Determining the role of CLN3 in the eye

摘要 由CLN3基因突变引起的幼年型神经元蜡样脂褐素沉着症(JNCL)导致 儿童早期的神经变性和视力丧失。JNCL的视力丧失是由于视网膜变性所致。 具体地说，在视网膜中，JNCL既影响感光感光细胞，也影响其潜在的 上皮细胞，RPE。此外，JNCL视网膜中的光感受器细胞丢失与视力丧失有关。 然而，JNCL光感受器细胞丢失的分子机制(S)目前尚不清楚。 人诱导多能干细胞(HiPSC)视网膜细胞在本实验室的初步研究 提示CLN3在维持关键的RPE结构(微绒毛)和功能(棚的吸收)方面的新作用 光感受器外节或POS)，对光感受器的生存和视力至关重要。 具体地说，JNCL-hiPSC-RPE显示POS微绒毛组织紊乱/气球膨胀和吞噬功能受损 与对照组HiPSC-RPE细胞相比。值得注意的是，与RPE微绒毛功能障碍和POS降低一致 JNCL-HiPSC-RPE细胞摄取，JNCL患者眼内RPE脂褐素减少(分解 POS消化产品)。综上所述，这些数据表明，CLN3是一种RPE微绒毛驻留蛋白， 调节JNCL的POS摄取和CLN3功能障碍导致POS吞噬功能受损 减少脂褐素在JNCL RPE细胞中的积聚。为了验证这一假设，在这个项目中，我们建议 对对照细胞和JNCL HiPSC-RPE细胞进行超微结构和分子生物学研究。具体地说，我们将首先 利用免疫组织化学、RNAScope和亚细胞分离技术确定肿瘤的定位 在原代(小鼠、猪、人)和对照组的RPE细胞中，CLN3与顶端微绒毛的比例。 RPE。接下来，我们将比较i)微绒毛结构和分子组成以及ii)POS结合和 对照与JNCL HiPSC-RPE细胞的内化。此外，为了关联降低的POS吞噬功能 通过JNCL-hiPSC-RPE细胞体内观察到的减少JNCL RPE中的脂褐素(自体荧光积聚)， 我们将评估平行培养中的自发荧光积累模式(细胞表面与细胞内)。 对照组和慢性POS喂养后(1~3个月)的JNCL hiPSC-RPE。我们还将在以下方面验证我们的发现 患者使用来自JNCL小鼠和猪模型的原代RPE/POS的HIPSC-RPE细胞。最后，我们将利用 在一项纵向研究中对JNCL患者进行活体视网膜成像，以记录其存在和数量 光感受器-RPE细胞层中的自体荧光/脂褐素与光感受器与RPE细胞的关系 患者在疾病发展过程中的损失。总之，从这项研究中获得的知识将提供 对CLN3在RPE生理学中的作用和II)RPE功能障碍的可能贡献的新见解 至JNCL视网膜病理生理学。 好了！

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. !