Hsp90-interacting Co-chaperones and their Family Proteins in Tau Regulation: Introducing a Novel Role for Cdc37L1.

Tau is a microtubule-associated protein that serves as a promoter of microtubule assembly and stability in neuron cells. In a collective group of neurodegenerative diseases called tauopathies, tau processing is altered as a result of gene mutations and post-translational modifications. In particular, in Alzheimer's disease (AD) or AD-like conditions, tau becomes hyperphosphorylated and forms toxic aggregates inside the cell. The chaperone heat shock protein 90 (Hsp90) plays an important role in the proper folding, degradation, and recycling of tau proteins and tau kinases. Hsp90 has many co-chaperones that aid in tau processing. In particular, a few of these co-chaperones, such as FK506-binding protein (FKBP) 51, protein phosphatase (PP) 5, cell division cycle 37 (Cdc37), and S100A1 have family members that are reported to affect Hsp90-mediated tau processing in either a similar or an opposite manner. Here, we provide a holistic review of these selected co-chaperones and their family proteins and introduce a novel Hsp90-binding Cdc37 relative, Cdc37-like-1 (Cdc37L1 or L1) in tau regulation. Overall, the proteins discussed here highlight the importance of studying family proteins in order to fully understand the mechanism of tau pathogenesis and to establish drug targets for the treatment of tauopathies.

Therapeutic Potential of the Hsp90/Cdc37 Interaction in Neurodegenerative Diseases.

Alzheimer's, Huntington's, and Parkinson's are devastating neurodegenerative diseases that are prevalent in the aging population. Patient care costs continue to rise each year, because there is currently no cure or disease modifying treatments for these diseases. Numerous efforts have been made to understand the molecular interactions governing the disease development. These efforts have revealed that the phosphorylation of proteins by kinases may play a critical role in the aggregation of disease-associated proteins, which is thought to contribute to neurodegeneration. Interestingly, a molecular chaperone complex consisting of the 90 kDa heat shock protein (Hsp90) and Cell Division Cycle 37 (Cdc37) has been shown to regulate the maturation of many of these kinases as well as regulate some disease-associated proteins directly. Thus, the Hsp90/Cdc37 complex may represent a potential drug target for regulating proteins linked to neurodegenerative diseases, through both direct and indirect interactions. Herein, we discuss the broad understanding of many Hsp90/Cdc37 pathways and how this protein complex may be a useful target to regulate the progression of neurodegenerative disease.

Stabilizing the Hsp70-Tau Complex Promotes Turnover in Models of Tauopathy.

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.

Identification of Apo B48 and other novel biomarkers in amyotrophic lateral sclerosis patient fibroblasts.

AIM: Amyotrophic lateral sclerosis (ALS) is a debilitating fatal neurodegenerative disorder. 90-95% of ALS cases are sporadic with no clear risk factors associated with the disease. Identification of biomarkers associated with ALS may lead to early detection and make it more amenable to therapeutic intervention. MATERIALS & METHODS: SILAC was used to quantitatively analyze the proteomes of ALS and control human fibroblasts. RESULTS: Out of total of 861 proteins identified, 33 were found to be differentially regulated. ApoB48 and Hsp20 were downregulated, while Fibulin-1 was upregulated. CONCLUSION: We report the differential regulation of these proteins in ALS fibroblasts, and their potential as novel biomarkers and possible drug targets for ALS.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

Identification and quantitative analysis of cellular proteins affected by treatment with withaferin a using a SILAC-based proteomics approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Withaferin A (WA) is a major bioactive compound isolated from the medicinal plant Withania somnifera Dunal, also known as "Ashwagandha". A number of published reports suggest various uses for WA including its function as an anti-inflammatory and anti-angiogenic drug molecule. The effects of WA at the molecular level in a cellular environment are not well understood. Knowledge of the molecular mechanism of action of WA could enhance its therapeutic value and may reveal novel pathways it may modulate. MATERIALS AND METHODS: In order to identify and characterize proteins affected by treatment with WA, we used SILAC- based proteomics analysis on a mouse microglial cell line (N9), which replicates phenotypic characteristics of primary microglial cells. RESULTS: Using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry (MS), a total of 2300 unique protein groups were identified from three biological replicates, with significant expression changes in 32 non-redundant proteins. The top biological functions associated with these differentially expressed proteins include cell death and survival, free radical scavenging, and carbohydrate metabolism. Specifically, several heat shock proteins (Hsps) were found to be upregulated, which suggests that the chaperonic machinery might be regulated by WA. Furthermore, our study revealed several novel protein molecules that were not previously reported to be affected by WA. Among them, annexin A1, a key anti-inflammatory molecule in microglial cells was found to be downregulated. Hsc70, Hsp90α and Hsp105 were found to be upregulated. We also found sequestosome1/p62 (p62) to be upregulated. We performed Ingenuity Pathway Analysis (IPA) and found a number of pathways that were affected by WA treatment. CONCLUSIONS: SILAC-based proteomics analysis of a microglial cell model revealed several novel proteins whose expression is regulated by WA and probable pathways regulated by WA.

