Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation.

In Alzheimer's disease (AD), the mechanisms of neuronal loss remain largely unknown. Although tau pathology is closely correlated with neuronal loss, how its accumulation may lead to activation of neurotoxic pathways is unclear. Here we show that tau increased the levels of ubiquitinated proteins in the brain and triggered activation of the unfolded protein response (UPR). This suggested that tau interferes with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and tau transgenic (rTg4510) mouse brains, but this was not always colocalized with tau. The increased levels of ubiquitinated protein were accompanied by increased levels of phosphorylated protein kinase R-like ER kinase (pPERK), a marker that indicates UPR activation. Depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including valosin-containing protein (VCP) and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3δ reporter, an ERAD substrate. Indeed, CD3δ accumulated in both in vitro and in vivo models of tau overexpression and AD brains. These data suggest that soluble tau impairs ERAD and the result is activation of the UPR. The reversibility of this process, however, suggests that tau-based therapeutics could significantly delay this type of cell death and therefore disease progression.

Imbalance of Hsp70 family variants fosters tau accumulation.

Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants can differentially regulate tau triage. Thus, efforts to promote Hsp72 expression and inhibit Hsc70 could be therapeutically relevant for tauopathies.

Cdc37/Hsp90 protein complex disruption triggers an autophagic clearance cascade for TDP-43 protein.

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.

Glucose-regulated protein 94 triage of mutant myocilin through endoplasmic reticulum-associated degradation subverts a more efficient autophagic clearance mechanism.

BACKGROUND: Mutant myocilin accumulates in the endoplasmic reticulum for unknown reasons. RESULTS: Glucose-regulated protein (Grp) 94 depletion reduces mutant myocilin by engaging autophagy. CONCLUSION: Grp94 triages mutant myocilin through ER-associated degradation, subverting autophagy. SIGNIFICANCE: Treating glaucoma could be possible by inhibiting Grp94 and reducing its novel client, mutant myocilin. Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that ß-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.

The Hsp90 kinase co-chaperone Cdc37 regulates tau stability and phosphorylation dynamics.

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3ß and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules.

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

Phosphorylation dynamics regulate Hsp27-mediated rescue of neuronal plasticity deficits in tau transgenic mice.

Molecular chaperones regulate the aggregation of a number of proteins that pathologically misfold and accumulate in neurodegenerative diseases. Identifying ways to manipulate these proteins in disease models is an area of intense investigation; however, the translation of these results to the mammalian brain has progressed more slowly. In this study, we investigated the ability of one of these chaperones, heat shock protein 27 (Hsp27), to modulate tau dynamics. Recombinant wild-type Hsp27 and a genetically altered version of Hsp27 that is perpetually pseudo-phosphorylated (3×S/D) were generated. Both Hsp27 variants interacted with tau, and atomic force microscopy and dynamic light scattering showed that both variants also prevented tau filament formation. However, extrinsic genetic delivery of these two Hsp27 variants to tau transgenic mice using adeno-associated viral particles showed that wild-type Hsp27 reduced neuronal tau levels, whereas 3×S/D Hsp27 was associated with increased tau levels. Moreover, rapid decay in hippocampal long-term potentiation (LTP) intrinsic to this tau transgenic model was rescued by wild-type Hsp27 overexpression but not by 3×S/D Hsp27. Because the 3×S/D Hsp27 mutant cannot cycle between phosphorylated and dephosphorylated states, we can conclude that Hsp27 must be functionally dynamic to facilitate tau clearance from the brain and rescue LTP; however, when this property is compromised, Hsp27 may actually facilitate accumulation of soluble tau intermediates.

Hsc70 rapidly engages tau after microtubule destabilization.

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7-9 interact with residues 312-342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.

The diarylheptanoid (+)-aR,11S-myricanol and two flavones from bayberry (Myrica cerifera) destabilize the microtubule-associated protein tau.

Target-based drug discovery for Alzheimer's disease (AD) centered on modulation of the amyloid ß peptide has met with limited success. Therefore, recent efforts have focused on targeting the microtubule-associated protein tau. Tau pathologically accumulates in more than 15 neurodegenerative diseases and is most closely linked with postsymptomatic progression in AD. We endeavored to identify compounds that decrease tau stability rather than prevent its aggregation. An extract from Myrica cerifera (bayberry/southern wax myrtle) potently reduced both endogenous and overexpressed tau protein levels in cells and murine brain slices. The bayberry flavonoids myricetin and myricitrin were confirmed to contribute to this potency, but a diarylheptanoid, myricanol, was the most effective anti-tau component in the extract, with potency approaching the best targeted lead therapies. (+)-aR,11S-Myricanol, isolated from M. cerifera and reported here for the first time as the naturally occurring aglycone, was significantly more potent than commercially available (±)-myricanol. Myricanol may represent a novel scaffold for drug development efforts targeting tau turnover in AD.

