate of histone deacetylase 6 (HDAC6), we sought to map all HDAC6-responsive sites in tau and determine how acetylation in a site-specific manner affects tau's biophysical properties in vitro Our findings indicate that several acetylation sites in tau are responsive to HDAC6 and that acetylation on Lys-321 (within a KCGS motif) is both essential for acetylation-mediated inhibition of tau aggregation in vitro and a molecular tactic for preventing phosphorylation on the downstream Ser-324 residue. To determine the functional consequence of this HDAC6-regulated phosphorylation event, we examined tau's ability to promote microtubule assembly and found that phosphorylation of Ser-324 interferes with the normal microtubule-stabilizing function of tau. Tau phosphorylation of Ser-324 (pSer-324) has not previously been evaluated in the context of tauopathy, and here we observed increased deposition of pSer-324-positive tau both in mouse models of tauopathy and in patients with Alzheimer's disease. These findings uncover a novel acetylation-phosphorylation switch at Lys-321/Ser-324 that coordinately regulates tau polymerization and function. Because the disease relevance of this finding is evident, additional studies are needed to examine the role of pSer-324 in tau pathobiology and to determine whether therapeutically modulating this acetylation-phosphorylation switch affects disease progression in vivo.

Keywords: Alzheimer disease; Tau protein (Tau); acetylation; aggregation; histone deacetylase 6 (HDAC6); neurodegenerative disease; phosphorylation; tauopathy.

Figure 1.

Pseudoacetylation of Lys-321 inhibits tau aggregation. a, WT and pseudoacetylated mutant KXGS recombinant tau proteins (K259Q/K290Q/K321Q/K353Q (4KQ) and K290Q/K321Q (2KQ)) were incubated with dextran sulfate to stimulate polymerization, which was detected by thioflavin S. The single K321Q mutant and the compound K290Q/K321Q and K259Q/K290Q/K321Q/K353Q mutants, which both include the K321Q mutation, exhibited the greatest inhibition of tau aggregation. b, acetylation-mimicking mutations were introduced on non-KXGS sites, and polymerization induced by dextran sulfate and detected by thioflavin S was compared with WT tau. All data are presented as mean ± S.D. ****, p < 0.0001; ***, p < 0.001; *, p < 0.05.

Figure 2.

Acetylation state of Lys-321 regulates tau filament formation. a–c, EM was used to compare dextran-sulfate induced filament assembly of WT tau (a) with mutant K321Q (b) or K321R (c) tau proteins. d–f, quantitation of EM analysis demonstrated that whereas the average length of WT and K321Q/R tau filaments per field was not different (F = 0.38, p = 0.69) (d), the total filament length per field (F = 47.9, p < 0.0001) (e) and the average number of filaments per field (F = 54.22, p < 0.0001) (f) were significantly reduced with K321Q mutant tau. All data are presented as mean ± S.D. (error bars). ****, p < 0.0001.

Figure 3.

Pseudoacetylation of Lys-321 prevents phosphorylation of Ser-324, which inhibits tau function. a, schematic diagram illustrating the location of the K321Q mutation within the third microtubule binding repeat domain (R3). b, HEK293T cells were cotransfected with WT or K321Q tau along with either GFP or MARK2, as indicated. Cell lysates were then prepared, and phosphorylation of KXGS motifs was evaluated by immunoblot. c, the impact of Ser-324 phosphorylation on tau function was assessed by monitoring microtubule assembly in the presence of WT and pseudophosphorylated (S324D/E) and dephosphorylated (S324A) mutant tau proteins. Error bars, S.D.

Figure 4.

Phosphorylation of Ser-324 is detected in the AAV model of tauopathy. a–f, representative images demonstrating the absence of pSer-324 immunolabeling in GFP-AAV–injected mice (a and b), whereas pSer-324–positive tau species are detected in Tau^P301L-AAV–injected mice in both the cortex (c) and CA1 field of the hippocampus (d), which exhibit high levels of human tau expression in this model (e and f). See Ref. for additional details regarding the characterization of this model. Scale bar, 50 μm.

Figure 5.

Phosphorylation of Ser-324 in AD brain. a, pSer-324, 12E8 (pSer-262/356), and total human tau (E1) levels in frontal cortex from patients with AD compared with control cases were monitored by immunoblotting. b–e, immunohistochemistry was used to evaluate pSer-324 in paraffin-embedded tissue sections from the cortex of control (b) and AD patients (c–e). Scale bar, 50 μm.

Figure 6.

HDAC6 inhibition reduces pSer-324 in primary neuronal cultures. a, pSer-324, total human tau (E1), acetylated tubulin, and GAPDH levels were monitored by immunoblotting lysates prepared from primary neurons transduced with P301L-AAV. b, quantitation of the pSer-324–positive signal was normalized to total human tau (E1) to control for changes in tau levels. The experiment was repeated three times in duplicate (see supplemental Fig. S6), and statistical significance was assessed by Student's t test (t = 4.906, p = 0.0006). c, changes in total human tau levels were assessed by quantifying E1 immunoreactivity normalized to GAPDH to control for protein loading (t = 3.679, p = 0.004). d, inhibition of HDAC6 following ACY-738 treatment was monitored by evaluating acetylation of tubulin, which was normalized to GAPDH to control for protein loading (t = 13.65, p < 0.0001). Error bars, S.D.