An optimized InCell Western screening technique identifies hexachlorophene as a novel potent TDP43 targeting drug.

TAR DNA binding protein (TDP43) is a DNA- and RNA-binding protein that is implicated in several neurodegenerative disorders termed as "TDP43 proteinopathies" including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and fronto-temporal lobe dementia (FTLD). We have developed an InCell Western (ICW) technique for screening TDP targeting drugs in 96 well plates. We tested 281 compounds and identified a novel compound hexachlorophene (referred to as B10) that showed potent reduction in TDP43 levels. The effect of B10 on TDP protein level was validated in two different cellular models: endogenous TDP43 expressing N9 microglial cells and TDP43-over-expressing HEK293 and HeLa cells. We also analyzed effect of B10 on various pathological forms of TDP such as the C25 cleaved fragment that localizes to the cytosol, insoluble high molecular weight species, and ALS-linked mutants. Our data suggest that B10 effectively reduces all forms of TDP. Overall, our data suggest that B10 could serve as a potential drug molecule for the treatment of AD, ALS and other TDP43 proteinopathies.

Withaferin A Regulates LRRK2 Levels by Interfering with the Hsp90- Cdc37 Chaperone Complex.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has been found to be mutated in patients with familial and sporadic Parkinson's disease, Alzheimer's disease and Crohn's disease. While the functions of LRRK2 are still largely unclear and mutations in LRRK2 are associated with adverse gain-of-function activities such as increased kinase activity, increased levels of LRRK2 alone are associated with toxicity in neurons. Consequently, exploring mechanisms to decrease levels of LRRK2 using pharmacological inhibitors would be highly advantageous. Previous work has shown that the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Cdc37 interact with and stabilize LRRK2. In the current study, we explore the regulation of LRRK2 by withaferin A (WA), a potent inhibitor of the interaction between Hsp90 and Cdc37. We report that treatment of the microglial cell line N9 with WA causes a decrease in cellular levels of LRRK2 in a dose- and time-dependent manner. We also find that treatment with WA disrupts the interaction between Hsp90, its co-chaperone Cdc37 and LRRK2, which leads to the destabilization and decreased levels of LRRK2. Additionally, treatment with celastrol, which is also an inhibitor of the Hsp90-Cdc37 complex, decreased LRRK2 levels. Interestingly, treatment with WA in the presence of celastrol enhanced the clearance of LRRK2. Overall, our data suggest that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex, which may have implications in the search for therapeutic strategies for Alzheimer's disease, Parkinson's disease and other LRRK2 proteinopathies.

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement.

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.

Synthesis, stereochemical analysis, and derivatization of myricanol provide new probes that promote autophagic tau clearance.

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.

Preparation and characterization of methylene blue nanoparticles for Alzheimer's disease and other tauopathies.

Methylene blue (MB) has been shown to slow down the progression of the Alzheimer's disease (AD) and other tauopathies; however distribution of MB into the brain is limited due its high hydrophilicity. In this study, we aimed to prepare novel hydrophobic glutathione coated PLGA nanoparticles to improve bioavailability of MB in the brain. Glutathione coated poly-(lactide-co-glycolide) (PLGA-b-PEG) nanoparticles (NPs) were prepared and tested in two different cell culture models of AD expressing microtubule associated protein tau (tau). The NPs showed a particle size averaging 136.5±4.4nm, which is suitable for the blood brain barrier (BBB) permeation. The in vitro release profile of the NPs exhibited no initial burst release and showed sustained drug release for up to 144 hours. Interestingly, treatment of newly formulated MB-NPs showed a potent reduction in both endogenous and over expressed tau protein levels in human neuroblastoma SHSY-5Y cells expressing endogenous tau and transfected HeLa cells over-expressing tau protein, respectively. Furthermore, in vitro BBB Transwell™ study showed significantly higher permeation of MB-NP compared to the MB solution through the co culture of rat brain endothelial 4 (RBE4) and C6 astrocytoma cells (p<0.05). The proposed MB loaded nanoparticles could provide a more effective treatment option for AD and many other related disorders.

Reconstructing the Hsp90/Tau Machine.

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. Recent work demonstrates 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 (AD). The Hsp90 co-chaperone repertoire has diverse effects on tau stability; some co-chaperones stabilize tau while others facilitate its clearance. We propose that each of these proteins may be novel therapeutic targets. While targeting Hsp90 directly may be deleterious at the organismal level, perhaps targeting individual co-chaperone activities will be more tolerable.

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.

Allosteric heat shock protein 70 inhibitors rapidly rescue synaptic plasticity deficits by reducing aberrant tau.

BACKGROUND: The 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. METHODS: Tauopathy 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. RESULTS: Tau 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. CONCLUSIONS: Therapeutics 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.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.

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.

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.

Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.

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.

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.