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.