Therapeutic Takedown: Hsp90 Inhibitors Tackle Tau

A pair of recent papers show that inhibitors of the chaperone heat shock protein 90 (Hsp90) enhance the degradation of mutated or hyperphosphorylated tau protein in animal models of neurodegenerative disease. The inhibitors help cells clear toxic tau species while leaving normal tau untouched. The results suggest that Hsp90 inhibitors could be useful for treating tauopathies, and potentially other neurodegenerative diseases that result from the accumulation of misfolded or modified proteins in neurons.

The idea of using Hsp90 inhibitors to usher toxic proteins to their demise comes from the cancer world, where oncologists first noticed that Hsp90 selectively stabilized mutated or aberrantly modified oncoproteins. When Hsp90 encounters a damaged protein, it can send it down one of two pathways, either refolding the protein, or targeting it for degradation by the proteasome. Small molecules that block the Hsp90 ATPase activity, and thus its folding capacity, proved to enhance the degradation of oncoproteins, and show selective toxicity for tumor cells. A novel, nontoxic Hsp90 inhibitor (17AAG), a derivative of the antibiotic geldanamycin, is currently in early-phase clinical trials in a number of cancers.

The parallels to neurodegenerative disease are clear. First, in most neurodegenerative diseases, aberrant proteins accumulate in cells with all-too-familiar results. Second, Hsp90 and its cofactor, the ubiquitin ligase CHIP, regulate levels of the microtubule-associated protein tau, a protein which itself can precipitate neurodegeneration. Now, several labs have provided a much-needed proof of concept for the therapeutic potential of 17AAG and its relatives in neurological disease, by testing Hsp90 inhibitors directly in several mouse models of tauopathy.

The most recent report, from Paul Greengard at the Rockefeller University and Gabriela Chiosis of Memorial Sloan Kettering Cancer Center, both in New York, shows that a brain-permeable Hsp90 inhibitor reduces the expression of P301L mutant tau in a mouse model of inherited frontotemporal dementia. Reporting in the May 21 issue of PNAS online, they show that their inhibitor also decreases the levels of phosphorylated and aggregated tau in mice expressing unmutated human tau, a model for the tau pathology that occurs in Alzheimer disease.

For their studies, first author Wenjie Luo and colleagues used both 17AAG and PU24FCl, a novel inhibitor developed in their lab. Both bind to Hsp90 and inhibit its ATPase activity, altering its association with client proteins and promoting their proteasomal degradation. In cells, the inhibitors reduced levels of mutant tau by enhancing their degradation, while it did not affect normal tau proteins. Interestingly, the Cdk5 kinase activator, p35, was also a client protein for Hsp90, and the inhibitor stimulated its degradation. This supplies an alternative pathway for decreasing phospho-tau by indirectly inhibiting Cdk5.

To test inhibitors in vivo, the researchers turned to another of their own compounds. PU-DZ8 is a brain-permeable Hsp90 inhibitor. After a single intraperitoneal dose, Luo and colleagues detected a significant decrease in soluble, insoluble, and hyperphosphorylated P301L mutant tau in the brain within 4 hours, which was maintained up to 36 hours. They also saw a transient decrease in p35 protein. Following up on this, they treated mice for 30 days and found sustained decreases in the tau proteins and p35.

In another model, Luo and colleagues tested the effects of inhibitors on mice expressing human wild-type tau, which develop AD-like tangle pathology. They saw no changes in the levels of wild-type tau, but did see sustained decreases in p35, and in levels of tau phosphorylated at the Cdk5 site S202. Thus, although the inhibitors did not increase degradation of tau in this model, their effects on p35 and tau phosphorylation leave the door open to their use in AD, where tau pathology stems from modified, not mutated, tau.

