Microglial phagocytosis of bipolar cells triggers inner retinal degeneration in Rs1-KO mice.

BACKGROUND: X-linked juvenile retinoschisis (XLRS) is a hereditary retinal disorder caused by mutations in the RS1 gene that leads to the formation of cavities in the inner nuclear layer (INL) and progressive vision loss, characterized by a disproportionate reduction of the b-wave compared to the a-wave in electroretinography (ERG). While previous research has largely focused on photoreceptor degeneration in XLRS, the specific roles of other cell populations, particularly bipolar cells and microglia, in the early stages of the disease have remained less well understood. Thus, this study aimed to elucidate the early cellular and molecular mechanisms of retinal degeneration in XLRS, with a particular focus on the role of microglia and bipolar cells. METHODS: Retinal structure and function were assessed in CRISPR/Cas9 Rs1-exon2 knockout (Rs1(−/y)) mice at 8 and 24 weeks using histology, spectral-domain optical coherence tomography (SD-OCT), ERG, and optokinetic response. To analyze cell-specific changes, we performed TUNEL assay, immunofluorescence, flow cytometry, and single-cell RNA sequencing (scRNA-seq) with trajectory analysis. RESULTS: Rs1(−/y) mice successfully recapitulated classic XLRS features, including INL schisis, reduced b/a-wave ERG ratio, and early vision loss. TUNEL assay and histological analysis revealed that cell death initiated in the INL at 8 weeks and progressed to the outer nuclear layer (ONL), while microglia displayed a progressive transition from a ramified to an ameboid morphology. scRNA-seq demonstrated a significant loss of cone bipolar cells, especially OFF-cone subtypes, which preceded photoreceptor degeneration. Importantly, microglial activation and enhanced phagocytosis of OFF-cone bipolar cells were observed prior to photoreceptor loss. This phagocytic process was found to be mediated by phosphatidylserine and complement C3b, independent of caspase-3 pathways. CONCLUSIONS: Our findings demonstrate that bipolar cell degeneration, driven by microglial phagocytosis of stressed yet viable OFF-cone bipolar cells, is an early and critical pathological event in XLRS that precedes photoreceptor loss. This process involves "eat-me" signals and complement activation independent of classical apoptosis. These results provide a new perspective on XLRS pathogenesis and suggest that therapeutic strategies targeting bipolar cells and microglial activity could offer promising avenues for early intervention. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12964-026-02692-8.