Project Summary Abstract While glaucoma is presently treated by lowering intraocular pressure (IOP), additional prevention of visual impairment may come from altering the detrimental tissue responses to IOP. Initial reports from our team, now confirmed by others, show that treatments affecting the cellular and connective tissues of the optic nerve head (ONH) are neuroprotective. The earliest effects on retinal ganglion cell axons at the ONH in glaucoma are mediated by astrocytes and microglia that respond to IOP-generated mechanical stress. Using mouse models and human glaucoma eyes, we have identified key junctional complexes at the interface between astrocytes and sclera at the ONH that are altered by IOP increase. Astrocytes at this location—the unmyelinated optic nerve (UON)—are relatively unique in structure to withstand scleral hoop stress and the translaminar pressure gradient. We are engaged in detailed study of the components of these junctional complexes and how they sense and translate stress into both beneficial and detrimental responses in glaucoma. The elements of this junctional pathway are known to involve Rho-kinase (ROCK) and its study and manipulation are key elements in the proposal. Astrocyte responses are now classified as protective or toxic in brain disease, and initial studies suggest these occur in glaucoma, in part mediated by interaction with microglia. A specific inhibitor of microglial activation of toxic astrocyte development (NLY01) will be studied for its mechanism of action and potential as therapy. We have developed and will utilize quantitative, rigorous assays for the effects on astrocytes of alteration in their structure and function, including ROCK inhibitors and the effects of transforming growth factor β pathway alterations. To facilitate the development of translational agents in later full glaucoma models, we developed a quantitative assay for axonal transport obstruction after 3 day IOP elevation. In a second major aim, astrocyte gene expression changes in the key UON region with experimental glaucoma will be fully studied in primary astrocytes, whole eye explants, and living mouse eyes. Both whole regional tissue digests and single cell RNAseq methodology is being employed to identify both how and in which cells the changes occur. The protein expression changes attendant to these alterations will be related to mechanosensation and mechanotranslation of astrocyte behavior and compared to quantitative structural changes at the microscopic level. Specific activators and inhibitors of the identified pathways will be used to point to potential treatment approaches. Several transgenic mouse models will be employed: 1) a strain with deficient laminin which is a key component of the connection of astrocytes to sclera; 2) an angiopoetin1 knockout that produces a new glaucoma model of higher IOP; and 3) a strain we have exploited to observe strain changes in the ONH in explants that has uniformly fluorescent as...