Differential roles of the uridine-P2Y6R signaling axis in mouse models of retinitis pigmentosa with distinct genetic mutations

Retinitis pigmentosa (RP) exhibits complex genetic heterogeneity, and the downstream pathogenic mechanisms underlying different etiologies remain unclear. This study aimed to explore the differential mechanisms by which the metabolite uridine and its receptor P2Y6R regulate microglial activation and photoreceptor apoptosis in different RP animal models.

① Uridine content in the retina was measured using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in rd1 mice (aged 28 d, weighing 13 to 18 g, n=3), rd10 mice (aged 45 d, weighing 20 to 25 g, n=3) and age-matched C57 BL/6J control mice (n=3/group), as well as RCS rats (aged 60 d, weighing 200 to 250 g, n=4) and age-matched RCS-rdy control rats (n=4). Retinal pyrimidine metabolites were analyzed using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) at various disease stages in rd10 mice (aged 18 d, weighing 7 to 10 g; aged 25 d, weighing 11 to 15 g; aged 45 d; n=5), with age-matched C57 BL/6J mice serving as controls (n=5). ② Five-week-old C57 BL/6J mice received unilateral subretinal injection of uridine (100 mmol/L, 2 μL), with contralateral eyes injected with PBS as controls. After 7 d, electroretinography (ERG), outer nuclear layer (ONL) thickness analysis, immunofluorescence staining, and TUNEL assay were performed (n=3). ③ RT-qPCR was used to detect mRNA expression of P2Y receptor family members (P2ry1, P2ry2, P2ry4, P2ry6, P2ry12) in the retina of C57 BL/6J mice following subretinal injection of uridine or PBS (n=3). ④ The P2Y6R inhibitor MRS2578 (3 mg/kg per day) or vehicle (corn oil containing 0. 6% dimethyl sulfoxide) was administered to rd10 mice (aged 14 d, weighing 7 to 10 g, n=3) and RCS rats (aged 15 d, weighing 25 to 30 g, n=3) prior to degeneration onset. ERG, ONL thickness, photoreceptor apoptosis, and microglial number and morphology were evaluated. ⑤ RT-qPCR was performed to detect the expression of uridine synthesis and metabolism-related enzymes (Upp1, Upp2, Uck1, Uck2) in the retina of 45-day-old rd10 and C57 BL/6J mice (n=3).

① Compared with age-matched C57 BL/6J control mice, retinal uridine content was significantly elevated in rd1 and rd10 mice (P<0. 05), whereas no significant change was observed in RCS rats compared with age-matched RCS-rdy control rats. Retinal uridine levels in rd10 mice progressively increased with disease progression compared with age-matched C57 BL/6J control mice (P<0. 0001). ② Subretinal injection of uridine in C57 BL/6J mice resulted in significant reductions in a-wave and b-wave amplitudes (P<0. 05), ONL thinning (P<0. 0001), increased microglial activation (P<0. 01) with migration toward the ONL, and increased TUNEL-positive cells (P<0. 01). ③ After uridine injection, the mRNA expression of P2ry6 was upregulated in the retina of C57 BL/6J mice (P<0. 05). ④ Compared with the vehicle-treated group, MRS2578 treatment significantly improved the amplitudes of the a-wave and b-wave (P<0. 05), increased the ONL thickness, reduced the number of TUNEL-positive cells (P<0. 05), and promoted the reversal of microglia from an amoeboid-activated state to a resting, ramified state in rd10 mice. In contrast, in RCS rats, MRS2578 treatment failed to significantly inhibit microglial activation or delay retinal degeneration compared with vehicle-treated controls. ⑤ Compared with age-matched C57 BL/6J control mice, rd10 mice exhibited upregulated expression of Upp1 (P<0. 05), Upp2 (P<0. 01), and Uck1 (P<0. 01) in the retina, with decreased UMP and uracil levels.

The uridine-P2Y6R signaling axis exacerbates retinal degeneration through microglial activation in the rd10 mouse model of primary photoreceptor degeneration, whereas this mechanism is absent in the RCS rat model of primary retinal pigment epithelial dysfunction. These findings reveal the heterogeneity of retinal degeneration mechanisms across different etiological backgrounds, suggesting that targeting the uridine-P2Y6R axis may represent a potential therapeutic strategy for specific RP subtypes.