Publications

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.

The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer's disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood-brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression.

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

The ATP-dependent 90 kDa heat shock protein, Hsp90, is a major regulator of protein triage, from assisting in nascent protein folding to refolding or degrading aberrant proteins. Tau, a microtubule associated protein, aberrantly accumulates in Alzheimer's disease (AD) and other neurodegenerative diseases, deemed tauopathies. Hsp90 binds to and regulates tau fate in coordination with a diverse group of co-chaperones. Imbalances in chaperone levels and activity, as found in the aging brain, can contribute to disease onset and progression. For example, the levels of the Hsp90 co-chaperone, FK506-binding protein 51 kDa (FKBP51), progressively increase with age. In vitro and in vivo tau models demonstrated that FKBP51 synergizes with Hsp90 to increase neurotoxic tau oligomer production. Inversely, protein phosphatase 5 (PP5), which dephosphorylates tau to restore microtubule-binding function, is repressed with aging and activity is further repressed in AD. Similarly, levels of cyclophilin 40 (CyP40) are reduced in the aged brain and further repressed in AD. Interestingly, CyP40 was shown to breakup tau aggregates in vitro and prevent tau-induced neurotoxicity in vivo. Moreover, the only known stimulator of Hsp90 ATPase activity, Aha1, increases tau aggregation and toxicity. While the levels of Aha1 are not significantly altered with aging, increased levels have been found in AD brains. Overall, these changes in the Hsp90 heterocomplex could drive tau deposition and neurotoxicity. While the relationship of tau and Hsp90 in coordination with these co-chaperones is still under investigation, it is clear that imbalances in these proteins with aging can contribute to disease onset and progression. This review highlights the current understanding of how the Hsp90 family of molecular chaperones regulates tau or other misfolded proteins in neurodegenerative diseases with a particular emphasis on the impact of aging.

Traumatic brain injury (TBI) caused by improvised explosive devices (IEDs) is a growing problem in military settings, but modeling this disease in rodents to pre-clinically evaluate potential therapeutics has been challenging because of inconsistency between models. Although the effects of primary blast wave injury have been extensively studied, little is known regarding the effects of noncontact rotational TBIs independent of the blast wave. To model this type of injury, we generated an air cannon system that does not produce a blast wave, but generates enough air pressure to cause rotational TBI. Mice exposed to this type of injury showed deficits in cognitive and motor task acquisition within 1-2 weeks post-injury, but mice tested 7-8 weeks post-injury did not retain any deficits. This suggests that the effects of a single, noncontact rotational TBI are not long lasting. Despite the transient nature of the behavioral deficits, increased levels of phosphorylated tau were observed at 2 and 8 weeks post-injury; however, this tau did not adopt typical pathological structures that have been observed in other TBI models that incorporate blast waves. This was possibly attributed to the fact that this injury was insufficient to induce changes in microglial activation, which was not affected at 2 or 8 weeks post-injury. Taken together, these data suggest that exposure to noncontact, rotational head injury only produces transient cognitive anomalies, but elicits some minor lasting neuropathological changes.

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

Single nucleotide polymorphisms in the FKBP5 gene increase the expression of the FKBP51 protein and have been associated with increased risk for neuropsychiatric disorders such as major depression and post-traumatic stress disorder. Moreover, levels of FKBP51 are increased with aging and in Alzheimer disease, potentially contributing to disease pathogenesis. However, aside from its glucocorticoid responsiveness, little is known about what regulates FKBP5 In recent years, non-coding RNAs, and in particular microRNAs, have been shown to modulate disease-related genes and processes. The current study sought to investigate which miRNAs could target and functionally regulate FKBP5 Following in silico data mining and initial target expression validation, miR-511 was found to suppress FKBP5 mRNA and protein levels. Using luciferase p-miR-Report constructs and RNA pulldown assays, we confirmed that miR-511 bound directly to the 3'-UTR of FKBP5, validating the predicted gene-microRNA interaction. miR-511 suppressed glucocorticoid-induced up-regulation of FKBP51 in cells and primary neurons, demonstrating functional, disease-relevant control of the protein. Consistent with a regulator of FKBP5, miR-511 expression in the mouse brain decreased with age but increased following chronic glucocorticoid treatment. Analysis of the predicted target genes of miR-511 revealed that neurogenesis, neuronal development, and neuronal differentiation were likely controlled by these genes. Accordingly, miR-511 increased neuronal differentiation in cells and enhanced neuronal development in primary neurons. Collectively, these findings show that miR-511 is a functional regulator of FKBP5 and can contribute to neuronal differentiation.

Peptidyl-prolyl cis/trans isomerases (PPIases), a unique family of molecular chaperones, regulate protein folding at proline residues. These residues are abundant within intrinsically disordered proteins, like the microtubule-associated protein tau. Tau has been shown to become hyperphosphorylated and accumulate as one of the two main pathological hallmarks in Alzheimer's disease, the other being amyloid beta (Ab). PPIases, including Pin1, FK506-binding protein (FKBP) 52, FKBP51, and FKBP12, have been shown to interact with and regulate tau biology. This interaction is particularly important given the numerous proline-directed phosphorylation sites found on tau and the role phosphorylation has been found to play in pathogenesis. This regulation then affects downstream aggregation and oligomerization of tau. However, many PPIases have yet to be explored for their effects on tau biology, despite the high likelihood of interaction based on proline content. Moreover, Pin1, FKBP12, FKBP52, cyclophilin (Cyp) A, CypB, and CypD have been shown to also regulate Ab production or the toxicity associated with Ab pathology. Therefore, PPIases directly and indirectly regulate pathogenic protein multimerization in Alzheimer's disease and represent a family rich in targets for modulating the accumulation and toxicity.

INTRODUCTION: The blood-brain barrier (BBB) is damaged in tauopathies, including progressive supranuclear palsy (PSP) and Alzheimer's disease (AD), which is thought to contribute to pathogenesis later in the disease course. In AD, BBB dysfunction has been associated with amyloid beta (Aß) pathology, but the role of tau in this process is not well characterized. Since increased BBB permeability is found in tauopathies without Aß pathology, like PSP, we suspected that tau accumulation alone could not only be sufficient, but even more important than Aß for BBB damage.

RESULTS: Longitudinal evaluation of brain tissue from the tetracycline-regulatable rTg4510 tau transgenic mouse model showed progressive IgG, T cell and red blood cell infiltration. The Evans blue (EB) dye that is excluded from the brain when the BBB is intact also permeated the brains of rTg4510 mice following peripheral administration, indicative of a bonafide BBB defect, but this was only evident later in life. Thus, despite the marked brain atrophy and inflammation that occurs earlier in this model, BBB integrity is maintained. Interestingly, BBB dysfunction emerged at the same time that perivascular tau emerged around major hippocampal blood vessels. However, when tau expression was suppressed using doxycycline, BBB integrity was preserved, suggesting that the BBB can be stabilized in a tauopathic brain by reducing tau levels.

CONCLUSIONS: For the first time, these data demonstrate that tau alone can initiate breakdown of the BBB, but the BBB is remarkably resilient, maintaining its integrity in the face of marked brain atrophy, neuroinflammation and toxic tau accumulation. Moreover, the BBB can recover integrity when tau levels are reduced. Thus, late stage interventions targeting tau may slow the vascular contributions to cognitive impairment and dementia that occur in tauopathies.

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.