Sarah N. Fontaine

Accelerated neurodegeneration through chaperone-mediated oligomerization of tau

Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5-/- mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimers disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

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.

Age-associated epigenetic upregulation of the FKBP5 gene selectively impairs stress resiliency.

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.

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

Background: High levels of constitutive heat shock protein 70 (Hsc70) preserve Tau levels, possibly contributing to neuropathology. Results: A dominant negative Hsc70 (DN-Hsc70) variant mimics small-molecule inhibition and facilitates Tau clearance by altering the associated interactome. Conclusion: DN-Hsc70 recruits a pro-degradation chaperome complement to the Tau-Hsc70 complex. Significance: Selective Hsc70 inhibition could have a therapeutic benefit in tauopathies including Alzheimer disease. 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.

Cellular factors modulating the mechanism of tau protein aggregation

Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones.

Exploiting the interaction between Grp94 and aggregated myocilin to treat glaucoma

Gain-of-function mutations in the olfactomedin domain of the MYOC gene facilitate the toxic accumulation of amyloid-containing myocilin aggregates, hastening the onset of the prevalent ocular disorder primary open-angle glaucoma. Aggregation of wild-type myocilin has been reported in other glaucoma subtypes, suggesting broader relevance of misfolded myocilin across the disease spectrum, but the absence of myocilin does not cause disease. Thus, strategies aimed at eliminating myocilin could be therapeutically relevant for glaucoma. Here, a novel and selective Grp94 inhibitor reduced the levels of several mutant myocilin proteins as well as wild-type myocilin when forced to misfold in cells. This inhibitor rescued mutant myocilin toxicity in primary human trabecular meshwork cells. Mechanistically, in vitro kinetics studies demonstrate that Grp94 recognizes on-pathway aggregates of the myocilin olfactomedin domain (myoc-OLF), accelerates rates of aggregation and co-precipitates with myoc-OLF. These results indicate that aberrant myocilin quaternary structure drives Grp94 recognition, rather than peptide motifs exposed by unfolded protein. Inhibition of Grp94 ameliorates the effects of Grp94-accelerated myoc-OLF aggregation, and Grp94 remains in solution. In cells, when wild-type myocilin is driven to misfold and aggregate, it becomes a client of Grp94 and sensitive to Grp94 inhibition. Taken together, the interaction of Grp94 with myocilin aggregates can be manipulated by cellular environment and genetics; this process can be exploited with Grp94 inhibitors to promote the clearance of toxic forms of myocilin.

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

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 Alzheimers 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.

Targeting the ER-autophagy system in the trabecular meshwork to treat glaucoma

A major drainage network involved in aqueous humor dynamics is the conventional outflow pathway, which is gated by the trabecular meshwork (TM). The TM acts as a molecular sieve, providing resistance to aqueous outflow, which is responsible for regulating intraocular pressure (IOP). If the TM is damaged, aqueous outflow is impaired, IOP increases and glaucoma can manifest. Mutations in the MYOC gene cause hereditary primary open-angle glaucoma (POAG) by promoting the abnormal amyloidosis of the myocilin protein in the endoplasmic reticulum (ER), leading to ER stress-induced TM cell death. Myocilin accumulation is observed in approximately 70-80% of all glaucoma cases suggesting that environmental or other genetic factors may also promote myocilin toxicity. For example, simply preventing myocilin glycosylation is sufficient to promote its abnormal accretion. These myocilin amyloids are unique as there are no other known pathogenic proteins that accumulate within the ER of TM cells and cause toxicity. Moreover, this pathogenic accumulation only kills TM cells, despite expression of this protein in other cell types, suggesting that another modifier exclusive to the TM participates in the proteotoxicity of myocilin. ER autophagy (reticulophagy) is one of the pathways essential for myocilin clearance that can be impacted dramatically by aging and other environmental factors such as nutrition. This review will discuss the link between myocilin and autophagy, evaluating the role of this degradation pathway in glaucoma as well as its potential as a therapeutic target.

Neurodegeneration and the Heat Shock Protein 70 Machinery: Implications for Therapeutic Development

The growing prevalence of individuals diagnosed with neurodegenerative disorders has brought into sharp relief the lack of treatment options for individuals struggling with these diseases. As more is discovered about the mechanisms of these neurodegenerative conditions, increasing evidence indicates a common theme in these proteinopathies is altered cellular protein homeostasis. In particular, the interactions of disease-associated proteins with the major cellular chaperones, heat shock proteins90 kDa and 70 kDa (Hsp90/Hsp70), are changed. Therefore, a promising strategy for therapeutic intervention is chemical inhibition and modification of these molecular chaperone proteins. Here we review the rationale behind therapeutic strategies targeting Hsp70 and its complement of co-chaperones, and describe the current literature regarding the use of small molecule inhibitors of Hsp70 in models of neurodegenerative disease.

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 Alzheimers 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.