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. !
Juvenile neuronal ceroid lipofuscinosis (JNCL) affects the retina in the eye and leads to vision loss in early childhood. However, the disease mechanism responsible for vision loss in JNCL is not known. In this proposal, we will use cellular and imaging studies on patient-derived retinal cells and JNCL patient retina to determine the disease mechanism underlying vision loss in JNCL. This knowledge has the potential to lead to a rational drug therapy for JNCL.