DRAM2: on the crossroad between trans-Golgi network and lysosomes?

背景：自噬是一个关键的动态平衡过程，受损的细胞成分被隔离到自噬小体中，并被溶酶体降解。自噬功能受损与炎症、神经退行性疾病和年龄相关疾病有关，包括老年性黄斑变性(AMD)。DNA损伤调节自噬调节器2(DRAM2)的双等位基因突变导致视网膜营养不良的发生，并伴有早期黄斑视锥感光细胞受累。为了更好地了解自噬在视网膜细胞功能中的作用，我们从两名DRAM2基因不同突变的患者中培养出了诱导多能干细胞(IPSCs)。使用CRISPR/Cas9原位基因编辑技术纠正这些DRAM2突变，产生了患者特有的等基因IPSCs。对DRAM2突变和等基因对照的光感受器和RPE细胞的并列比较显示，DRAM2蛋白的严重丢失和关键溶酶体水解酶(CTSD、NPC2、TPP1、PPT1)的显著下调，这些酶通过分解蛋白质、脂类和相关糖参与自噬的晚期阶段。反过来，透射式电子显微镜和脂体学分析揭示了RPE的脂质积累和丢失以及光感受器的活性。综上所述，我们提出假设：1)DRAM2在溶酶体关键酶从跨高尔基体网络到溶酶体的囊泡运输中起关键作用；2)DRAM2缺乏/功能障碍导致溶酶体酶的运输受损，进而导致溶酶体功能障碍以及光感受器和RPE细胞中脂质/废物的堆积；3)未经处理的废物积累导致光感受器和RPE细胞的进行性丢失。3)检测补充DRAM2对光感受器和RPE细胞中脂质和废物积累的影响。实验方法：采用本课题组建立的方法，对光感受器和RPE细胞进行DRAM2 IPSCs的体外培养和分化，并对其进行等基因对照。酵母双杂交结合定点突变和定向免疫共沉淀试验将用于鉴定和验证DRAM2相互作用蛋白。结合共聚焦显微镜和差速离心法以及密度梯度细胞器分离的分离式GFP系统标记内源DRAM2将被用来研究DRAM2的加工和通过内质网和反高尔基网络运输到晚期内小体和溶酶体。重要的是，基于AAV的DRAM2补充将被测试以恢复光感受器和RPE细胞中功能DRAM2的表达，以逆转废物积累和活性丧失。

Background: Autophagy is a key homeostatic process where damaged cellular components are sequestered into autophagosomes and degraded by the lysosomes. Impairment of autophagy has been implicated in inflammatory, neurodegenerative and age-related diseases including age related macular degeneration (AMD). Bi-allelic mutations in the DNA damage regulated autophagy modulator 2 (DRAM2) result in development of retinal dystrophy with early macular cone photoreceptor involvement. To better understand the role of autophagy in retinal cell function, we have generated induced pluripotent stem cells (iPSCs) from two patients with different mutations in the DRAM2 gene. Correction of these DRAM2 mutations using CRISPR/Cas9 in situ gene editing techniques yielded patient-specific isogenic iPSCs. Side by side comparison of DRAM2 mutated and isogenic control photoreceptors and RPE cells demonstrated a profound loss of DRAM2 protein and a significant downregulation of key lysosomal hydrolases (CTSD, NPC2, TPP1, PPT1) that are involved in the late stages of autophagy by breaking down proteins, lipids and associated sugars. In turn, lipid accumulation and loss of RPE and photoreceptor viability were revealed by transmission electron microscopy and lipidomic analyses. Together these data have led us to hypothesise that: 1) DRAM2 is critically involved in vesicular transport of key lysosomal enzymes from the trans-Golgi network to the lysosomes; 2) DRAM2 deficiency/dysfunction results in impaired transport of lysosomal enzymes which in turn causes lysosome dysfunction and lipid/waste accumulation in photoreceptors and RPE cells; 3) unprocessed waste accumulation causes progressive photoreceptor and RPE cell loss.Building upon these pilot data, we here propose to 1) assess DRAM2 processing and trafficking in photoreceptors and RPE cells; 2) dissect the DRAM2-associated functional module; and 3) test the effect of DRAM2 supplementation in abolishing lipid and waste accumulation in photoreceptors and RPE cells.Experimental approach: In vitro culture and differentiation of DRAM2 iPSCs and isogenic controls to photoreceptors and RPE cells using protocols established in our groups. Yeast two hybrid combined with site directed mutagenesis and directed co-immunoprecipitation assays will be used to identify and validate DRAM2-interacting proteins. Tagging of endogenous DRAM2 with a split-GFP system combined with confocal microscopy and differential centrifugation and density-gradient dependent organelle isolation will be used to address DRAM2 processing and trafficking through the endoplasmic reticulum and trans-Golgi network to late endosomes and lysosomes. Importantly AAV based supplementation of DRAM2 will be tested to restore expression of functional DRAM2 in photoreceptor and RPE cells to reverse waste accumulation and loss of viability.