Glaucoma is complex of devastating blinding diseases that together represent the primary cause of irreversible blindness in the U.S. There is substantial evidence that pathological mechanical stimulation mediated by an increase in intraocular pressure (IOP) plays a causal role in the etiology of glaucoma. The present proposal is to characterize the molecular mechanisms which might underlie the cellular response in this disease but could also play a key function in other diseases that involve mechanical stress in the retina, such as diabetic retinopathy, ischemia and macular edema. We found that a mechanosensitive cation channel, TRPV4, is selectively localized to retinal ganglion cells (RGCs) and in Muller glial cells. Because these are the two cell types that are specifically targeted in glaucoma, we hypothesize that mechanosensitive channels mediate the effects of pathological increases in IOP. The central focus of the proposal is to characterize this transduction mechanism in RGCs by combining biophysical, cellular and translational approaches. Studies proposed in Aim 1 will establish the molecular mechanism of mechanosensitive channel activation and desensitization, their role in calcium transport, cellular physiology and RGC survival. We will test conditions that mimic RGC injury in "low-tension" pathologies and test a number of models under which mechanosensitive channels might contribute to excitotoxic RGC injury. The proposed studies will also capitalize on preliminary work which shows remarkable effectiveness of non toxic small molecule antagonists in blocking pressure-stimulated loss of RGCs in vitro and in vivo glaucoma models.
Aim 2 of the proposed research builds on the characterization of pressure-sensitive channels in Aim 1 to study how these mechanisms regulate the swelling response of RGCs and retinal astroglia. Although cells typically swell in response to normal light-evoked neuronal activity, swelling is exacerbated in pathological conditions such as ischemia and diabetes, and can be highly neurotoxic. The proposed studies will explore the molecular complexes that involve swelling-activated calcium channels, water channels, calcium waves and regulatory volume decrease mechanisms. Thus, the goal of proposed research is to establish an intuitive conceptual and experimental framework that helps unify our understanding of retinal IOP transduction, cell swelling, and volume sensing and calcium homeostasis in retinal cells. By doing so, it will help predict the effects of mechanical forces that act through direct hydrostatic compression of cellular membranes as well as determine molecular mechanisms that are activated by tensile stretching, pulling and swelling. We will then test these predictions using mouse models of inducible and chronic glaucoma. This may help to refine our understanding of mechanical injury in vision disorders such as glaucoma, diabetic retinopathy and ischemia and contribute to developing effective neuroprotective treatments.
Excessive mechanical stimuli associated with increased intraocular pressure and cell swelling represents the main risk factor for developing retinal edema, ischemia and glaucoma. This application will test the hypothesis that retinal cells respond to such stimuli through specialized 'mechanosensitive'ion channels which, by increasing the intracellular concentration of calcium, regulate their pathophysiology. The proposed studies will elucidate the molecular mechanisms that regulate the function of these channels under low- and high-pressure conditions and determine whether suppression of these channels using non toxic small molecule antagonists protects the retina from neuronal degeneration in mouse glaucoma models.
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