The results jibe with work from researchers at the Mayo Clinic in Jacksonville, Florida, that appeared in March in the Journal of Clinical Investigation. In that paper, Chad Dickey, Leonard Petrucelli, and collaborators show the effects of their own Hsp90 inhibitor in the hTau mouse. They find a decrease in aberrant phospho-tau species in the mice after treatment with EC102, a brain permeable inhibitor. They do not see effects on normal tau.

Dickey and colleagues also present an extensive mechanistic analysis of the action of the inhibitors using siRNA knockdown of a number of proteins in the Hsp90 refolding and degradation pathways. The picture that emerges is one where Hsp90 serves as a fork in the road for phospho-tau: on the one side is refolding, the other degradation. Possibly, by attempting to refold problematic tau, Hsp90 protects it from degradation. Inhibition of Hsp90’s ATPase stops that futile effort, and steers tau toward the proteasome.

In further support of the use of Hsp90 inhibitors in AD, Dickey and colleagues show that in affected tissue from the temporal cortex of human AD brain, there is a form of Hsp90 that binds EC102 with an affinity 1,000 times higher than Hsp90 from unaffected tissues or control brain. This very high-affinity form of the chaperone has also been seen in some cancer cells, and was a large part of the reason why the inhibitors were pursed for anticancer applications.

ATPase inhibitors have a second effect on Hsp90, where they act to release the HSF1 transcription factor and induce the expression of additional chaperone proteins including Hsp70. Previous work has shown an inverse relationship between aggregated tau and Hsp70/90 levels in brain, and data from the Greenberg lab a few years ago indicated that upregulating chaperone expression decreased tau aggregates and was neuroprotective (Dou et al., 2003). While data from the Dickey and Luo studies suggest that the effects of inhibitors are mainly direct, and do not require Hsp70 induction, it is conceivable that increasing the levels of Hsp70 or 90 chaperones could contribute to mitigating tau toxicity.

Clearly more mysteries in the Hsp90-tau story will need to be sorted out to find clinical application of the inhibitors. As Dmitry Goryunov and Ronald Liem of Columbia University write in a commentary accompanying the Dickey et al. paper, “The relative contributions of protein refolding, degradation, and aggregation to the neutralization of toxic tau are far from being elucidated.” However, they add, “The notion that the chaperone machinery is a promising target for pharmacologic intervention in AD and other tauopathies has just received a robust boost.”

Neither paper shows evidence for behavioral improvements in the mice, an important additional piece of information to provide full proof of concept. In an encouraging side note, 17AAG, was recently shown to prevent neurodegeneration and improve motor function in a mouse model of spinal and bulbar muscular atrophy (SBMA) caused by a polyQ-expanded androgen receptor (see ARF related news story). The treatment enhanced proteasome-mediated degradation of the polyQ AR, and improved the animals’ mobility and survival.

Attacking Hsp90 from another angle, Brian Blagg and colleagues at the University of Kansas in Lawrence have developed alternative inhibitors that interact with a second ATP-binding site in the C-terminal portion of the protein. In a recent paper (Ansar et al., 2007), they show that a compound targeting this site induces Hsp70 expression and protects cells against the toxicity of amyloid-β peptides in vitro. (Blagg’s Kansas colleague Elias Michaelis talked about the compound, an analog of the antibiotic novobiocin at the recent Alzheimer Therapy Development Foundation meeting (see ARF related news story).—Pat McCaffrey

Comments

  1. Chaperones are molecular machines designed to maintain proteins in a properly folded state (1). Misfolded or misassembled proteins that cannot be corrected by chaperones are ubiquitinated and thereby targeted for degradation by the proteasome (2). We and others proposed that the chaperone and proteasome systems may act in concert in clearing the toxic forms of phosphorylated tau (p-tau) that are associated with tauopathies, particularly Alzheimer disease (3-5). This same approach might also work for other neurodegenerative diseases.

