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Determining the role of CLN3 in the eye

确定 CLN3 在眼睛中的作用

基本信息

批准号：
10357934
负责人：
Ruchira Singh
金额：
$ 45.82万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10357934
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 Evolution Excision Eye Family suidae Functional disorder Genotype Health Homeostasis Human Image Immunohistochemistry Impairment Ingestion Knowledge Laboratories Lead Light Link Lipofuscin Longitudinal Studies Mediating 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 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 juvenile neuronal ceroid lipofuscinosis mouse model multimodality novel porcine model retinal imaging retinal progenitor cell uptake visual cycle

项目摘要

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