Using two antibodies specific for ac-tau that we developed, we found that tau is acetylated and that acetylation of tau contributes to the accumulation of p-tau. In primary neurons, tau acetylation was elevated by an FTDP-17-linked mutation and by low levels of oligomeric Aβ. Tau acetylation was enhanced in patients at early and moderate Braak stages of tau pathology, before NFTs form. Moreover, we demonstrated that SIRT1 deacetylates tau and that acetyltransferase p300 mediates its acetylation. SIRT1 deficiency elevated ac-tau in vivo. Inhibition of SIRT1 blocked tau polyubiquitination and tau turnover, resulting in p-tau accumulation. In contrast, deacetylating tau by inhibiting p300 with a highly specific small-molecule inhibitor effectively eliminated p-tau.
Our ac-tau-specific antibodies Ab708 or 9AB did not recognize nonacetylated recombinant tau, allowing us to specifically detect ac-tau in vivo. The mutagenesis study showed that Ab708 recognizes the acetylated lysines at positions 163, 174 and 180, and possibly other acetylated lysines. Mass spectrometry detected p300-induced tau acetylation at 23 lysines. However, p300 likely acetylates other lysines in the 13% of the tau sequence we did not analyze. Importantly, the sites acetylated in vivo probably would differ from those mapped under cell-free conditions. In addition, depending on neuronal subtypes and cellular conditions, different sets of lysines could be acetylated. Systematic mass spectrometry will be needed to map the acetylation sites of tau under different pathophysiological conditions.
p300 induced tau acetylation both in vitro
and in vivo
. Exactly how p300 acetylates tau in vivo
remains to be determined. Although p300 is mainly a nuclear protein, it can be cytosolic, acting as an E4 ubiquitin ligase for p53 (Shi et al., 2009
). Since small amount of tau also may be present in the nucleus in some cell types (Wang et al., 1993
), p300 might acetylate tau directly. However, p300 could also induce tau acetylation indirectly by unknown mechanisms. Inhibition of p300 with siRNA or the small molecule C646 reduced ac-tau levels significantly. C646 induced a much stronger decetylation effects than p300 siRNA, perhaps because C646 may also inhibit CBP, which shares 90% homology with p300 in the acetyltransferase domain (Liu et al., 2008
). Due to the functional redundancy of CBP and p300, inhibition of both by C646 is likely to result in much stronger effects than inhibition of p300 only with siRNA.
Our study suggests that SIRT1 interacts with tau in vivo
and directly deacetylates tau under cell-free conditions. Inhibition of SIRT1 with siRNA or the small molecule EX527 increased tau acetylation, and deleting SIRT1 elevated ac-tau in mouse brains. However, whether SIRT1 directly deacetylates tau in vivo
remains to be established. SIRT1 shuttles between the nucleus and cytosol, and its subcellular localization is regulated by pathophysiological stimuli. SIRT1 is localized in the cytoplasm of embryonic and adult neural precursor cells, but transiently translocated in the nucleus in response to differentiation stimuli, resulting in reduction of cytosolic SIRT1 activities during differentiation (Hisahara et al., 2008
). We found that levels of ac-tau were increased in primary neurons during maturation and correlated negatively with SIRT1 levels. These findings support the model that increased tau acetylation during neural maturation could be mediated by a reduction in cytosolic SIRT1 activity. The subcellular localization of SIRT1 is also modulated by stress and apoptosis (Greiss et al., 2008
; Jin et al., 2007
), providing potential mechanisms by which SIRT1 regulates tau acetylation and tauopathy during neuronal injury.
In human brains, elevated tau acetylation preceded the accumulation of NFTs, supporting the model that tau acetylation is an early event in tau-mediated neurodegeneration (). Our findings also suggest that tau acetylation is increased by stress due to Aβ accumulation or by mutations associated with tauopathy. However, the exact mechanisms underlying increased tau acetylation are undefined. Some likely mechanisms include deficiency in SIRT1 levels or SIRT1 activities in the cytosol, enhanced p300 levels or p300 activity, or alterations in tau conformation that blocks the access/binding to deacetylases or enhances the access/binding to acetyltransferases. These mechanisms are not mutually exclusive. Inhibition of p300 diminished ac-tau in primary neurons expressing TauP301L, consistent with the notion that P301L-induced hyperacetylation requires active p300. In AD brains, SIRT1 levels are reduced, and this reduction correlates with the amount of tau aggregates (Julien et al., 2009
), consistent with a role of SIRT1 deficiency in Aβ-induced tau hyperacetylation. SIRT1 was found to reduce Aβ generation by activating transcription of a gene encoding α-secretase (Donmez et al., 2010
). SIRT1 deficiency could also exacerbate the accumulation of Aβ, which could increase tau acetylation and tau phosphorylation even further. Other stress pathways induced during neuronal injury may also contribute to tau hyperacetylation and hyperphosphorylation.
Little is known how acetylation affects the functions of tau. We focused on the role of acetylation in ubiquitin-dependent degradation and effects on p-tau. However, acetylation of tau is likely to play other important roles in the development of tau-mediated neuropathology. We identified at least 13 putatively acetylated lysines in the microtubule-binding domains, raising the possibility that acetylation of tau could affect its ability to bind to and to stabilize microtubules, leading to neuronal dysfunction. The positively charged proline-rich regions are tightly bound to negatively charged microtubule surface (Amos, 2004
; Kar et al., 2003
). Since acetylation neutralizes charges in the microtubule-binding domain, aberrant acetylation might interfere with the binding of tau to microtubule, leading to tau dysfunction (). These residues appear to be variably acetylated by p300 in vitro
, consistent with a dynamic acetylation process that is amenable to modulation in vivo
The cross talk of tau acetylation with tau ubiquitination or phosphorylation may have implications for tau-mediated neurodegeneration (). Our findings suggest that tau acetylation directly contributes to accumulation of p-tau, a hallmark of tauopathy. Besides affecting p-tau turnover, tau acetylation may also modulate the activities of kinases involved in tau phosphorylation (). How kinase activities are modulated by tau acetylation is unknown. Regardless of the exact mechanisms, SIRT1 deficiency, high Aβ levels, or FTDP-17-linked mutations could lead to hyperacetylation of lysines on p-tau, preventing it from being ubiquitinated and degraded via the UPS pathway. In contrast, inhibition of p300 diminished ac-tau and effectively eliminated p-tau, suggesting that interfering with tau acetylation may be a new approach to reduce tauopathy.