    Hsp90 inhibitors bind to the ATPase domain of Hsp90, which prevents any further attempts at refolding the client proteins, instead promoting their proteasomal degradation. Recently, we were the first to demonstrate that pharmacologic manipulation of Hsp90 activity through the use of Hsp90 inhibitors was able to selectively reduce “abnormal” p-tau species in cultured cells (3,6) and in mice (4). We identified EC102 as an Hsp90 inhibitor with the optimal properties to facilitate blood-brain barrier permeability. Once-daily peripheral administration of this compound into transgenic tau mice for one week significantly and specifically reduced only abnormal tau species in the brains of these mice, preserving “normal” tau. In cell culture, we described a mechanism whereby knocking down Hsp90 with siRNA abolished the EC102 effect, indicating that Hsp90 is required as a structural component of a tau-refolding/degradation complex and therefore a "client" of Hsp90. We also highlight the role of the tau ubiquitin ligase CHIP as critical for this process, consistent with our previous findings in CHIP null mice (7). Most remarkable was our finding that the Hsp90 complex isolated from only those areas of the AD brain that accumulate amyloid and tau had a 1,000-fold higher affinity for Hsp90 inhibitors. Unaffected regions from these same patients as well as control brains did not have this high affinity complex. These results suggest that Hsp90 antagonists might selectively target areas of injury and act like a "magic bullet" for treatment of Alzheimer disease.

    The most recent contribution to the area of Hsp90 inhibitor therapy for Alzheimer disease from Gabriela Chiosis’ group largely confirms these data. Their work strengthens the notion that Hsp90 inhibitors can preferentially degrade only abnormal tau species, the production of which is certainly enhanced by mutations in tau. They also describe the efficacy of these drugs over a relatively short dosing period in two distinct mouse models of tauopathy; the same hTau model used in our previous work, that overexpresses all six isoforms of wild-type human tau, and the JNPL3 mouse model that overexpresses a single human tau isoform harboring the P301L mutation that causes FTDP-17, a heritable form of amyloid-negative tauopathy. They also suggest that tau exists in a complex with Hsp90 and p35, a component of the Cdk5 complex which is known to aberrantly phosphorylate tau; however, this complex seems to predominate in CNS regions that lack tau pathology in the JNPL3 model.

    Collectively, these studies provide the foundation for future investigation of these compounds to treat Alzheimer disease, using alternative transgenic mouse models. Specifically, determining whether Hsp90 inhibition can delay the pathological progression and memory loss in an inducible mouse model of tauopathy (rTg4510) will establish the framework for introduction to the clinic. With the recent advance in the field demonstrating that an amyloid-depositing mouse model does not develop memory deficits on a tau knockout background (8), it is clear that tau has finally emerged as a legitimate target for AD therapeutics.

    References:

    Dickey CA, Petrucelli L. Current strategies for the treatment of Alzheimer's disease and other tauopathies. Expert Opin Ther Targets. 2006 Oct;10(5):665-76. PubMed.

    Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13-21. PubMed.

    Dickey CA, Dunmore J, Lu B, Wang JW, Lee WC, Kamal A, Burrows F, Eckman C, Hutton M, Petrucelli L. HSP induction mediates selective clearance of tau phosphorylated at proline-directed Ser/Thr sites but not KXGS (MARK) sites. FASEB J. 2006 Apr;20(6):753-5. PubMed.

    Dickey CA, Kamal A, Lundgren K, Klosak N, Bailey RM, Dunmore J, Ash P, Shoraka S, Zlatkovic J, Eckman CB, Patterson C, Dickson DW, Nahman NS, Hutton M, Burrows F, Petrucelli L. The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J Clin Invest. 2007 Mar;117(3):648-58. PubMed.

    Dou F, Netzer WJ, Tanemura K, Li F, Hartl FU, Takashima A, Gouras GK, Greengard P, Xu H. Chaperones increase association of tau protein with microtubules. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):721-6. PubMed.

