Summary Glaucoma is a common neurodegenerative disorder characterized by a progressive loss of retinal ganglion cells (RGCs) that can lead to visual impairment and blindness. Age, elevated intraocular pressure (IOP), and genetics are the main risk factors. In a search for neuroprotective factors, we identified astrocytic SPP1 (Secreted phosphoprotein 1). SPP1 deletion increases vulnerability of RGCs to elevated IOP, while SPP1 overexpression preserves RGC numbers and rescues visual function. Using a transgenic reporter/conditional Spp1 knock-out strain we developed during a previous grant period, we found that in astrocytes, SPP1 promotes phagocytosis and secretion of neurotrophic factors while inhibiting production of neurotoxic and pro-inflammatory factors. Interestingly, SPP1 overexpression also significantly slows the RGC loss and visual function decline that occurs during normal aging even in the absence of glaucoma. To move SPP1 closer to a therapeutic use in humans, it is important to understand the mechanisms of action. We found that in astrocytes SPP1 up-regulates transcription of genes related to oxidative phosphorylation, functionally enhances mitochondrial respiration, and promotes the integrity of mitochondrial microstructure. One of the top SPP1-responsive genes is Vdac1 (voltage-dependent anion channel 1), a protein in the outer membrane of mitochondria that promotes ATP delivery from mitochondria into cytoplasm in astrocytes. One of the goals of this application is therefore to test whether the neuroprotective activity of SPP1 is mediated by its effect on mitochondrial function and Vdac1 expression. In addition to SPP1 expression in astrocytes, SPP1 is also expressed in retinal alpha RGC (but not in other RGCs). Deletion of Spp1 from alpha RGCs leads to a significantly worse outcome for all RGC survival, not just that of the alpha cells themselves. We hypothesize that alpha RGCs, in addition to their function as retinal output neurons, also form part of a protective system and secrete SPP1 for the benefit of neighboring (SPP1-negative) RGCs. We hypothesize that this effect is mediated by microglia. Microglia activation is an important cause of RGC degeneration in glaucoma and our preliminary data indicate that microglia from Spp1 knock-out retinas show stronger morphological signs of activation. In response to increased IOP, microglia from Spp1-negative mice also express more inflammatory markers. Finally, for therapeutic use, a small molecule that up-regulates the expression of SPP1 by astrocytes and alpha RGCs may be more suitable than the protein itself. We identified several such molecules (e.g., retinoic acid, thyroid hormone, and vitamin C). Though none of these is a likely candidate for a glaucoma therapy in humans, these proof of principle experiments indicate that a more systematic search using a small molecule library is promising. We will use astrocytes from our transgenic reporter/conditional knock-out strain to screen...