Graduation Year

2016

Document Type

Thesis

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Chad Dickey, Ph.D.

Committee Member

Yu Chen, Ph.D.

Committee Member

Daniel Lee, Ph.D.

Committee Member

Vladimir Uversky, Ph.D.

Keywords

myocilin, Grp94, glaucoma, chaperones, autophagy

Abstract

Glaucoma is a neurodegenerative protein misfolding disorder classified by increases in IOP, damage to retinal ganglion cells (RGCs), optic nerve (ON) head damage, and progressive irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form of glaucoma, constituting over 90% of clinical cases. POAG is observed in patients where normal outflow channels, mainly the trabecular meshwork (TM), are exposed at the angle formed by the iris and cornea. However, due to TM cellular dysfunction, aqueous outflow resistance is increased preventing normal circulation of aqueous humor. Recent studies have shown that in 2-4% of POAG cases, increased intracellular levels of a secreted glycoprotein, called myocilin, are present in the TM.

Myocilin is a 504aa glycoprotein, with an unknown precise function. Recent studies have postulated the importance of myocilin in oligodendrocyte differentiation, axonal myelination, and early apoptosis of retinal cells in development, but exact function is still widely debated. However, it is important to note that pathology associated with myocilin is only seen during POAG. Also, only cells of the TM exhibit toxicity when overexpressing mutant myocilin. In the normal eye, myocilin is secreted from the ER of TM cells, however, mutations in the MYOC gene lead to an aggregation-prone form of the protein, which is inefficiently processed and degraded from the ER, leading to build-up and associated toxicity. There are over 70 known MYOC mutations associated with glaucoma, with over 90% occurring in the C-terminal OLF domain. Some of the more common, pathological mutations are: I477N, Y437H, P370L, W286R, N480K. All of these mutations have been observed in patients with glaucoma, and all lead to build-up and aggregation of the mutant protein within the ER of TM cells.

Recently, work out of our lab has discovered an interaction between mutant forms of myocilin and the chaperone Grp94. Grp94 is the resident Hsp90 isoform of the ER. Grp94 is an important chaperone in ER quality control, aiding in the output of properly folded secretory and membrane-bound proteins. Besides protein folding, other roles of Grp94 in the ER include: calcium buffering, roles in ER quality control (including targeting misfolded proteins for ERAD), peptide binding, and roles in ER stress. Generally, terminally misfolded proteins in the ER are degraded through ERAD; the Grp94 mediated shuttling of misfolded proteins to the ER trans-membrane machinery for ubiquitination and subsequent translocation to the cytosol for proteasomal degradation. However, in the case of POAG, ERAD is inefficient in mutant myocilin degradation, causing protein accumulation within the ER.

In this study, we demonstrate that specific Grp94 inhibition of interaction with mutant or misfolded myocilin leads to myocilin degradation and subsequent lowering of protein accumulation in the TM, thus reducing downfield pathology associated with POAG. Grp94 preserves mutant myocilin in the ER of TM cells leading to protein accumulation and aggregation precipitating TM cellular dysfunction. We showed in various in vitro cell assays that Grp94 inhibition leads to a reduction in intracellular protein levels, while alleviating TM cellular toxicity. Furthermore, in the Tg-MYOCY437H mouse model of POAG, we showed that topical ocular administration of a specific Grp94 inhibitor alleviated glaucomatous pathologies, including elevated IOP, myocilin accumulation in the TM, reduced scotopic/photopic visual responses, and RGC health and viability. Finally, we have proven the importance of ER-stress pathway malfunction in the development of POAG pathology, while also discovering the involvement of the autophagy mechanism for myocilin degradation following Grp94 inhibition.

Overall, this work proves that Grp94 is an important regulator of myocilin pathology during POAG. While there are no current therapeutics on the market that directly target the underlying POAG disease mechanism, specific Grp94 inhibition shows great promise and should be considered for human clinical trials. If successful, specific targeted Grp94 inhibition could be the first curative therapeutic options for patients suffering from myocilin-associated POAG.

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