Shiley Eye Institute, University of California, San Diego, 9415 Campus Point Drive, La Jolla, CA, 92093, USA.
Department of Biochemistry, Faculty of Medicine, UNAM, Mexico City, Mexico.
Sci Rep. 2022 Sep 10;12(1):15273. doi: 10.1038/s41598-022-19351-w.
Mutations in the Membrane-type frizzled related protein (Mfrp) gene results in an early-onset retinal degeneration associated with retinitis pigmentosa, microphthalmia, optic disc drusen and foveal schisis. In the current study, a previously characterized mouse model of human retinal degeneration carrying homozygous c.498_499insC mutations in Mfrp (Mfrp) was used. Patients carrying this mutation have retinal degeneration at an early age. The model demonstrates subretinal deposits and develops early-onset photoreceptor degeneration. We observed large subretinal deposits in Mfrp mice which were strongly CD68 positive and co-localized with autofluorescent spots. Single cell RNA sequencing of Mfrp mice retinal microglia showed a significantly higher number of pan-macrophage marker Iba-1 and F4/80 positive cells with increased expression of activation marker (CD68) and lowered microglial homeostatic markers (TMEM119, P2ry13, P2ry13, Siglech) compared with wild type mice confirming microglial activation as observed in retinal immunostaining showing microglia activation in subretinal region. Trajectory analysis identified a small cluster of microglial cells with activation transcriptomic signatures that could represent a subretinal microglia population in Mfrp mice expressing higher levels of APOE. We validated these findings using immunofluorescence staining of retinal cryosections and found a significantly higher number of subretinal Iba-1/ApoE positive microglia in Mfrp mice with some subretinal microglia also expressing lowered levels of microglial homeostatic marker TMEM119, confirming microglial origin. In summary, we confirm that Mfrp mice carrying the c.498_499insC mutation had a significantly higher population of activated microglia in their retina with distinct subsets of subretinal microglia. Further, studies are required to confirm whether the association of increased subretinal microglia in MfrpKI/KI mice are causal in degeneration.
Mfrp 基因中的突变导致早发性视网膜变性，伴有视网膜色素变性、小眼球、视盘玻璃疣和黄斑裂孔。在本研究中，使用了一种先前描述的携带 Mfrp 基因（Mfrp）中 c.498_499insC 突变的同源纯合子的人类视网膜变性小鼠模型。携带该突变的患者在早期就会出现视网膜变性。该模型表现出视网膜下沉积物，并出现早发性光感受器变性。我们观察到 Mfrp 小鼠中存在大量的视网膜下沉积物，这些沉积物强烈表达 CD68，并与自发荧光斑点共定位。Mfrp 小鼠视网膜小胶质细胞的单细胞 RNA 测序显示，与野生型小鼠相比，具有更多的泛巨噬细胞标志物 Iba-1 和 F4/80 阳性细胞，并且激活标志物（CD68）表达增加，小胶质细胞稳态标志物（TMEM119、P2ry13、P2ry13、Siglech）表达降低，证实了视网膜免疫染色中观察到的小胶质细胞激活，表明小胶质细胞在视网膜下区域激活。轨迹分析确定了一小群具有激活转录组特征的小胶质细胞，这些细胞可能代表 Mfrp 小鼠中表达更高水平 APOE 的视网膜下小胶质细胞群体。我们使用视网膜冷冻切片的免疫荧光染色验证了这些发现，并发现 Mfrp 小鼠中存在更多的视网膜下 Iba-1/ApoE 阳性小胶质细胞，其中一些视网膜下小胶质细胞也表达降低的小胶质细胞稳态标志物 TMEM119，证实了小胶质细胞的起源。总之，我们证实携带 c.498_499insC 突变的 Mfrp 小鼠的视网膜中有明显更多的激活小胶质细胞，并且其视网膜下小胶质细胞存在不同的亚群。还需要进一步的研究来确认 MfrpKI/KI 小鼠中增加的视网膜下小胶质细胞是否与变性有关。
