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Heat shock protein 70 (Hsp70) is a chaperone that normally scans the proteome and initiates the turnover of some proteins (termed clients) by linking them to the degradation pathways. This activity is critical to normal protein homeostasis, yet it appears to fail in diseases associated with abnormal protein accumulation. It is not clear why Hsp70 promotes client degradation under some conditions, while sparing that protein under others. Here, we used a combination of chemical biology and genetic strategies to systematically perturb the affinity of Hsp70 for the model client, tau. This approach revealed that tight complexes between Hsp70 and tau were associated with enhanced turnover while transient interactions favored tau retention. These results suggest that client affinity is one important parameter governing Hsp70-mediated quality control.

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

We previously discovered that one specific scalemic preparation of myricanol (1), a constituent of Myrica cerifera (bayberry/southern wax myrtle) root bark, could lower the levels of the microtubule-associated protein tau (MAPT). The significance is that tau accumulates in a number of neurodegenerative diseases, the most common being Alzheimer's disease (AD). Herein, a new synthetic route to prepare myricanol using a suitable boronic acid pinacol ester intermediate is reported. An X-ray crystal structure of the isolated myricanol (1) was obtained and showed a co-crystal consisting of (+)-aR,11S-myricanol (2) and (-)-aS,11R-myricanol (3) coformers. Surprisingly, 3, obtained from chiral separation from 1, reduced tau levels in both cultured cells and ex vivo brain slices from a mouse model of tauopathy at reasonable mid-to-low micromolar potency, whereas 2 did not. SILAC proteomics and cell assays revealed that 3 promoted tau degradation through an autophagic mechanism, which was in contrast to that of other tau-lowering compounds previously identified by our group. During the course of structure-activity relationship (SAR) development, we prepared compound 13 by acid-catalyzed dehydration of 1. 13 had undergone an unexpected structural rearrangement through the isomyricanol substitution pattern (e.g., 16), as verified by X-ray structural analysis. Compound 13 displayed robust tau-lowering activity, and, importantly, its enantiomers reduced tau levels similarly. Therefore, the semisynthetic analogue 13 provides a foundation for further development as a tau-lowering agent without its SAR being based on chirality.

BackgroundThe microtubule-associated protein tau accumulates in neurodegenerative diseases known as tauopathies, the most common being Alzheimer's disease. One way to treat these disorders may be to reduce abnormal tau levels through chaperone manipulation, thus subverting synaptic plasticity defects caused by tau's toxic accretion.MethodsTauopathy models were used to study the impact of YM-01 on tau. YM-01 is an allosteric promoter of triage functions of the most abundant variant of the heat shock protein 70 (Hsp70) family in the brain, heat shock cognate 70 protein (Hsc70). The mechanisms by which YM-01 modified Hsc70 activity and tau stability were evaluated with biochemical methods, cell cultures, and primary neuronal cultures from tau transgenic mice. YM-01 was also administered to acute brain slices of tau mice; changes in tau stability and electrophysiological correlates of learning and memory were measured.ResultsTau levels were rapidly and potently reduced in vitro and ex vivo upon treatment with nanomolar concentrations of YM-01. Consistent with Hsc70 having a key role in this process, overexpression of heat shock protein 40 (DNAJB2), an Hsp70 co-chaperone, suppressed YM-01 activity. In contrast to its effects in pathogenic tauopathy models, YM-01 had little activity in ex vivo brain slices from normal, wild-type mice unless microtubules were disrupted, suggesting that Hsc70 acts preferentially on abnormal pools of free tau. Finally, treatment with YM-01 increased long-term potentiation in tau transgenic brain slices.ConclusionsTherapeutics that exploit the ability of chaperones to selectively target abnormal tau can rapidly and potently rescue the synaptic dysfunction that occurs in Alzheimer's disease and other tauopathies.

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

The heat shock protein 70 (Hsp70) family of molecular chaperones has important functions in maintaining proteostasis under stress conditions. Several Hsp70 isoforms, especially Hsp72 (HSPA1A), are dramatically upregulated in response to stress; however, it is unclear whether these family members have biochemical properties that are specifically adapted to these scenarios. The redox-active compound, methylene blue (MB), has been shown to inhibit the ATPase activity of Hsp72 in vitro, and it promotes degradation of the Hsp72 substrate, tau, in cellular and animal models. Here, we report that MB irreversibly inactivates Hsp72 but not the nearly identical, constitutively expressed isoform, heat shock cognate 70 (Hsc70; HSPA8). Mass spectrometry results show that MB oxidizes Cys306, which is not conserved in Hsc70. Molecular models suggested that oxidation of Cys306 exposes Cys267 to modification and that both events contribute to loss of ATP binding in response to MB. Consistent with this model, mutating Cys267 and Cys306 to serine made Hsp72 largely resistant to MB in vitro, and overexpression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Furthermore, mutating Cys267 and Cys306 to the pseudo-oxidation mimic, aspartic acid, mirrored MB treatment: the C267D and C306D mutants had reduced ATPase activity in vitro, and overexpression of the C267/306D double mutant significantly reduced tau levels in cells. Together, these results suggest that redox sensing by specific cysteine residues in Hsp72, but not Hsc70, may be an important component of the chaperone response to oxidative stress.

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 min after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer's disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.

The RNA-binding protein, trans-active response DNA-binding protein 43 (TDP-43), is normally found in the nucleus, but in amyotrophic lateral sclerosis, frontal temporal dementia, and some cases of Alzheimer disease it is cleaved and mislocalized to the cytosol, leading to accumulation. The mechanisms contributing to this are largely unknown. Here, we show that part of the normal clearance cascade for TDP-43 involves the Cdc37/Hsp90 complex. An Hsp90 inhibitor that disrupts the Cdc37/Hsp90 complex reduced TDP-43 levels to a greater extent than a standard Hsp90 ATPase inhibitor. When Cdc37 was depleted, TDP-43 underwent proteolytic clearance that was dependent on nuclear retrotranslocation and autophagic uptake. Accumulation of the microtubule-associated protein tau prevented the clearance of cleaved TDP-43, but not its production. This caused cleaved TDP-43 to accumulate, a feature observed in the brain of persons with Alzheimer disease. Clearance of cleaved TDP-43 was also prevented by knockdown of the autophagic inducer beclin1. Thus, in cells where TDP-43 clearance is normally needed, a system that employs manipulation of the Hsp90 complex and autophagy exists. But when tau accumulation is occurring, cleaved TDP-43 can no longer be cleared, perhaps explaining the emergence of these co-pathologies.

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

BackgroundIt has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.ResultsHere, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.ConclusionsNeuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

Abstract Background It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection. Results Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced. Conclusions Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.