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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.
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 (1–3). 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 (5–7). 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 (8–10). 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 (13–16). 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.
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.
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).
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.
Conflicts of Interest
No potential conflicts of interest to disclose.