Age-associated epigenetic upregulation of the FKBP5 gene selectively impairs stress resiliency.

Single nucleotide polymorphisms (SNPs) in the FK506 binding protein 5 (FKBP5) gene combine with traumatic events to increase risk for post-traumatic stress and major depressive disorders (PTSD and MDD). These SNPs increase FKBP51 protein expression through a mechanism involving demethylation of the gene and altered glucocorticoid signaling. Aged animals also display elevated FKBP51 levels, which contribute to impaired resiliency to depressive-like behaviors through impaired glucocorticoid signaling, a phenotype that is abrogated in FKBP5-/- mice. But the age of onset and progressive stability of these phenotypes remain unknown. Moreover, it is unclear how FKBP5 deletion affects other glucocorticoid-dependent processes or if age-associated increases in FKBP51 expression are mediated through a similar epigenetic process caused by SNPs in the FKBP5 gene. Here, we show that FKBP51-mediated impairment in stress resiliency and glucocorticoid signaling occurs by 10 months of age and this increased over their lifespan. Surprisingly, despite these progressive changes in glucocorticoid responsiveness, FKBP5-/- mice displayed normal longevity, glucose tolerance, blood composition and cytokine profiles across lifespan, phenotypes normally associated with glucocorticoid signaling. We also found that methylation of Fkbp5 decreased with age in mice, a process that likely explains the age-associated increases in FKBP51 levels. Thus, epigenetic upregulation of FKBP51 with age can selectively impair psychological stress-resiliency, but does not affect other glucocorticoid-mediated physiological processes. This makes FKBP51 a unique and attractive therapeutic target to treat PTSD and MDD. In addition, aged wild-type mice may be a useful model for investigating the mechanisms of FKBP5 SNPs associated with these disorders.

The role of FKBP5 in mood disorders: action of FKBP5 on steroid hormone receptors leads to questions about its evolutionary importance.

Research on the FKBP5 gene and FKBP51 protein has more than doubled since the discovery that polymorphisms in this gene could alter treatment outcomes and depressive behavior in humans. This coincided with other data suggesting that the stress hormone axis contributes to the development of numerous mental illnesses. As a result, FKBP51 now lies at the heart of the research of many stress related psychiatric disorders, which has led to advances in the understanding of this protein and its role in humans and in animal models. Specifically, FKBP5-/- mice and a naturally existing overexpression of FKBP5 in 3 genera of new world monkeys have helped understand the effects of FKBP5 in vivo. This review will highlight these finding as well as discuss the current evolutionary need for the FKBP5 gene.

Accelerated neurodegeneration through chaperone-mediated oligomerization of tau.

Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5-/- mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimer's disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.

Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

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.

Neuronal life span versus health span: principles of natural selection at work in the degenerating brain.

Impaired nutrient delivery to the brain due to decreased blood flow contributes to cognitive decline and dementia in Alzheimer's disease (AD). Considering this, many studies have suggested that neuroprotective agents like those used in stroke could prevent AD onset or progression by promoting cell survival. However, research in the past decade suggests that the culprit behind the cognitive loss in AD models is actually the soluble tau accumulating inside of surviving neurons. In fact, tau reductions improve cognition in mouse models of AD, even those that only deposit amyloid plaques. There is emerging evidence that neuroprotection alone in these AD models may be insufficient to restore neuron function and cognition. Only when soluble tau is reduced on a neuroprotective background could memory be rescued. Thus, once a neuron begins to accumulate tau, it may survive in a malfunctioning capacity, leading to impaired electrical signaling and memory formation in the brain. These data imply that multiple drugs may be necessary to ameliorate the different disease components. In fact, strategies to preserve neurons without affecting the soluble protein burden within neurons may accelerate the disease course.

A new anti-depressive strategy for the elderly: ablation of FKBP5/FKBP51.

The gene FKBP5 codes for FKBP51, a co-chaperone protein of the Hsp90 complex that increases with age. Through its association with Hsp90, FKBP51 regulates the glucocorticoid receptor (GR). Single nucleotide polymorphisms (SNPs) in the FKBP5 gene associate with increased recurrence of depressive episodes, increased susceptibility to post-traumatic stress disorder, bipolar disorder, attempt of suicide, and major depressive disorder in HIV patients. Variation in one of these SNPs correlates with increased levels of FKBP51. FKBP51 is also increased in HIV patients. Moreover, increases in FKBP51 in the amygdala produce an anxiety phenotype in mice. Therefore, we tested the behavioral consequences of FKBP5 deletion in aged mice. Similar to that of naïve animals treated with classical antidepressants FKBP5-/- mice showed antidepressant behavior without affecting cognition and other basic motor functions. Reduced corticosterone levels following stress accompanied these observed effects on depression. Age-dependent anxiety was also modulated by FKBP5 deletion. Therefore, drug discovery efforts focused on depleting FKBP51 levels may yield novel antidepressant therapies.

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

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.

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden.

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.