Identification of a Small-Molecule Modulator of Astrocyte Reactivity for Optic Nerve Protection

Purpose: Injury of the optic nerve leads to retinal ganglion cells (RGCs) apoptosis and irreversible vision loss, in which reactive astrocytes play a central role. The aim of this study is to modulate pathological reactive astrocytes to reduce the progression of optic nerve degeneration. Methods: Given the therapeutic potential of small molecules to modulate astrocyte reactivity, we used a drug-screening platform to identify small molecules, and evaluated their capacity to regulate astrocyte phenotypes and preserve RGCs after optic nerve crush (ONC). The primary astrocytes from neonatal C57BL/6J mouse cortices, A1 astrocytes, are induced by TNF, IL-1α, and C1q (TIC), both of them are confirmed at transcript and protein levels. High-throughput screening using SiPer, a computational screening platform, together with DRUG-seq2, yielding candidate small molecules, whose effects on A1/A2 transitions were assessed by RT-qPCR, RNA sequencing (RNA-seq), Western blotting, and immunofluorescence. In vivo, an ONC model received intravitreal compound delivery. RGC survival and astrocyte phenotypes were evaluated by retinal flat-mounts and immunofluorescence. Results: Primary astrocytes exposed to TIC acquired A1 phenotype, characterized by upregulated C3, GBP2, H2-d1, and H2-t23, and induced RGC cytotoxicity. Transcriptomic drug screening identified proteasome inhibition as a potential strategy to suppress pathological reactive astrocytes. Marizomib, a blood-brain barrier (BBB)-permeable proteasome inhibitor, downregulated A1 markers and upregulated A2 neuroprotective genes. In an ONC model, Marizomib reduced GBP2-positive astrocytes and, at a lower dose, a modest increase in RGC survival was observed at 14 days post-ONC. Conclusions: We developed a small-scale drug-screening platform and identified Marizomib as a modulator of astrocyte phenotypes. Its therapeutic potential was validated both in vitro and in vivo, providing a new chemical tool to modulate astrocyte reactivity for future therapeutic exploration.