Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease

Abstract

PurposeCLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link betweenCLN3mutation and POS phagocytosis defect.MethodsIsogenic control andCLN3mutant stem cell lines were generated by CRISPR-Cas9­mediated biallelic deletion of exons 7 and 8. A transgenicCLN3Δ7-8/Δ7-8(CLN3) Yucatan miniswine was also used to study the impact ofCLN3Δ7-8/Δ7-8mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis ofCLN3Δ7/8and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age.ResultsCLN3Δ7-8/Δ7-8RPE (CLN3RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosedCLN3Δ7-8/Δ7-8POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased inCLN3miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age.ConclusionsCLN3Δ7-8/Δ7-8mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.