Chad A. Dickey - US grants

[unreadable] DESCRIPTION (provided by applicant): The development of therapeutics for Alzheimer's disease (AD) is of critical importance to the United States due to the enormous expected growth of the elderly population within the next 2 decades. While much of the effort toward pharmacological intervention in this disease has been directed toward amyloid, recent evidence has emerged suggesting that the other central pathological component, tau, may be a necessary therapeutic target, particularly in patients who already have disease symptoms. Here, we have developed a unique methodology that allows for the rapid analysis of tau levels, providing an efficient way to monitor the efficacy of compounds directed toward intracellular targets such as tau. By using this technique we propose to 1) identify new compounds that alter tau and begin to define their mechanism of action, and 2) better characterize drugs that upregulate molecular chaperone expression a protective cascade that has been implicated in the removal of pathological tau species and which has also been implicated in other diseases linked to abnormal protein accumulation. Those compounds with the greatest promise based on in vitro analyses will then be used in animal models for tauopathy that are exclusive to the Mayo Clinic. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): A primary pathological component of Alzheimer's disease (AD) is the formation of neurofibrillary tangles (NFT) composed of hyper-phosphorylated tau (p-tau), a process closely linked to neurodegeneration. Expediting the removal of these p-tau species may be a highly relevant therapeutic stratagem. We have shown that inhibition of the ATPase activity of Hsp90 degrades p-tau independent of de novo chaperone transcription by heat shock factor-1; however multiple degradation pathways have been described for the tau protein, and these may hold equal importance with regard to the mechanisms underlying tau accumulation. Therefore we have begun investigating the mechanisms used by both the constitutive chaperone complex and novel independent pathways of degradation to facilitate the removal of abnormal p-tau. The Hsp90 complex typically works in concert with various interchangeable components (i.e.E3 ubiquitin ligases, prolyl isomerases, etc) culminating in either complete or partial re-folding of the substrate, or its degradation. While several components specifically involved in p-tau degradation have been identified, we have found that the re-folding co-chaperone, P23, may also regulate tau biology, acting rather to prevent its degradation. This interaction would provide new evidence that AD pathogenesis is due in part to the mis-folding of the inherently linear tau protein, an event perhaps precipitated by amyloid accretion. In addition, the unique family of small heat shock proteins may act in an entirely different way to promote tau degradation. Therefore, in the mentored phase of this award, I plan to develop my skills in the administration of genetic material to the murine brain, focusing on viral mediated delivery of shRNAs and genes of interest. A major focus of this phase will be the delivery of the Hsp27 by AAV to tau transgenic mice to determine the impact that this would have on tau pathology. In the latter phase of the award, we will investigate two novel pathways that regulate tau degradation; one mediated primarily by Hsp27 and the other mediated by the mature Hsp90 complex. We plan to further investigate the impact that the bifurcation of the Hsp90 pathway might have on AD pathogenesis, exploring how restorative co-chaperones might not only prevent tau degradation, but may also promote its aggregation. In addition, we plan to examine the role that amyloid may have in promoting tau dysfunction to either impair or facilitate its processing via the chaperone network, perhaps providing a novel mechanism of AD onset. PUBLIC DESCRIPTION Alzheimer's disease is the result of abnormal protein accumulation in the brain with the primary risk factor being age. Our goal is to identify ways in which these proteins accumulate and perhaps identify new drug targets for the treatment of Alzheimer's disease. Specifically, we intend to focus on the removal of the proteins once they have already started to accumulate in an effort to reverse the progression of the disease rather than prevent it. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Recent evidence suggests that intermediate oligomers of the microtubule-associated protein tau are more neurotoxic in tauopathies than densely packed ?-sheet fibrils. However, this remains unproven because relevant mechanisms to trap distinct assemblies of tau aggregates have been lacking. Here we will fill these gaps in our knowledge by using the Hsp90/co-chaperone machinery to control tau structure and assembly to prove how tau aggregate structure relates to its toxicity. Our team showed that tau physically interacts with the chaperone Hsp90, providing the first 3-dimensional structure of a client complexed with Hsp90. While Hsp90 levels are largely static in the aging brain, a group of co-chaperones that can interface with tau through Hsp90 are much more dynamic; some rise and others fall during a lifetime. We have found that Hsp90 and one of the rising co-chaperones, the cis/trans peptidyl-prolyl isomerase (PPIase) FK506 binding protein 51 (FKBP51), coordinate to provoke tau pathogenesis by reducing tau ?-sheet amyloidosis. This corresponded with increased oligomerization and neurotoxicity in tau transgenic mice. Thus, we speculate that the Hsp90 complex controls whether tau aggregates into toxic or benign species depending on the associated co- chaperones. In fact, we now have evidence that just as FKBP51 levels increase in the aging brain, so do the levels of a second Hsp90-associated PPIase, cyclophilin 40 (CyP40/PPID), to an even greater extent than FKBP51. And just like FKBP51, CyP40 reduces tau aggregation and produces amorphous intermediates. Now, we have also discovered that two other co-chaperones, Aha1 and FKBP52, which decrease in the aging brain, actually enhance the ?-sheet propensity of tau. With these tools, we can now test the hypothesis that tau toxicity arises due to structural changes in tau assemblies brought on by the Hsp90/co-chaperone system. To test this, we will determine if Hsp90/co-chaperone complexes that promote tau oligomer formation inevitably lead to toxicity. We will then determine if Hsp90/co-chaperone complexes that stimulate tau amyloidosis prevent its toxicity. Lastly we will determine the impact of Hsp90/co-chaperone complexes that favor tau amyloid or oligomer production on functional deficits in a mouse model of tauopathy. We anticipate that we will identify ways to regulate tau aggregation using the dynamic Hsp90 complex, which will allow us to home in on structures of toxic tau intermediates. We also will determine whether distinct Hsp90/co-chaperone complexes can differentially triage aberrant tau in the brain, possibly allowing us to improve the specificity of therapeutics targeting this mechanism.

