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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cell Pharmacol. Author manuscript; available in PMC 2010 June 2.
Published in final edited form as:
Mol Cell Pharmacol. 2010 January 1; 2(2): 43–46.
PMCID: PMC2879647
NIHMSID: NIHMS203127

Hsp70 ATPase Modulators as Therapeutics for Alzheimer’s and other Neurodegenerative Diseases

Abstract

Neurodegenerative diseases caused by abnormal accumulation of the microtubule associated protein tau (MAPT, tau) are collectively called tauopathies. The most devastating tau related disorder is Alzheimer’s disease (AD). Molecular chaperones such as heat shock proteins (Hsp) have emerged as critical regulators of tau stability. Several studies from our group and others have shown that the chaperone network can be targeted for the development of therapeutic strategies for AD and other neurodegenerative diseases. Here we will discuss a recent paper and current work from our laboratory where we have manipulated the ATPase activity of the 70-kDa heat shock protein (Hsp70) to regulate tau turnover. A high-throughput screening assay revealed several compounds that activated or inhibited Hsp70’s ATPase activity. Inhibitors dramatically and rapidly reduced tau levels, whereas activators stabilized tau, both in cells and brain tissue. Moreover, increased levels of Hsp70 improved ATPase inhibitor efficacy, suggesting that therapies aimed at inducing Hsp70 levels followed by inhibition of its ATPase activity may be a very effective strategy to treat AD. These findings demonstrate that Hsp70 ATPase activity can be targeted to modify the pathologies of AD and other tauopathies.

Keywords: Tau, Alzheimer’s disease, Chaperones, Heat shock proteins, Therapeutic, Hsp70, ATPase

A number of studies have suggested that the tau protein plays a central role in the pathogenesis of AD and other related neurodegenerative diseases, commonly known as tauopathies (13). The normal function of tau is to promote assembly and stabilization of microtubules; this is specifically critical in neurons for axonal transport. Dysfunction of tau either by genetic or environmental factors leads to its aggregation into neurofibrilliary tangles (NFTs) in the brain of AD and other tauopathy patients (4). In the case of AD, accumulation of amyloid plaques composed of Aβ peptides, have been largely shown to initiate cellular events that result in tau aggregation (57). Due to this fact, most of the biotech and pharmaceutical industry efforts have focused on Aβ-based therapeutic targets (6). However, pioneering work in recent years has shown that neurodegeneration and cognitive dysfunction are critically linked to tau accumulation (810). Moreover, the recent failure of Aβ lowering agents, such as tramiprosate (11) and flurbiprofen (2) in phase III clinical trials, suggests that there is a need to pursue other therapeutic approaches, including those that reduce the levels of pathological tau.

Our group and several others have shown molecular chaperones, such as heat shock proteins Hsp70 and Hsp90 play a significant role in tau processing (1316). Increased levels of Hsp70 and Hsp90 were found to promote tau solubility and microtubule binding in various cellular models (13). Hsp90 and Hsp70 exchange and hydrolyze ATP, which regulates substrate binding and release (17). Thus, recent efforts have shifted from regulating heat shock protein levels to regulating their ATPase function. Pharmacologic inhibition of Hsp90 ATPase function significantly reduced the intracellular levels of the disease-associated tau species (18, 19); however the ATPase function of cytosolic Hsp70 had not been targeted for chemical design, until recently described by our group (20, 21).

Screening of 2800 bioactive compounds by using a newly described robust and reliable high-throughput system for Hsp70 ATPase modulators revealed several inhibitors and activators (22). Approximately 80% reduction in Hsp70 enzymatic function was observed by two distinct chemical classes of identified inhibitors: benzothiazines (methylene blue, MB and azure C, AC) and flavones (myricetin, MY). Identified activators 115-7c and SWO2 belonging to the dihydropyrimidine family caused an approximate 45% increase in Hsp70 activity. Surprisingly, one of the identified inhibitors, MB, was also found in our previous cell-based screening assay as a potent tau reducer (23). Having this battery of new compounds and our experience in handling both tau and molecular chaperones, we endeavored to explore the effect of ATPase modulators on tau protein levels.

We generated a stable HeLa cell line overexpressing tau for the characterization of the compounds identified above. The treatment of cells with inhibitors MB, AC and MY showed significant reduction in total tau and phospho-tau levels, while activator 115-7c and SWO2 shown an increase in tau levels in a dose dependent manner. We have now validated tau reduction by MB in primary neurons. Primary neurons were obtained from the cortex and hippocampus of wild type mice. Neurons were grown for 10 days on poly-L-lysine-coated plate in neurobasal medium with B27 supplement as described (24). Treatment with various doses of MB in primary neuron showed a dose dependent reduction in tau very similar to tau stable HeLa cells (Figure 1). We also found that tau levels from acute mouse brain slice cultures from wildtype and tau transgenic mice were significantly and rapidly reduced. The effect of these drugs was selective for tau regulation. Two other neurodegenerative disease related proteins (α-synuclein from Parkinson’s disease and TAR-DNA binding protein from amyotrophic lateral sclerosis), were not affected by Hsp70 ATPase inhibition.

