||Progressive retinal ganglion cell (RGC) degeneration in glaucoma, the leading cause of permanent vision loss, is commonly caused by elevated intraocular pressure (IOP). Neuroprotective treatments complementing current IOP-reducing therapies could improve glaucoma management (Chapters 1, 3). IOP elevations induce glial reactivity (Chapter 4) and dysregulate RGC calcium (Chapters 2-3, 5-7), contributing to RGC degeneration (Chapters 5, 7), but it is unknown how glaucomatous forces perturb RGC and glial Ca2+ homeostasis. We discovered that mouse RGCs and Müller glia respond to osmotic pressure and tensile stretch with a cytosolic Ca2+ elevation that is primarily mediated by opening of the mechanosensitive cation channel transient receptor potential vanilloid 4 (TRPV4; Chapters 5-7). We therefore hypothesized that TRPV4 activation by glaucomatous forces drives RGC excitotoxicity. Consistent with this, intraocular injection of a selective TRPV4 agonist (GSK1016790A) induced mouse RGC loss (Chapter 7). This was prevented by systemic administration of a selective TRPV4 antagonist (HC-067047). Sustained exposure to glaucomatous mechanical strain caused RGC apoptosis, which was rescued by Ca2+ chelation or pharmacological/genetic TRPV4 antagonism, indicating that Ca2+ influx via TRPV4 is required for mechanical excitotoxicity (Chapter 7). Furthermore, RGCs and Müller glia swell during the progression of glaucoma and other blinding conditions, indicating the presence of aberrant osmotic gradients and loss of volume control. We found that RGC and Müller iv cell swelling is exacerbated by TRPV4-dependent Ca2+ influx. Swelling differentially activated TRPV4 in neurons and glia, the later of which required phospholipase A2- dependent production of 5,6-EET, an endogenous TRPV4 agonist. The water channel aquaporin 4 (AQP4) facilitated water entry, which enhanced glial TRPV4 activation (Chapter 6). Finally, we found that TRPV4 antagonism in mouse and primate glaucoma models lowered IOP to normal levels, potentially by promoting fluid drainage from the eye via the trabecular meshwork (TM). Although IOP elevation for eight weeks caused mouse RGC loss, this was prevented by daily treatment with a TRPV4 antagonist (Chapter 7). TRPV4 inhibition, therefore, simultaneously lowers IOP and increases RGC resilience. This, together with our finding that TRPV4 is expressed in human RGCs, Müller glia and TM cells (Chapters 6, 7), makes TRPV4 an attractive therapeutic target for prevention of glaucoma.