Muller glial cell pathophysiology during glaucoma onset

PROJECT SUMMARY The goal of this proposal is to understand how glial cell pathophysiology contributes to disease progression in glaucoma, a neurodegenerative eye disease characterized by dysfunction and death of retinal ganglion cells (RGCs), the output neurons of the retina. Elevated intraocular pressure (IOP) is the most common risk factor for glaucoma, yet the mechanisms linking high IOP and RGC loss remain poorly understood. Here, we focus on an important but previously unexamined aspect of disease progression: how early structural remodeling of Müller glial cells contributes to degeneration of RGCs. In glaucoma, as in other retinal diseases, Müller cells undergo a variety of molecular and morphological changes referred to as reactive gliosis. However, little is known regarding how Müller cells respond to ocular hypertension early in disease or how gliosis leads to RGC degeneration. We will use a combination of optical imaging, transgenic mice, viral techniques, and patch-clamp electrophysiology in an ex vivo eyecup preparation to fill these gaps in our knowledge. Aberrant Ca2+ signaling during disease onset may be an important upstream event that occurs prior to overt signs of Müller cell gliosis. In Aim 1A, we will test the hypothesis, suggested by our preliminary findings, that elevated pressure induces excessive Ca2+ signaling in Müller cell peri-synaptic processes within the inner plexiform layer (IPL) of the retina. We will use two-photon (2P) microscopy to compare Ca2+ signals in Müller glia in eyes with normal pressure vs. eyes in which IOP has been chronically elevated in a microbead occlusion model of glaucoma. In Aim 1B, we will directly test whether changes in Ca2+ signaling can drive morphological changes in Müller cells by selectively manipulating Müller cell intracellular [Ca2+]. Experiments in Aim 2A will use the microbead occlusion model and 2P imaging of fluorescently labeled Müller glia and RGCs to investigate whether ocular hypertension alters the physical relationship between Müller glial fine processes and RGC dendrites. In our preliminary work examining the morphology of Müller cells in tissue from eyes with chronically elevated IOP, we found that Müller cell processes retract within specific sublayers of the IPL. We will test the hypothesis, suggested by these preliminary findings, that Müller cell processes selectively withdraw from certain RGC subtypes soon after onset of ocular hypertension. An important function of Müller cell peri-synaptic processes is to regulate extracellular glutamate near synapses. Withdrawal of Müller glia could contribute importantly to disease progression by promoting RGC death via excitotoxic damage. Experiments in Aim 2B will examine this possibility by using two-photon Ca2+ imaging to test the hypothesis that elevated IOP increases light-evoked N-methyl-D-aspartate-type glutamate receptor activity in the dendrites of specific RGC subtypes. In both Aims, we will focus specifically on early-stage disease by conducting experiments within the first 1-2 weeks after IOP elevation. By identifying pathophysiological changes in Müller cells that occur early in disease progression, this work will help reveal novel ways to diagnose and treat glaucoma before RGC degeneration leads to irreversible vision loss.