Temporal modulation of microglial repopulation attenuates retinal degeneration in retinitis pigmentosa

Retinitis pigmentosa (RP) is an inherited retinal degenerative disorder characterized by the progressive loss of retinal pigment epithelial cells and rod and cone photoreceptors and irreversible vision loss, with no effective therapies currently available. Microglia, the resident immune cells in the retina, are known to aggravate neurodegeneration when chronically activated. Here, we investigated the impact of temporally controlled microglial depletion and repopulation on retinal degeneration in rd10 mice. Using the CSF1R inhibitor PLX5622, we achieved efficient microglial depletion during the peak of degeneration (P21), followed by spontaneous microglial repopulation. Short-term-repopulated microglia displayed a ramified morphology and homeostatic transcriptomic signatures, including the downregulation of disease-associated microglia (DAM) genes and suppression of neurodegeneration-related pathways, effects that were correlated with preserved visual function and photoreceptor survival. However, long-term repopulated microglia progressively exhibited DAM phenotypes, which coincided with diminished therapeutic efficacy and exacerbated neurodegeneration. Single-cell RNA sequencing confirmed the dynamic transcriptional transitions of repopulated microglia, their altered regulon activity, and their functional association with other immune cells. To counteract microglial reactivation, we developed a sequential 2-round depletion-repopulation strategy, which restored microglial homeostasis, reduced the expression of the proinflammatory cytokine IL-1β, preserved outer nuclear layer thickness, and sustained visual function. Our findings highlight the time-dependent plasticity of microglial phenotypes and suggest that temporally optimized microglial modulation is a promising therapeutic strategy for retinal neurodegeneration in RP. • Morphological and scRNA-seq analyses reveal that short-term microglial repopulation restores homeostatic morphology and visual function, while long-term repopulation leads to microglial reactivation and functional decline. • CD45 + scRNA-seq determines IFNG-IFNGR1/2-HIF1α networks as drivers of microglial reactivation in long-term microglial repopulation. • Immune cell-cell interactions strengthen in long-term microglial repopulation, with increased IFNG-IFNGR1/2 and TNFSF14-LTβR signaling from infiltrating immune cells, consistent with inflammatory activation. • A sequential 2 × DR strategy resets microglial programs, lowers IL-1β, and sustains retinal circuit function.