Identifying and leveraging strategies of inherently resilient retinal neurons to treat degeneration

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ABSTRACT Retinal ganglion cells (RGCs) are the sole connection between the eye and the brain. They are particularly susceptible to degeneration, and their damage and death leads to vision loss in conditions like glaucoma, diabetic retinopathy, optic nerve glioma, and optic neuritis. Most treatments for these diseases are not focused on specifically rescuing RGCs, but on relieving apparent drivers of disease progression. For example, current glaucoma treatments focus on reducing elevated intraocular pressure (IOP), but are not effective in the majority of patients. Further, many glaucoma patients also have RGC degeneration without IOP elevations. Thus, new treatments to preserve RGCs in degenerative diseases represent an important unmet clinical need. Although RGC cell death leads to vision loss, RGC death in degenerative conditions is incomplete even in severely affected patients and robust animal models. Understanding how some RGCs natively persist in degenerative conditions can inform the development of new treatment strategies. To identify native coping strategies, we will directly observe cellular traits of individual RGCs prior to and during the course of degeneration, focusing on cellular homeostasis. We have established longitudinal, in vivo, 2-photon imaging of genetically encoded biosensors in RGCs to directly observe energetic and Ca2+ homeostasis at single RGC resolution repeatedly over a protracted period of time. This approach allows for measurements that would normally require either end point sample collection, pooling of RGCs from multiple retinae, or both; limitations that obscure population heterogeneity and individual cell dynamics. We will characterize baseline heterogeneity of energetic and Ca2+ homeostasis, along with dynamics following axon injury and directly relate these measurements with RGC survival or death. Mechanisms of homeostasis are highly relevant to a range of degenerative diseases but have yet to be thoroughly investigated in models of RGC degeneration. Our preliminary data indicate that mouse RGCs that natively survive optic nerve crush have salient features of energetic and Ca2+ homeostasis that can be distinguished from the RGC population as a whole prior to induction of degeneration. These results strongly suggest that homeostatic set-points influence RGC survival outcomes in a severe degeneration model. Further, we will conduct experiments to preserve RGCs in optic nerve crush models by manipulating these pathways to mimic the properties of resilient RGCs using both gene overexpression or repression interventions. Doing so we can validate which of our observations are correlative or causative. The goals of our proposal are thus to: more thoroughly define the homeostatic fingerprint of well surviving RGCs; determine how axotomy induced degeneration impinges on homeostasis of well-surviving versus poorly-surviving RGCs; and translate this information into interventions that preserve RGCs that would otherwise degenerate. Taken together our experiments will identify and validate new approaches towards protection of RGCs.