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

DRAM2：位于跨高尔基体网络和溶酶体之间的十字路口？

• 批准号: 2882796
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2882796
• 关键词: DRAM2; crossroad; between; trans; Golgi

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.

背景资料：自噬是一个关键的稳态过程，其中受损的细胞成分被隔离到自噬体中并被溶酶体降解。自噬的损伤与炎症、神经退行性和年龄相关性疾病（包括年龄相关性黄斑变性（AMD））有关。DNA损伤调节的自噬调节剂2（DRAM 2）中的双等位基因突变导致视网膜营养不良的发展，伴有早期黄斑锥光感受器受累。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DRAM 2基因突变不同的患者中产生了诱导多能干细胞（iPSC）。使用CRISPR/Cas9原位基因编辑技术校正这些DRAM 2突变产生了患者特异性等基因iPSC。DRAM 2突变和同基因对照光感受器和RPE细胞的并排比较证明了DRAM 2蛋白的严重损失和关键溶酶体水解酶（CTSD、NPC 2、TPP 1、PPT 1）的显著下调，所述溶酶体水解酶通过分解蛋白质、脂质和相关糖参与自噬的晚期阶段。反过来，通过透射电子显微镜和脂质组学分析揭示了脂质积累和RPE和感光细胞活力的丧失。这些数据一起使我们假设：1）DRAM 2关键地参与关键溶酶体酶从trans-Golgi网络到溶酶体的囊泡转运; 2）DRAM 2缺陷/功能障碍导致溶酶体酶的转运受损，这反过来导致溶酶体功能障碍和脂质/废物在光感受器和RPE细胞中的积累; 3）未处理的废物积累导致进行性的感光细胞和RPE细胞损失。基于这些初步数据，我们在这里建议1）评估DRAM 2在感光细胞和RPE细胞中的加工和运输; 2）解剖DRAM 2相关的功能模块;和3）测试补充DRAM 2在消除光感受器和RPE细胞中的脂质和废物积累中的作用。实验方法：使用我们组中建立的方案，体外培养和分化DRAM 2 iPSC和同基因对照至光感受器和RPE细胞。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DRAM 2相互作用蛋白。标记内源性DRAM 2与分裂GFP系统结合共聚焦显微镜和差速离心和密度梯度依赖性细胞器分离将用于解决DRAM 2加工和运输通过内质网和trans-Golgi网络到晚期内体和溶酶体。重要的是，将测试基于AAV的DRAM 2补充以恢复感光细胞和RPE细胞中功能性DRAM 2的表达，从而逆转废物积累和活力丧失。