PROJECT SUMMARY Recent studies of clinical/postmortem and experimental samples have accumulated evidence that supports a connection of inflammatory responses to neurodegeneration in glaucoma. Although inflammatory aspects of glaucomatous neurodegeneration are attracting increasing attention, how neuroinflammation is regulated in glaucoma, and how we can prevent neurodegenerative inflammation remain unknown. Here, we present experimental data leading us to hypothesize that NF-κB plays a key role in activating the glia-driven neuroinflammation in the glaucomatous retina and optic nerve, and that targeting this transcriptional activator mechanism is a logical strategy to provide immunomodulation and avoid secondary injury processes. With respect to intimate inter-relationship between astroglia and microglia in induction of the inflammatory/neurotoxic phenotype, NF-κB, the key transcriptional activator of inflammatory mediators, should be critical for both glial subtypes and their interaction in neuroinflammation in glaucoma. To test this hypothesis, we will comparatively study glial subtype-targeted conditional transgenic lines (in which the NF-κB canonical pathway is inactivated by drug-induced deletion of IκKβ in astroglia or microglia) and background controls with or without ocular hypertension (induced by anterior chamber microbead/viscoelastic injections). We will longitudinally analyze glial subtype-targeted transgenic effects on a diverse set of inflammatory outcomes at different sites of injury (retina, optic nerve head, and optic nerve axons). The studied outcomes will include the glial inflammatory activation phenotype and cell type-specific effects of glial subtype-targeted IκKβ deletion. Besides a preselected set of molecules, freshly isolated samples of astroglia and microglia will be analyzed by quantitative mass spectrometry to also gain high-throughput molecular information about transgenic effects on NF-κB-regulated inflammation pathways, other transcriptional targets of NF-κB, and molecules relevant to glial neurosupport functions. Since glia drive both innate and adaptive immune responses in glaucoma, additional analysis will include T-cell reactivity and autoantibody production. To determine whether transgenic inhibition of glial NF-κB (and neuroinflammation) protects neurons from immunogenic injury in mouse glaucoma, we will also analyze neuron structure (by RGC and axon counts) and function (by PERG). We expect that this project, complementing the astroglia- or microglia- targeted transgenic lines with molecular analysis of astroglia-specific and microglia-specific responses (in isolated cell type-specific samples) will allow us to determine the role of NF-κB in individual contribution and inter- relationship of astroglia and microglia in neuroinflammation (in the retina, optic nerve head, and axons), characterize specific molecular components, and value glial NF-κB as a treatment target to restore immune homeostasis and provi...