Impaired TRVP4-eNOS signaling in TM contributes to glaucoma

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Abstract Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Elevated intraocular pressure (IOP) due to dysfunction of trabecular meshwork (TM) tissue is a hallmark of POAG. However, the pathological mechanisms leading to TM dysfunction and IOP elevation are poorly understood. TM has an intrinsic ability to maintain IOP homeostasis by sensing the changes in the flow of aqueous humor (AH). In this regard, we recently showed that Ca2+ influx through transient receptor potential vanilloid 4 ion channels in the TM (TRPV4TM channels) lowers IOP via activation of endothelial nitric oxide synthase (eNOS)–NO signaling. Importantly, we showed that TRPV4TM-eNOSTM signaling is impaired in glaucomatous primary human TM cells. The major goals of this application are to elucidate the pathological mechanisms that impair TRPV4TM-eNOSTM signaling in glaucoma and to target them for rescuing the TM function. Chronic endoplasmic reticulum (ER) stress is a crucial contributor to TM dysfunction and IOP elevation in glaucoma. In our preliminary studies, we observed that chronic ER stress activates inducible NOS (iNOS), an enzyme commonly associated with the formation of oxidant molecule peroxynitrite (PN). PN levels are elevated in TM tissues from POAG donor eyes and exogenous PN reduced TRPV4TM channel activity in human primary TM cells. Moreover, induction of ER stress also lowered TRPV4TM channel activity. Therefore, we hypothesize that PN-induced inhibition of TRPV4TM-eNOSTM signaling contributes to TM dysfunction and IOP elevation in glaucoma. The major objectives of this application are to determine whether chronic ER stress leads to TM dysfunction and IOP elevation via PN-induced lowering of TRPV4TM-eNOSTM signaling in glaucoma, and to target this pathology for the treatment of glaucoma. In Aim 1, we will determine whether chronic ER stress lowers TRPV4 channel activity in TM cells. In Aim 2, we will determine whether PN levels are elevated in glaucoma and whether PN lowers TRPV4 channel activity in TM. We will also determine whether chronic ER stress underlies elevated PN levels in glaucoma. In Aim 3, we will target PN pathology to lower elevated IOP in mouse and human models of ocular hypertension. This proposal utilizes complementary expertise of Dr. Zode’s laboratory in glaucoma research and ER stress, and Dr. Sonkusare’s laboratory in TRP ion channel imaging and electrophysiology. This study will utilize state-of-art Ca2+ imaging, patch-clamp, eNOS activity, and nitric oxide measurements in primary human TM cells and TM tissues, human perfusion cultured donor eyes, and mouse models of glaucoma. Successful completion of the proposed studies will provide novel pathological mechanisms and therapeutic targets for the treatment of general POAG.