Abstract
Purpose :
Reactive optic nerve astrocytes protect retinal ganglion cell (RGC) survival and visual function in glaucoma and traumatic optic neuropathy. To better understand the molecular mechanism of astrocyte-mediated neuroprotection, we conducted gene expression profiling of the optic nerve head and identified Secreted Phosphoprotein 1 (SPP1, also called osteopontin) as one of the leading candidate genes. Here, we directly test the hypothesis that SPP1 acts as a neuroprotective factor that promotes RGC survival, and improves visual function in glaucoma and traumatic optic nerve injury.
Methods :
We generated a new transgenic mouse line that combines GFP expression from the endogenous Spp1 locus with the incorporation of loxP sites for conditional deletion of the Spp1 gene and activation of red fluorescent protein after cre recombination. This strain was crossed to B6.GFAP-cre mice to generate an astrocyte-specific Spp1 knock out. Using the microbead model of glaucoma and optic nerve crush, we tested how RGC numbers and visual function (pattern ERG, visual acuity) were affected by astrocyte-specific knock-out of Spp1. Immunohistochemistry, qPCR, and RNA-sequencing were used to identify signaling pathways and molecular mechanisms of neuroprotection. Mitochondrial respiration was measured in cultured astrocytes using a Seahorse metabolic analyzer.
Results :
SPP1 expression was increased in optic nerve astrocytes in glaucoma and optic nerve traumatic injury and was driven by the TGF-β1/Runx1/E2f1 signaling pathway. Conditional knock-out of Spp1 in astrocytes led to more pronounced RGC loss and visual impairment in glaucoma and optic nerve traumatic injury. RNA-sequencing showed that in Spp-/- astrocytes genes associated with neuroinflammation were up-regulated, whereas neurotrophic factors, genes associated with phagocytic activity, synaptogenesis, and especially oxidative phosphorylation were down-regulated. Conversely, exogenous SPP1 strongly induced VDAC1 expression and promoted oxidative phosphorylation in astrocytes.
Conclusions :
SPP1induces a neuroprotective state in astrocytes, promotes mitochondrial function and VDAC1 expression, and protects RGCs and vision function in glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.