David E. Kang, PhD - US grants

? 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.

The microtubule-associated protein tau (MAPT) aggregates and accumulates in multiple neurodegenerative diseases, including Alzheimer?s disease (AD). Abnormal tau accumulation leads to oligomerization and formation of neurofibrillary tangles associated with neuronal loss, synaptic dysfunction, and cognitive impairments. While tau undergoes different post-translational modifications including phosphorylation, acetylation, and ubiquitination, ubiquitination is critical for tau turnover via the ubiquitin-proteasome system and autophagy-lysosome pathways. Tau is known to be ubiquitinated by various E3 ligases, including CHIP, TRAF6, and MARCH7. However, very little is known about the role of deubiquitinases (DUBs) in the regulation of tau function, turnover, or tauopathy. The human genome encodes >90 DUBs. Ubiquitin specific peptidases (USPs) are the largest family of DUBs comprising ~50 members in humans. Of these, 27 are expressed in the CNS. Our results from an unbiased screen of CNS-expressed DUBs identified USP11 and USP13 as positive regulators of tau. By taking advantage of mouse models and human postmortem tissues together with molecular, cell biological, imaging, biochemical, electrophysiological, behavioral, viral, histochemical, and recombinant protein toolsets, this proposal will 1. validate the role of USP11 in tau pathogenesis as a function of age and sex in vivo, and 2. determine the mechanistic basis of USP11 in tau stability, aggregation, and toxicity in genetically modified neurons and in vitro systems. Successful conclusion of these studies will determine the significant contribution of USP11, and its DUB activity, to tauopathy in humans and mice as a function of aging and sex. USP11 levels could in part account for potential sex differences in severity of tauopathy in humans and mice. Moreover, these results will provide novel mechanistic insights to USP11 DUB activity, in concert with RanBP9, in tau modification and toxicity.

Project Summary The CHCHD10 gene coding for a mitochondrial protein is mutated in familial and sporadic Frontotemporal Dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), and mixed FTD-ALS. The estimated prevalence of CHCHD10 mutations is 7.7% among FTD in the Chinese population and 0.68-2.6% among FTD-ALS patients of European descent, making CHCHD10 the second most frequently mutated gene in FTD and FTD-ALS. We know that the FTD-ALS CHCHD10S59L mutation and the ALS CHCHD10R15L mutation promotes CHCHD10 aggregation and mitochondrial dysfunction. However, as only 1 patient with CHCHD10 mutation (ALS-linked CHCHD10R15L) and no FTD patient with CHCHD10 mutation has come to autopsy, we do not know the pathological signatures of CHCHD10-driven pathogenesis and to what extent such signatures are present in sporadic diseases (i.e. FTD, FTD-ALS, AD). As misfolded proteins tend to clog and inhibit the proteasome, such misfolded and aggregation-prone proteins are frequently translocated into mitochondria as an alternative pathway for proteostasis. We recently generated transgenic (Tg) mice expressing wild type (WT) CHCHD10WT, CHCHD10R15L, or CHCHD10S59L driven by the neuronal mouse PrP promoter, which show clear pathophysiological phenotypes. By taking advantage of mouse models and human postmortem tissues together with molecular, biochemical, histochemical, proteomics, electrophysiological, and behavioral toolsets, this proposal will test the overarching hypothesis that the loss of endogenous CHCHD10 (as seen in sporadic ADRDs) and FTLD/ALS-linked CHCHD10 mutations drive diverse pathological signatures resulting from disruptions in mitochondrial proteostasis and autophagic clearance of proteotoxically challenged mitochondria, and that restoration of WT CHCHD10 represents a viable strategy to mitigate proteotoxic burden and disease outcomes. Aim 1 will define the role of wild type CHCHD10 in mitigating pathological phenotypes in vivo. Aim 2 will identify and validate the neuropathological signatures of FTLD/ALS-linked CHCHD10 mutations. Aim 3 will determine the role of mutant CHCHD10 in mitophagy flux in vivo.