Recent evidence suggests that neuronal protein degradation machinery may be impaired in neurodegenerative diseases such as AD, triggering the aberrant accumulation of tau proteins. This has been attributed, in part, to a decline in proteasomal and lysosomal function (11
). An additional potential mechanism, however, is age-associated loss of activity and expression of chaperone and associated proteins (35
). The results of the present study support this hypothesis and suggest that enhancing the activity of endogenous chaperones through Hsp90 inhibition facilitates reductions in p-tau accumulation and selectively targets aberrant p-tau species that are associated with neurotoxicity. These data suggest that the surveillance mechanism of the chaperone network is highly sensitive and tightly regulated, rather than simply an unregulated “garbage disposal” for aberrant or unfolded protein species (36
). In addition, the results of these studies suggest that the administration of Hsp90 inhibitors may provide a useful clinical approach to the treatment of AD and other tauopathies.
The dynamic and conserved chaperone complex (Figure ) that facilitates the removal of abnormal proteins such as p-tau typically works in concert with various interchangeable components (i.e., E3 ubiquitin ligases, prolyl isomerases, and other cochaperones). This process culminates either in the partial or complete refolding of the substrate or in its proteasomal degradation. Our results indicate that p-tau is a client of Hsp90 and its chaperone network. Constitutive levels of Hsp90 were required for p-tau degradation (Figure A). In addition, abnormal tau (hyperphosphorylated) facilitated binding to Hsp90 (Figure C). We have previously shown that phosphorylation of tau is constitutive, and neuronal processing of the aberrant protein is a continuous process (12
). Taken together, these results suggest that subtle impairment in the chaperone system that may occur with aging, coupled with even minor overproduction of abnormal client proteins, may result in aberrant accumulation and aggregation client proteins such as p-tau.
Our results demonstrate that the degradation of p-tau follows a processing sequence through the chaperone cycle depicted in Figure . CHIP plays a central role in this succession of events: it has been shown to degrade Hsp70 client proteins as well as Hsp70 itself, thus acting as both a ubiquitin ligase and a stress sensor (19
). CHIP also positively regulates HSF1 activity (38
). In the present study, CHIP was shown to be a central regulator of constitutive and inducible chaperones. Deletion of CHIP in vivo reduced constitutive expression of the Hop chaperone (Figure B), an essential component for transferring proteins from the Hsp70 machine to the Hsp90 complex (23
). Postdevelopmental alterations of a single chaperone could interrupt renaturation of a client protein and subsequently lead to neurotoxic protein accumulation. Our results suggest that CHIP plays a central role in mediating proper protein triage decisions, given its vast regulatory role through protein interaction and ubiquitination mechanisms.
Cellular processing of aberrant proteins includes repair, degradation, or both. The selected pathway may be lineage specific. For example, in nondividing neurons, in which apoptosis is not a viable option, the chosen protein processing pathway must be stable and sustainable for decades. Combinations of repair and degradation may also be used. Our present results and others’ indicate that Hsp90 serves a somewhat passive scaffolding function, allowing other cochaperones (including P23 and Pin1) to interact with damaged client proteins. P23 maintains mature Hsp90/client complexes in a partially active state and can ultimately lead to complete refolding of the client protein (25
). Pin1 is a tau-specific prolyl isomerase involved in tau dephosphorylation (24
). When these cochaperones are individually suppressed, they promote p-tau degradation (Figure ). These results provide the first evidence to our knowledge that proteins involved in the refolding of abnormal Hsp90 substrates may compete with chaperones involved in degradation. In addition, Hsp90 inhibitors and both P23 and Pin1 siRNAs promote a similar fate for client proteins in that they subvert attempts to refold proteins, thereby favoring degradation. This work suggests that the chaperone/client protein complex initially attempts to restore protein function, but should that fail, the system resorts to a protein degradation pathway.
It was previously demonstrated that CHIP antagonizes the interaction of P23 with Hsp90 (40
). The present results suggest that by removing P23, CHIP is able to freely and noncompetitively bind Hsp90, thus facilitating degradation. In addition, Pin1–/–
mice demonstrate pathological tau accumulation (24
), suggesting that without Pin1 and its associated dephosphorylation and refolding, the degradation machinery may become overburdened, leading to accumulation of tau. These findings provide evidence for new substrates potentially involved in tau biology and imply a bifurcation of the chaperone network.