    Dickey CA, Eriksen J, Kamal A, Burrows F, Kasibhatla S, Eckman CB, Hutton M, Petrucelli L. Development of a high throughput drug screening assay for the detection of changes in tau levels -- proof of concept with HSP90 inhibitors. Curr Alzheimer Res. 2005 Apr;2(2):231-8. PubMed.

    Dickey CA, Yue M, Lin WL, Dickson DW, Dunmore JH, Lee WC, Zehr C, West G, Cao S, Clark AM, Caldwell GA, Caldwell KA, Eckman C, Patterson C, Hutton M, Petrucelli L. Deletion of the ubiquitin ligase CHIP leads to the accumulation, but not the aggregation, of both endogenous phospho- and caspase-3-cleaved tau species. J Neurosci. 2006 Jun 28;26(26):6985-96. PubMed.

    Roberson ED, Scearce-Levie K, Palop JJ, Yan F, Cheng IH, Wu T, Gerstein H, Yu GQ, Mucke L. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science. 2007 May 4;316(5825):750-4. PubMed.

    View all comments by Leonard Petrucelli

  2. In this paper, Luo and colleagues showed that PU24FCI inhibited Hsp90 activity and accelerated tau degradation. It is quite surprising that this drug treatment in JNPL3 mice reduced the amount of sarcosyl-insoluble tau within hours. This result suggests the possibility that the drug treatment can resolve pre-existing PHF-tau aggregates through enhancing tau degradation.

    Santacruz and colleagues showed previously that after turning off mutant tau expression, NFT formation continued while memory impairment and neuron loss were rescued (SantaCruz et al., 2005). These older results suggest that before but not during NFT formation, tau may be modified to affect neuronal function. Because PU24FCI treatment reduced both soluble and insoluble tau levels, Hsp90 may recognize a modified form of tau, which may be toxic, and reduce its level. If so, I wonder if PU24FCI treatment can prevent the severe motor dysfunction that is known to develop in the JNPL3 mouse strain?

    References:

    Santacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, Guimaraes A, DeTure M, Ramsden M, McGowan E, Forster C, Yue M, Orne J, Janus C, Mariash A, Kuskowski M, Hyman B, Hutton M, Ashe KH. Tau suppression in a neurodegenerative mouse model improves memory function. Science. 2005 Jul 15;309(5733):476-81. PubMed.

    View all comments by Akihiko Takashima

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References

News Citations

  1. Toxicity of Polyglutamine Expansion Follows Normal Channels
  2. New York: Back to School—R and D in Academia

Paper Citations

  1. Dou F, Netzer WJ, Tanemura K, Li F, Hartl FU, Takashima A, Gouras GK, Greengard P, Xu H. Chaperones increase association of tau protein with microtubules. Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):721-6. PubMed.
  2. Ansar S, Burlison JA, Hadden MK, Yu XM, Desino KE, Bean J, Neckers L, Audus KL, Michaelis ML, Blagg BS. A non-toxic Hsp90 inhibitor protects neurons from Abeta-induced toxicity. Bioorg Med Chem Lett. 2007 Apr 1;17(7):1984-90. PubMed.

Further Reading

Primary Papers

  1. Dickey CA, Kamal A, Lundgren K, Klosak N, Bailey RM, Dunmore J, Ash P, Shoraka S, Zlatkovic J, Eckman CB, Patterson C, Dickson DW, Nahman NS, Hutton M, Burrows F, Petrucelli L. The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J Clin Invest. 2007 Mar;117(3):648-58. PubMed.
  2. Goryunov D, Liem RK. CHIP-ping away at tau. J Clin Invest. 2007 Mar;117(3):590-2. PubMed.
  3. Luo W, Dou F, Rodina A, Chip S, Kim J, Zhao Q, Moulick K, Aguirre J, Wu N, Greengard P, Chiosis G. Roles of heat-shock protein 90 in maintaining and facilitating the neurodegenerative phenotype in tauopathies. Proc Natl Acad Sci U S A. 2007 May 29;104(22):9511-6. PubMed.

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