Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration

Rab and associated effector proteins are involved in all steps of cargo transport through subcellular compartments during the endocytic pathway that participates in multiple cellular processes including signal transduction, protein recycling and degradation. We identified Rabgef1 (also called Rabex-5) as the only differentially expressed Rab-associated gene that exhibits strong concordance with photoreceptor maturation by mining the retinal transcriptome data. The retinas of Rabgef1-/- mice exhibit defects in photoreceptor morphology, followed by cell death, and almost complete loss of both rod and cone photoreceptor function within 3 weeks after birth. Like other cells/tissues, Rabgef1 interacts with Rabaptin5 in the retina and its loss leads to reduced early endosomes in the photoreceptor inner segments as indicated by EEA1 immunostaining. Rabgef1-/- photoreceptors abnormally accumulate autophagosomes, and proteomic profiling of endosome-enriched fractions demonstrate significant depletion of phototransduction and mitochondrial proteins. Transcriptome analysis of developing retina before the degeneration is evident reveals energy metabolism modules and gene regulatory landscape that likely suggest the trajectory of cellular changes leading to photoreceptor degeneration. Our results directly implicate endocytic pathway in outer segment biogenesis and photoreceptor development and provide strong evidence that endocytosis defects in the retina can trigger upregulation of autophagic mechanisms. We also suggest that Rabgef1 and components of the endocytic and autophagic pathways should be considered as candidates for human retinopathies. Retinal transcriptome of Rabgef1 WT and KO mutants were profiles at three different ages: P6, P10, P14. All samples had at least two replicates. Transcriptome landscape were compared to determine the pathway changes associated with Rabgef1 loss of function.Rab蛋白（Rab）及其相关效应蛋白参与了内吞途径（endocytic pathway）中货物经由亚细胞区室转运的全部步骤，而该内吞途径参与包括信号转导、蛋白质回收与降解在内的多种细胞生物学过程。我们通过挖掘视网膜转录组数据，鉴定出Rabgef1（亦称Rabex-5）是唯一与光感受器细胞（photoreceptor）成熟呈现显著相关性的差异表达Rab相关基因。Rabgef1-/-小鼠的视网膜表现出光感受器细胞形态异常，随后出现细胞死亡，并在出生后3周内几乎完全丧失视杆细胞与视锥细胞的功能。与其他细胞/组织类似，Rabgef1在视网膜中可与Rabaptin5相互作用，其缺失会导致光感受器内节的早期内体数量减少，这一现象可通过EEA1免疫染色得以验证。Rabgef1-/-光感受器细胞会异常积累自噬体；对富集内体的组分进行蛋白质组谱分析显示，光转导相关蛋白与线粒体蛋白出现显著耗竭。在退行性变表型显现前对发育中的视网膜进行转录组分析，发现其能量代谢模块与基因调控图谱或可揭示驱动光感受器细胞退行性变的细胞变化轨迹。本研究结果直接将内吞途径与光感受器外节生物发生及发育过程关联起来，并提供了强有力证据，表明视网膜内吞缺陷可触发自噬机制的上调。我们同时提出，Rabgef1以及内吞与自噬途径的相关组分，可作为人类视网膜病变的候选研究靶点。我们在三个不同时间点（P6、P10、P14）对Rabgef1野生型（WT）与敲除（KO）突变体的视网膜转录组进行了分析；所有样本均至少设置2次生物学重复。通过对比转录组图谱，我们确定了与Rabgef1功能丧失相关的通路变化。