Molecular determinants of cytotoxicity due to mutant myocilin misfolding - Glaucoma is a heterogeneous ocular disease that affects nearly 70 million individuals worldwide. Despite the availability of symptomatic treatments and surgeries, ~10% of patients reach permanent blindness, and treatments do not address the underlying cause of the disease. In most glaucoma subtypes, elevated intraocular pressure (IOP) is the causal risk factor. Elevated IOP originates from trabecular meshwork (TM) dysfunction. Neuron-like TM cells maintain the TM extracellular matrix, an anterior eye segment tissue, and TM cell loss an established pathological hallmark of a glaucomatous TM. Genetics also support TM cell death as being an early event in glaucoma development. Coding mutations in myocilin are closely associated with the early-onset autosomal-dominant inherited form of glaucoma and comprise the strongest genetic link to glaucoma (4-10% of all glaucoma and ~30% of juvenile onset). Expressed robustly in TM cells, mutant myocilin cannot be secreted and instead mutant myocilin is sequestered in the endoplasmic reticulum (ER) where it is cytotoxic. TM cell loss hastens IOP elevation, leading to an accelerated timeline for retinal cell death and optic nerve damage in the posterior eye, which are the proximal causes of vision loss in glaucoma. Molecular pathways contributing to TM cytotoxicity are not established, however, hindering the development of disease- modifying therapies. We will use our newly developed baker’s yeast (Saccharomyces cerevisiae) model of myocilin-associated glaucoma to decipher the molecular mechanisms underlying the pathophysiology of TM cell loss in glaucoma. S. cerevisiae is a model system that has been used to decipher mechanisms underlying the pathophysiology of numerous human proteostasis disorders, including Parkinson’s, Alzheimer’s, and Huntington’s diseases, and has led to new therapeutic targets. Our S. cerevisiae model faithfully replicates the gain-of-function mechanism underlying glaucoma pathogenesis: when expressed in the ER of S. cerevisiae, mutant myocilin is cytotoxic, and, depending on the experimental design, wild-type myocilin can either be benign or moderately cytotoxic. Further, targeted screening of S. cerevisiae deletion clones associated with proteostasis has identified gene-specific modulation of cytotoxicity, with new targets beyond those identified previously from experiments using mammalian cells. We propose three Specific Aims to define the cytotoxicity mechanisms caused by myocilin misfolding: (1) screen the S. cerevisiae gene deletion library to identify clones that restore cell viability; (2) probe the mechanisms underlying preliminary hits obtained in preparation of Aim 1 and new hits that emerge as Aim 1 is completed; (3) conduct a high throughput screen to identify small molecules that rescue cytotoxicity due to mutant myocilin misfolding. The expected outcomes are gene products and pathways that sensitize cells to myocilin toxicity and small molecule leads for rescuing TM cell death. In the long term, this work will identify new targets for disease-modifying therapies for glaucoma.