These previous findings, as well as our present results, demonstrate how the cellular chaperone machinery may mediate p-tau degradation; however, it remains unclear how the chaperone complex is able to distinguish between aberrant and normal p-tau species. Tau is normally a natively unfolded protein, yet early in the pathogenesis of AD, the microtubule-binding domain (MTBD) and the N terminus of tau interact to form a hairpin motif recognized by the Alz-50 and MC-1 antibodies (41
). This serves as a tau misfolding event and is thought to be initiated by phosphorylation (42
). We previously reported that CHIP binds and ubiquitinates tau within the MTBD (11
). Phosphorylation of tau by MARK2/PAR-1 occurs at the KXGS motifs (S262 and S356). Both sites are also within the MTBD (16
). In addition, p-tau from AD brains was recently shown to be polyubiquitinated at lysine residues that lie in close proximity to the MARK2/PAR-1 consensus sites (43
The present study demonstrates (Figure ) that CHIP is unable to bind or ubiquitinate tau that is phosphorylated or mutated at these KXGS residues, regardless of polarity (phosphorylated serine being hydrophilic, while alanine is hydrophobic). Therefore, KXGS may be a recognition consensus for both MARK2/PAR-1 and CHIP, which would result in potential competitive binding. On the other hand, recent data suggest that nitration of N-terminal tyrosines promotes aggregation and misfolding of tau protein (44
). Tau folding in AD may normally occur by a sterically favorable interaction of the nitrated tyrosine with the –OH group of serine residues within the KXGS motif. Disruption of these serines could therefore prevent misfolding and consequently also block chaperone/CHIP binding and activity. Regardless, tau phosphorylated by MARK2 has an extended half-life (45
), perhaps due to inhibition of degradation mechanisms. This species may thus serve as a catalyst for subsequent pathogenic events. This could suggest an antagonistic relationship between CHIP and MARK2 with regard to tau turnover.
Our results demonstrated that Hsp90 inhibition did not affect degradation of S262/S356 p-tau. These results suggest that (a) CHIP is intimately linked to tau degradation following Hsp90 inhibition and that (b) the involvement of CHIP in this process makes the pharmacologic inhibition of Hsp90 (with drugs such as EC102) extremely specific for promoting degradation of only aberrant p-tau species, leaving normal p-tau species unaffected. This is supported further by a similar pattern of specificity of the chaperones facilitating Hsp90 inhibitor–mediated degradation for mutant huntingtin protein in Huntington’s disease (47
) and the in vivo efficacy of Hsp90 inhibitors in a polyglutamine expansion disorder with mutant androgen receptors (48
In the present study, we extended these observations and showed that following systemic administration, a novel Hsp90 inhibitor (EC102) can cross the blood-brain barrier and rapidly reduce levels of soluble p-tau species (16
). The Htau mouse model we used produces all 6 splice variants of the human tau protein (29
), represents a rodent model for the study of the progressive pathogenesis of tauopathies such as AD, and may serve as a reasonable in vivo model for testing pharmacologic inhibitors of soluble p-tau accumulation.
Our results demonstrated rapid and selective degradation of aberrant p-tau in the brains of Htau mice following administration of EC102 (Figure ). Neither normal tau species nor other Hsp90 client proteins (Cdk5 or Akt) were affected by the drug. In addition, these results suggest that an intermittent dosing scheme may be possible, but exact treatment regimens remain to be determined. The potential utility of EC102 is further supported by the results of binding affinity studies in human AD brain tissue, in which EC102 demonstrated 1,000-fold greater affinity for Hsp90 from affected areas (temporal cortex) than from unaffected areas (cerebellar cortex) and controls from the same areas. Thus, the higher affinity present in AD-affected brains is a result of disease (abnormal protein accumulation) rather than an intrinsic difference between brain regions. Our results demonstrate a potential for localization of therapeutic agents to areas of disease and suggest that clinically, lower concentrations of Hsp90 inhibitors could be used to treat AD, potentially limiting unwanted effects on Hsp90/client complexes in normal tissues. Finally, the presence of Hsp90 complexes with high affinity for EC102 in other neurodegenerative disorders may justify the use of Hsp90 inhibitor therapy in other diseases.
In summary, this work demonstrates that CHIP and the Hsp90 chaperone system are essential for the proper degradation of p-tau, a protein species that undergoes continuous turnover in the neuron (12
). Minor perturbations in the chaperone system with aging may retard turnover of tau, leading to aggregation and subsequent neurodegeneration. The demonstration that Hsp90 inhibitors enhance Hsp90/CHIP-mediated p-tau degradation helps to clarify the mechanisms of tau metabolism and provides a possible therapeutic strategy for the clinical management of tauopathies.