Figure 1
Inhibition of Hsp70 ATPase activity reduces tau in the neuron

We found that the reductions in tau caused by inhibition of Hsp70 ATPase function were extremely rapid. Within 5 minutes of treatment, reductions in tau levels were observed in stably overexpressing tau HeLa cells, human [BE(2)M17 and SHSY5Y] and murine (Neuro2a) neuroblastoma cells with endogenous tau levels. Drug efficacy analysis across cell lines showed significant reduction in tau levels after 5 minutes following Hsp70 inhibitor treatment, and highly significant reductions after 60 minutes.

Based on previous studies suggesting that increasing Hsp70 levels leads to increased binding to tau (14), we explored whether high levels of Hsp70 would affect the efficacy of Hsp70 ATPase modulators. Cells overexpressing Hsp70 were treated with inhibitors and we found that increased efficacy when Hsp70 levels were increased. We had similar results from a study using the Hsp70-inducing compound, celastrol (25). Since then, we have demonstrated a similar phenomenon with Hsp70 ATPase activators. Indeed, Hsp70 overexpression increased efficacy of Hsp70 ATPase activators for increasing tau levels (Figure 2). Taken together these studies clearly suggest that by increasing the amount of Hsp70, the number of Hsp70/tau complexes also increases, at which point modulation of Hsp70 ATPase function may become more efficacious.

Figure 2
Increasing Hsp70 levels enhances the Hsp70 activator drug efficacy

Unlike Hsp90 inhibitors which induce expression of heat shock proteins (18, 26) we found that levels of heat shock proteins were not elevated following Hsp70 inhibition; in fact they were marginally decreased. Based on our data showing that as Hsp70 levels increase, so does Hsp70 ATPase modulator efficacy, perhaps combining Hsp90 with Hsp70 inhibition could be a highly effective drug regimen for treating AD. We speculated, however that Hsp70 inhibition may also be an effective treatment strategy for other related tau disorders. In this vein, Hsp70 inhibition showed significant and similar reduction in mutant tau species that are linked to amyloid-independent tauopathies, such as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17) or progressive supranuclear palsy (PSP). However, Hsp90 inhibitors were not effective against all mutant forms of tau. Thus, Hsp70 inhibition may be an effective strategy for any disease with some component of tau pathology.

A final caveat to our study was the impact that tau phosphorylation had on Hsp70 ATPase modulator efficacy. The hyperphosphorylation of tau in neurons is widely accepted to play a vital role in the molecular pathogenesis of AD and in neurodegeneration (27, 28). Thus, we investigated the effect of ATPase modulating drugs on disease related phospho-tau species. We tested two types of aberrant phospho-tau species here by over-expressing kinases; tau phosphorylated at proline-directed serine/threonine residues by GSK3β and tau phosphorylated at KXGS motifs by MARK2. Tau phosphorylated by MARK2 was shown to be degradation resistant, as previously described (15, 16). Conversely, tau phosphorylated by GSK3β was very sensitive to Hsp70 ATPase modulation.

Based on all of our data combined we propose the following model summarized in Figure 3. We hypothesize that tau is initially recognized by the Hsp70/Hsp40 complex. This then forms an intermediate complex with Hsp90 and HOP as described earlier (15). Inhibitors designed to target Hsp70 bypass the intermediate complex formation step and facilitate immediate tau degradation via the proteasome. Inhibitors designed to target Hsp90 require Hsp70 to facilitate the intermediate complex formation and then Hsp90 inhibitors can promote tau degradation. Hsp90 inhibition leads to activation of heat shock factor 1, which produces more chaperones that could then be targeted by Hsp70 ATPase modulators, leading to improved efficacy. These findings provide new insights into the sequence of tau degradation by the chaperone system. Moreover, our results demonstrate that the ATPase domain of Hsp70 could be a potent target for future drug discovery for the treatment of AD and other tauopathies. Indeed, one of the ATPase inhibitors identified during the course of our study, methylene blue, has passed phase II clinical trials and is now entering phase III trials of treatment of AD patients (29).

Figure 3
Schematic model showing distinctions between Hsp70 and Hsp90 inhibition with regard to tau biology

Acknowledgments

We would like to thank Dr. Jason E. Gestwicki and Dr. Erik R.P. Zuiderweg for their great collaboration and for providing compounds. We would like to thank Dr. Peter Davies (Albert Einstein COM, NY) for PHF1 (pS396/S404) antibody. This work was supported by the Rosalinde and Arthur Gilbert Foundation/American Federation for Aging Research, CurePSP, the Alzheimer’s Association grant IIRG-09-130689 and NIA grant R00AG031291.

Footnotes

Conflicts of Interest

No potential conflicts of interest to disclose.

