Determining the role of CLN3 in the eye

确定 CLN3 在眼睛中的作用

• 批准号: 9902470
• 负责人: Ruchira Singh
• 金额: $ 50.54万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902470
• 关键词: Affect; Animal Model; Apical; Atrophic; Binding; Blindness; CLN3 gene; Cadaver; Cell Death; Cell Fractionation; Cell physiology; Cell surface; Cells; Cellular Structures; Chronic; Clinical; Data; Dextrans; Digestion; Disease; Epithelial; Epithelial Physiology; Epithelium; Evolution; Excision; Eye; Family suidae; Functional disorder; Genotype; Health; Homeostasis; Human; Image; Immunohistochemistry; Impairment; Ingestion; Knowledge; Laboratories; Lead; Light; Link; Lipofuscin; Longitudinal Studies; Mediating; Modeling; Molecular; Mus; Mutation; Nerve Degeneration; Nutrient; Pathology; Patient imaging; Patients; Pattern; Phagocytosis; Pharmacotherapy; Phenotype; Photoreceptors; Play; Proteins; Recycling; Retina; Retinal Degeneration; Retinal Photoreceptors; Rodent; Role; Secondary to; Spielmeyer-Vogt Disease; Structure; Structure of retinal pigment epithelium; Techniques; Testing; Transportation; Vision; Vitamin A; Waste Products; cellular imaging; cellular microvillus; disease-causing mutation; early childhood; experimental study; feeding; gene correction; histological studies; imaging study; in vivo; induced pluripotent stem cell; insight; internal control; multimodality; novel; retinal imaging; retinal progenitor cell; uptake; visual cycle

ABSTRACT Juvenile neuronal ceroid lipofuscinosis (JNCL), caused by mutations in the CLN3 gene, leads to neurodegeneration and vision loss in early childhood. Vision loss in JNCL is due to retinal degeneration. Specifically, in the retina, JNCL affects both the light sensing photoreceptor cells and their underlying epithelium, RPE. Furthermore, photoreceptor cell loss in the JNCL retina has been linked to vision loss. However, the molecular mechanism(s) underlying photoreceptor cell loss in JNCL are currently not known. Preliminary studies in our laboratory utilizing human induced pluripotent stem cell (hiPSC)-derived retinal cells suggest a novel role of CLN3 in maintaining key RPE structure (microvilli) and function (uptake of shed photoreceptor outer segments or POS) that are essential for photoreceptor survival and therefore vision. Specifically, JNCL hiPSC-RPE display microvilli disorganization/ballooning and impaired phagocytosis of POS compared to control hiPSC-RPE cells. Notably, consistent with RPE microvilli dysfunction and reduced POS uptake in JNCL hiPSC-RPE cells, RPE in the eyes of JNCL patients have reduced lipofuscin (breakdown products of POS digestion). Together, these data suggest that “CLN3 is a RPE microvilli resident protein that regulates POS uptake and CLN3 dysfunction in JNCL leads to impaired POS phagocytosis and subsequently decreased lipofuscin accumulation in JNCL RPE cells.” To test this hypothesis, in this project, we propose to employ ultrastructural and molecular studies on control and JNCL hiPSC-RPE cells. Specifically, we will first utilize immunohistochemistry, RNAScope and subcellular fractionation techniques to confirm the localization of a proportion of CLN3 in RPE cells to the apical microvilli in primary (mouse, porcine, human) and control hiPSC- RPE. Next, we will compare i) microvilli structure and molecular composition and ii) POS binding and internalization by control vs. JNCL hiPSC-RPE cells. Furthermore, to correlate the reduced POS phagocytosis by JNCL hiPSC-RPE cells to reduced lipofuscin (autofluorescence accumulation) seen in JNCL RPE in vivo, we will evaluate the pattern of autofluorescence accumulation (cell surface vs. intracellular) in parallel cultures of control and JNCL hiPSC-RPE after chronic POS feeding (1-3 months). We will also validate our findings on patient hiPSC-RPE cells using primary RPE/POS from a JNCL mouse and porcine model. Finally, we will utilize in vivo retinal imaging of patients with JNCL in a longitudinal study to document the presence and amount of autofluorescence/lipofuscin in the photoreceptor-RPE cell layer in relationship to photoreceptor vs. RPE cell loss in patients progressing through the disease. Altogether, the knowledge gained from this study will provide novel insights into i) the role of CLN3 in RPE physiology and ii) the plausible contribution of RPE dysfunction to JNCL retinal pathophysiology. !

摘要 幼年神经元蜡样质脂褐质沉积症（JNCL），由CLN 3基因突变引起，导致 神经退化和视力丧失。JNCL的视力丧失是由于视网膜变性造成的。 具体而言，在视网膜中，JNCL影响感光细胞和它们的底层细胞。 上皮，RPE。此外，JNCL视网膜中的感光细胞损失与视力丧失有关。 然而，JNCL中感光细胞损失的分子机制目前尚不清楚。 利用人诱导多能干细胞（hiPSC）衍生的视网膜细胞在我们实验室中进行的初步研究 提示CLN 3在维持关键RPE结构（微绒毛）和功能（脱落吸收）中的新作用 光感受器外节或POS），其对于光感受器存活和因此的视觉是必需的。 具体而言，JNCL hiPSC-RPE显示微绒毛解体/气球样变和POS吞噬功能受损。 与对照hiPSC-RPE细胞相比。值得注意的是，与RPE微绒毛功能障碍和POS减少一致 JNCL hiPSC-RPE细胞中的吸收，JNCL患者眼中的RPE减少了脂褐素（分解 POS消化产物）。总之，这些数据表明“CLN 3是一种RPE微绒毛驻留蛋白， 在JNCL中调节POS摄取和CLN 3功能障碍导致POS吞噬功能受损， 减少JNCL RPE细胞中脂褐素的积累。”为了验证这一假设，在本项目中，我们建议 对对照和JNCL hiPSC-RPE细胞进行超微结构和分子研究。具体来说，我们将首先 利用免疫组织化学、RNAScope和亚细胞分级技术来确定 RPE细胞中CLN 3与原代（小鼠、猪、人）和对照hiPSC中顶端微绒毛的比例。 RPE。接下来，我们将比较i）微绒毛结构和分子组成和ii）POS结合和 对照与JNCL hiPSC-RPE细胞的内化。此外，为了将减少的POS吞噬作用 通过JNCL hiPSC-RPE细胞减少体内JNCL RPE中观察到的脂褐素（自体荧光积累）， 我们将在平行培养中评价自发荧光积累的模式（细胞表面与细胞内） 慢性POS喂养（1-3个月）后对照和JNCL hiPSC-RPE的变化。我们还将验证我们的发现， 使用来自JNCL小鼠和猪模型的原代RPE/POS的患者hiPSC-RPE细胞。最后，我们将利用 在一项纵向研究中，JNCL患者的体内视网膜成像记录了 光感受器-RPE细胞层中的自体荧光/脂褐素与光感受器vs. RPE细胞的关系 患者在疾病中的损失。总之，从这项研究中获得的知识将提供 对i）CLN 3在RPE生理学中的作用和ii）RPE功能障碍的合理贡献的新见解 JNCL视网膜病理生理学。 !