Chemical Manipulation of Hsp70 ATPase Activity Regulates Tau Stability

Jinwal, Umesh K.; Miyata, Yoshinari; Koren, John; Jones, Jeffrey R.; Trotter, Justin H.; Chang, Lyra; O’Leary, John C.; Morgan, David; Lee, Daniel; Shults, Cody L.; Rousaki, Aikaterini; Weeber, Edwin J.; Zuiderweg, Erik R. P.; Gestwicki, Jason E.; Dickey, Chad A.

doi:10.1523/jneurosci.3345-09.2009

Cited by 206 publications

(300 citation statements)

References 43 publications

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“…This cellular activity is not as dramatic as that of MKT-077, which reduced tau levels by >80% at 30 μM ( Figure 4D); 23 however, reducing tau by only ∼50% is predicted to provide benefits in some AD models. 4 Importantly, we also confirmed that, like MKT-077 and other Hsp70 inhibitors, 12,41,42 YM-08 did not induce a stress response, based on the unchanged levels of stress inducible Hsp72 (HSPA1) in the treated cell lysates ( Figure 4C). Finally, we found that all of the truncated compounds had significantly reduced anti-tau activity ( Figure 4D), consistent with the in vitro binding studies and the proposed importance of each of the three ring systems.…”

Section: ■ Results and Discussionsupporting

confidence: 73%

“…7,11 In cellular models, inhibitors of the ATPase activity of Hsp70 have been shown to favor tau turnover and "re-set" its homeostasis. 12 Moreover, first-generation Hsp70 inhibitors, such as methylene blue (MB), improve learning and memory in mouse models of tauopathy, 13,14 suggesting that this strategy holds promise for the eventual treatment of AD and other tauopathies.…”

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confidence: 99%

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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

Miyata

Lee

et al. 2013

ACS Chem. Neurosci.

114

121

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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.

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Section: ■ Results and Discussionsupporting

confidence: 73%

mentioning

confidence: 99%

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

Miyata

Lee

et al. 2013

ACS Chem. Neurosci.

114

121

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“…Indeed, this compound had superior anti-tau activity compared with other previously described Hsp70 inhibitors (Fig. 5A) (6,11,32,33). Importantly, tubulin levels were unchanged by Hsp70 inhibition.…”

Section: Resultsmentioning

confidence: 66%

“…Hsc70-mediated stabilization of tau levels may be relevant to tauopathies, as high levels of Hsc70 are found in brains of AD patients relative to other Hsp70 isoforms (30). Compounds that inhibit Hsp90 and Hsp70 family proteins reduce tau levels (6,11,(31)(32)(33) and rescue synaptic plasticity defects in tauopathy mouse models (33). Based on this evidence, we sought to determine why Hsc70 and tau are so intimately involved with each other and perhaps determine the most effective way to exploit this interaction for therapeutic intervention to treat tauopathies.…”

Section: Introductionmentioning

confidence: 99%

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics

Fontaine¹,

Martin²,

Akoury³

et al. 2015

Human Molecular Genetics

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The pathological accumulation of abnormally hyperphosphorylated and aggregated tau, a neuronal microtubule (MT)-associated protein that functions to maintain MT stability, is implicated in a number of hereditary and sporadic neurodegenerative diseases including frontotemporal dementia and Alzheimer's disease. Targeting tau for the treatment of these diseases is an area of intense interest and toward that end, modulation of cellular molecular chaperones is a potential therapeutic target. In particular, the constitutive Hsp70 isoform, Hsc70, seems highly interconnected with tau, preserving tau protein levels and synergizing with it to assemble MTs. But the relationship between tau and Hsc70, as well as the impact of this interaction in neurons and its therapeutic implications remain unknown. Using a human dominant negative Hsc70 that resembles isoform selective inhibition of this important chaperone, we found for the first time that Hsc70 activity is required to stimulate MT assembly in cells and brain. However, surprisingly, active Hsc70 also requires active tau to regulate MT assembly in vivo, suggesting that tau acts in some ways as a co-chaperone for Hsc70 to coordinate MT assembly. This was despite tau binding to Hsc70 as substrate, as determined biochemically. Moreover, we show that while chronic Hsc70 inhibition damaged MT dynamics, intermittent treatment with a small molecule Hsp70 inhibitor lowered tau in brain tissue without disrupting MT integrity. Thus, in tauopathies, where MT injury would be detrimental to neurons, the unique relationship of tau with the Hsc70 machinery can be exploited to deplete tau levels without damaging MT networks.

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“…We examined the role of Hsp70 and CHIP in fraction II on the ubiquitination of C331A nNOS. Methylene blue has been shown to inhibit the ATPase activity of Hsp70 (29) and to be a useful reagent for defining Hsp70-dependent ubiquitination (30). As shown in Fig.…”

Section: C331a Nnos Is Preferentially Ubiquitinated By Fraction Ii-mentioning

confidence: 99%

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

Clapp

Peng

Morishima

et al. 2010

Journal of Biological Chemistry

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It is established that suicide inactivation of neuronal nitricoxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N G -nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.Nitric-oxide synthases (NOS) are cytochrome P450-like hemoprotein enzymes that catalyze the conversion of L-arginine to nitric oxide and citrulline by a process that requires NADPH and molecular oxygen (1). There are three major mammalian isoforms as follows: neuronal NOS (nNOS), 2 endothelial NOS, and inducible NOS. NOS is bidomain in structure with an oxygenase domain, which contains the binding site for the heme, L-arginine, and tetrahydrobiopterin, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH (2). NOS is a highly regulated enzyme requiring homodimerization and bound calmodulin for efficient electron transfer from the flavins to the heme moiety to enable synthesis of NO. Another mechanism of regulation is the ubiquitination and proteasomal degradation of NOS (3). Of particular pharmacological interest is the finding that certain drugs cause the suicide inactivation, covalent alteration, ubiquitination, and proteasomal degradation of nNOS (3-8). This phenomenon is not unique to nNOS as it is well documented that the suicide inactivation of other P450 cytochromes leads to covalent alteration, enhanced ubiquitination, and proteasomal turnover of the enzymes (9). The C terminus of Hsc70-interacting protein (CHIP) has been shown to be an E3 ligase that ubiquitinates cytochromes P450 3A4 and 2E1 as well as nNOS (10 -12). The...

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Chemical Manipulation of Hsp70 ATPase Activity Regulates Tau Stability

Cited by 206 publications

References 43 publications

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

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 active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

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