References

1. Iqbal K, Liu F, Gong CX, Alonso Adel C, Grundke-Iqbal I. Mechanisms of tau-induced neurodegeneration. Acta Neuropathol. 2009;118:53–69. [PMC free article] [PubMed]
2. Bunker JM, Kamath K, Wilson L, Jordan MA, Feinstein SC. FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells. J Biol Chem. 2006;281:11856–11863. [PubMed]
3. Trojanowski JQ, Ishihara T, Higuchi M, et al. Amyotrophic lateral sclerosis/parkinsonism dementia complex: transgenic mice provide insights into mechanisms underlying a common tauopathy in an ethnic minority on Guam. Exp Neurol. 2002;176:1–11. [PubMed]
4. Lee VM. Biomedicine. Tauists and beta-aptists united--well almost! Science. 2001;293:1446–1447. [PubMed]
5. Oddo S, Caccamo A, Kitazawa M, Tseng BP, LaFerla FM. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease. Neurobiol Aging. 2003;24:1063–1070. [PubMed]
6. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002;297:353–356. [PubMed]
7. Frautschy SA, Baird A, Cole GM. Effects of injected Alzheimer beta-amyloid cores in rat brain. Proc Natl Acad Sci U S A. 1991;88:8362–8366. [PubMed]
8. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991;82:239–259. [PubMed]
9. Mukaetova-Ladinska EB, Garcia-Siera F, Hurt J, et al. Staging of cytoskeletal and beta-amyloid changes in human isocortex reveals biphasic synaptic protein response during progression of Alzheimer's disease. Am J Pathol. 2000;157:623–636. [PubMed]
10. Roberson ED, Scearce-Levie K, Palop JJ, et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science. 2007;316:750–754. [PubMed]
11. Gauthier S, Aisen PS, Ferris SH, et al. Effect of tramiprosate in patients with mild-to-moderate Alzheimer's disease: exploratory analyses of the MRI sub-group of the Alphase study. J Nutr Health Aging. 2009;13:550–557. [PubMed]
12. Green RC, Schneider LS, Amato DA, et al. Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized controlled trial. JAMA. 2009;302:2557–2564. [PMC free article] [PubMed]
13. Dou F, Netzer WJ, Tanemura K, et al. Chaperones increase association of tau protein with microtubules. Proc Natl Acad Sci U S A. 2003;100:721–726. [PubMed]
14. Petrucelli L, Dickson D, Kehoe K, et al. CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet. 2004;13:703–714. [PubMed]
15. Dickey CA, Kamal A, Lundgren K, et al. The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J Clin Invest. 2007;117:648–658. [PMC free article] [PubMed]
16. Dickey CA, Dunmore J, Lu B, et al. HSP induction mediates selective clearance of tau phosphorylated at praline-directed Ser/Thr sites but not KXGS (MARK) sites. FASEB J. 2006;20:753–755. [PubMed]
17. Slepenkov SV, Witt SN. The unfolding story of the Escherichia coli Hsp70 DnaK: is DnaK a holdase or an unfoldase? Mol Microbiol. 2002;45:1197–1206. [PubMed]
18. Dickey CA, Eriksen J, Kamal A, et al. 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;2:231–238. [PubMed]
19. Luo W, Dou F, Rodina A, et al. Roles of heat-shock protein 90 in maintaining and facilitating the neurodegenerative phenotype in tauopathies. Proc Natl Acad Sci U S A. 2007;104:9511–9516. [PubMed]
20. Jinwal UK, Miyata Y, Koren J, 3rd, et al. Chemical manipulation of hsp70 ATPase activity regulates tau stability. J Neurosci. 2009;29:12079–12088. [PMC free article] [PubMed]
21. Koren J, 3rd, Jinwal UK, Jin Y, et al. Facilitating Akt clearance via manipulation of Hsp70 activity and levels. J Biol Chem. 285:2498–2505. [PMC free article] [PubMed]
22. Chang L, Bertelsen EB, Wisen S, Larsen EM, Zuiderweg ER, Gestwicki JE. High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK. Anal Biochem. 2008;372:167–176. [PubMed]
23. Dickey CA, Ash P, Klosak N, et al. Pharmacologic reductions of total tau levels; implications for the role of microtubule dynamics in regulating tau expression. Mol Neurodegener. 2006;1:6. [PMC free article] [PubMed]
24. Abisambra JF, Fiorelli T, Padmanabhan J, Neame P, Wefes I, Potter H. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease. PLoS One. 5:e8556. [PMC free article] [PubMed]
25. Westerheide SD, Bosman JD, Mbadugha BN, et al. Celastrols as inducers of the heat shock response and cytoprotection. J Biol Chem. 2004;279:56053–56060. [PubMed]
26. Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell. 1998;94:471–480. [PubMed]
27. Johnson GV, Stoothoff WH. Tau phosphorylation in neuronal cell function and dysfunction. J Cell Sci. 2004;117:5721–5729. [PubMed]
28. Hanger DP, Anderton BH, Noble W. Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends Mol Med. 2009;15:112–119. [PubMed]
29. Rafii MS, Aisen PS. Recent developments in Alzheimer's disease therapeutics. BMC Med. 2009;7:7. [PMC free article] [PubMed]