Certain mutations of MYOC, a gene expressed in trabecular meshwork (TM cells), are associated with the juvenile onset of POAG (primary open angle glaucoma). The gene product of MYOC, myocilin, is a secretory protein of unknown function. Targeted null mutations of MYOC show neither an elevated intraocular pressure nor manifest morphological abnormalities in the TM, indicating that the disease-causing mutations of MYOC produce proteins that cause negative effects in TM cells (called gain-of-function toxicity). What is the nature of this toxicity? What is the molecular mechanism underlying the toxicity? In this project, we hypothesize that certain mutations of MYOC result in unfolding of myocilin leading to its retention and aggregation in the endoplasmic reticulum (ER) of the TM cells. Persistent accumulation of secretory or membrane proteins in the ER lumen is known to cause """"""""ER stress."""""""" This stress elicits a unique but complex ER-to-nucleus signaling called """"""""Unfolded Protein Response"""""""" (UPR) consisting of mechanisms to restore homeostasis in the ER. A deficiency or overshoot in UPR is known to adversely affect some, if not all of the functions of the ER. Therefore we further hypothesize that retention of aberrant myocilin induces UPR which, in turn, leads to Ca 2+ deregulation in TM cells. A number of studies on the pharmacology of TM have clearly demonstrated that elevated intracellular Ca 2+ increases the contractility of the TM cells and decreases the outflow facility across TM. Thus, the specific aims of this project are to characterize UPR and its mechanisms resulting from overexpression/mutations in MYOC, and to determine the adverse effects of myocilin-induced UPR on Ca2+ homeostasis. UPR induced by overexpression of myocilin and mutant forms of MYOC will be examined in primary cultures of bovine TM and HEK-293T cell line, respectively. As a positive control of UPR, we will employ exogenous drugs known to cause protein unfolding in the ER (e.g., tunicamycin). We will characterize UPR in terms of activation of components of UPR signaling pathways and on activation of various ER-specific chaperones. Ca 2+ deregulation will be investigated by examining transcriptional activation of SERCA Ca2+ ATPase and structural components of capacitative calcium influx pathways. Our techniques and protocols include use of quantitative real-time PCR, Northern blotting, Western blotting, confocal microscopy, and coimmuniprecipitation. These studies will lead to the development of an essential knowledge base and influence research in the field of glaucoma pathophysiology, diagnostics, and therapeutics.
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