DESCRIPTION (provided by applicant): Over 100,000 people in the US suffer from primary open-angle glaucoma (POAG) caused by mutations in the MYOC gene. This form of POAG results from optic nerve damage caused by the death of a protective cell network called the trabecular meshwork (TM). TM cell death occurs in these cases because mutant myocilin abnormally accumulates into toxic aggregates. This mechanism is reminiscent of neurodegenerative diseases, such as Alzheimer's, Huntington's and Parkinson's, where abnormal proteins accumulate in neurons and lead to cell death. In fact, TM cells are long-lived just like neurons. Moreover, mutations that cause earlier POAG onset also make myocilin aggregate more readily, similar to proteins associated with neurodegenerative diseases. Thus, both types of diseases can be considered proteostasis disorders, meaning that long-lived cells (neurons and TM) progressively lose their ability to prevent the toxic accumulation of mutant proteins with age. Thus, strategies aimed at restoring proteostasis in TM cells could be beneficial for glaucoma, just as they have proven for neurodegenerative disease. Through a series of studies, we determined that the Grp94 chaperone (an Hsp90 isoform) that resides in the endoplasmic reticulum, mistakenly preserves mutant myocilin in cells. Importantly, Grp94 only affects misfolded myocilin: Properly folded and functioning myocilin is unaffected by Grp94 manipulation. Grp94 recognizes only myocilin that is misfolded due to either mutations or impaired glycosylation: But Grp94 is unable to clear this misfolded myocilin, and instead, preserves it, causing its toxic accumulation. Thus, myocilin misfolding disrupts proteostasis by mistakenly engaging the Grp94 chaperone. We have shown that the clearance of toxic myocilin can be accelerated simply by inhibiting Grp94! Our team has developed the first isoform selective Grp94 inhibitor termed BnIm. Because the list of Grp94-dependent substrates is small, compared to other Hsp90 isoforms, the toxicity profile for this Grp94 inhibitor also appears low. Therefore, we propose to validate and improve upon this Grp94 inhibitor for the treatment of myocilin-associated POAG by establishing structure-activity relationships of Grp94 inhibitors to elucidate mechanisms of misfolded myocilin triage. We will also evaluate the biological efficacy of Grp94 inhibitors towards mutant myocilin in disease relevant systems and then work to develop Grp94 inhibitors with greater efficacy and biological activity towards misfolded myocilin. These studies will result in a new suite of Grp94 modulators and demonstrate that Grp94 is a novel clinical target to treat glaucoma caused by misfolded myocilin. In addition, mechanisms identified herein that clarify how Grp94 regulates myocilin triage could provide new insights for other proteostasis diseases.

DESCRIPTION (provided by applicant): In this revised application, we will investigate how the FK506 binding protein 5 (FKBP5) is up- regulated to coordinate how the brain responds to stress. Since 2004, our team has worked to show that single nucleotide polymorphisms (SNPs) in the FKBP5 gene associate with increased risk of psychopathologies caused by stress, as highlighted in Nature Genetics 36:1319-25 2004 & JAMA 299:1291-305 2008. We have also shown that these risk SNPs increase the levels of FKBP5 through a mechanism that involves demethylation of the FKBP5 gene (Nature Neuroscience. 16:33-41 2013 & Journal of Clinical Investigation 123:4158-69 2013). There are currently 2 other mechanisms besides these SNPs that are known to increase FKBP5 levels in the brain: 1) Stimulation of the glucocorticoid receptor (GR) by the steroid hormone cortisol (corticosterone/CORT), and 2) modulation of the receptor tyrosine kinase EphB2 (Attwood et al. Nature 473:372-5 2011). In our previous work, we found that mice lacking the FKBP5 gene (FKBP5-/- mice) are protected from stress-induced depressive-like phenotypes, and that apart from this improved resiliency, these mice seem very normal. Acute suppression of FKBP5 in the amygdala has also been shown to protect mice from anxiety-like behavior, but there are still gaps in our knowledge about the function and regulation of FKBP5 in the brain. While we know that FKBP5 does reduce resiliency to stress, we do not know whether chronically increased FKBP5 levels in the brain can fully model impaired stress resiliency through a mechanism that is similar to humans carrying risk SNPs. We also do not know how FKBP5 overexpression impacts learning and memory despite clear connections between stress and cognitive function. We know that chronic stress disrupts cognitive processes and electrophysiological function of neurons. But we do not know if stress- induced deficits in cognition, plasticity, hippocampal volume or neurogenesis are mediated by FKBP5. Lastly, we know that FKBP5 expression is up-regulated by GR and EphB2 signaling, and we also know that demethylation of the FKBP5 gene in humans contributes to FKBP5 upregulation. But we do not know how other epigenetic modifying proteins contribute to FKBP5 expression and demethylation, nor do we know how the EphB2 receptor regulates FKBP5 expression through a similar methylation mechanism. To fill these gaps, we will examine the effects of FKBP5 overexpression on stress resiliency and cognitive function, examine the effects of FKBP5 on cognitive and neuronal deficits caused by chronic stress and investigate the mechanisms that control FKBP5 expression. We anticipate that these studies will show the importance of FKBP5 to the brain's stress response, leading to new insights about its role in psychopathologies and cognitive function. We will also define new upstream regulators of FKBP5 expression.