MFRP in the retina is a molecular hub that organizes the apical membrane of RPE cells by engaging in interactions with specific protein and lipid molecules

Membrane frizzled-related protein (MFRP) is an integral membrane protein present in the retinal pigment epithelium (RPE) and is essential for ocular development and the physiology of the retina. Mutations in MFRP are associated with autosomal recessive non-syndromic nanophthalmos, leading to severe hyperopia and early-onset retinitis pigmentosa. While several preclinical gene augmentation and gene editing trials hold promise for future therapies aimed at stopping degeneration and restoring retinal function, the molecular mechanisms underlying MFRP biology remain largely undefined. Here, we studied the biochemical properties of MFRP and the molecular consequences of its loss in a mouse model. Using transcriptomic and lipidomic approaches, we observed that DHA accumulation constitutes a primary defect in the MFRP-deficient RPE. In biochemical assays, we showed that MFRP undergoes extensive glycosylation and preferentially binds several classes of lipids, including phosphatidylserine and phosphatidylinositol 4-phosphate, as well as several other transmembrane proteins, notably adiponectin receptor 1 (ADIPOR1) and inward rectifier potassium channel 13 (KCNJ13). Moreover, MFRP determines ADIPOR1 and KCNJ13 subcellular localization in the RPE in vivo. This feature is affected upon MFRP deficiency and can be restored by gene therapy approaches. Overall, our observations suggest that MFRP constitutes an important interaction hub within the apical membrane of RPE cells, essential for protein trafficking and subcellular localization within the RPE and lipid homeostasis within the entire retina. RNA-seq profiling of RPE cells from 1 mo wt and retinal degeneration 6 (rd6) mice膜卷曲相关蛋白（Membrane frizzled-related protein，MFRP）是一类整合膜蛋白，表达于视网膜色素上皮（retinal pigment epithelium，RPE）中，对眼部发育及视网膜生理功能具有关键作用。MFRP突变常与常染色体隐性非综合征性小眼球症相关，可引发重度远视及早发性视网膜色素变性。尽管多项临床前基因增补与基因编辑试验为未来阻断视网膜变性、恢复视网膜功能的治疗方案带来了潜在可能，但MFRP相关生物学过程的分子机制仍未得到充分阐明。本研究借助小鼠模型，探究了MFRP的生化特性及其缺失所引发的分子层面效应。通过转录组学与脂质组学分析手段，我们发现二十二碳六烯酸（docosahexaenoic acid，DHA）的异常蓄积是MFRP缺陷型视网膜色素上皮细胞的核心缺陷。生化实验证实，MFRP可发生广泛的糖基化修饰，并能优先结合多类脂质，包括磷脂酰丝氨酸（phosphatidylserine）与4-磷酸磷脂酰肌醇（phosphatidylinositol 4-phosphate）；同时还可与多种其他跨膜蛋白特异性结合，其中尤为关键的是脂联素受体1（ADIPOR1）与内向整流钾通道13（KCNJ13）。此外，MFRP在体内可调控ADIPOR1与KCNJ13在视网膜色素上皮细胞中的亚细胞定位。MFRP缺失会破坏这一调控功能，而该功能可通过基因治疗手段得以修复。综上，本研究结果表明，MFRP是视网膜色素上皮细胞顶膜中重要的相互作用枢纽，其对视网膜色素上皮内的蛋白质运输与亚细胞定位，以及全视网膜的脂质稳态均不可或缺。本研究还对1月龄野生型（wild type，wt）小鼠与视网膜变性6型（rd6）小鼠的视网膜色素上皮细胞开展了RNA测序转录组分析。