A patient-specific stem cell model of Batten disease

Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, is a family of fatal, autosomal recessive neurodegenerative lysosomal storage disorders, mostly of childhood onset, consisting of mutations in 1 of 13 CLN genes. Batten patients have visual impairment that is followed by blindness, cognitive decline, motor imbalance, refractory seizures and premature death. The abnormal lysosomal storage of ceroid, a pathological autofluorescent material having similar biochemical properties to lipofuscin is a hallmark of Batten disease. Subunit C of the mitochondrial ATP synthase has been identified as a main component of storage material detected only in the central nervous system tissues in CLN2 and CLN3 mutations. Therefore, the study of the involvement of lysosomes and their interactions with other cell compartments, for example mitochondria and endoplasmic reticulum is essential to uncover relevant affected cellular pathways for drug development. As the central nervous system is severely affected in NCL, the establishment of human neuronal models will enable disease modeling and drug screening in Batten disease. CLN2 and CLN3 mutations represent the most commonly occurring forms of Batten disease in patients. Patient-specific stem cell models of Batten disease can be a powerful tool to explore disease mechanisms of Batten disease. Aims: The overarching aims of this thesis were: 1. To generate iPSCs from CLN2 and CLN3 patient fibroblasts followed by genome editing using CRISPR-Cas9 to correct a 1 kb deletion mutation in CLN3 iPSCs. 2. To investigate electrophysiological, ultrastructural and proteome differences in isogenic CLN3 iPSC-derived neurons. Methods: CLN2 and CLN3 fibroblasts were reprogrammed using episomal-based vectors to generate iPSCs. The reprogrammed iPSCs were validated for pluripotency expression, trilineage differentiation potential, copy number variation and integration of episomal vectors. To correct 1 kb deletion in CLN3 iPSCs, Cas9 ribonucleoprotein was delivered with a plasmid donor template carrying the wild-type 1 kb sequence and an allele-specific single guide RNA (sgRNA) to target the breakpoint sequence of the mutation. The edited clone was identified through puromycin selection. This was followed by Cre-Lox recombination to remove the puromycin resistance cassette from the genome. The corrected clone, CLN3-Cor was validated for pluripotency expression, trilineage differentiation potential, copy number variation and sgRNA off-target mutations. To characterize phenotypic differences of CLN3 cell line, the isogenic CLN3 iPSCs were differentiated into neurons. This study assessed electrophysiological activity using microelectrode array (MEA) from day 4 to day 42 of differentiation, global protein expression on day 14 and day 42 of differentiation through mass spectrometry, protein expression of LAMP1 and subunit C on day 14, 28 and 42 of differentiation through western blot, endocytosis using DQ-BSA Green assay, and ultrastructural changes through transmission electron microscopy (TEM) on day 42 of differentiation. Results: Quality control performed on CLN2 and CLN3 iPSCs, as well as CLN3-Cor iPSCs demonstrated that the iPSCs were pluripotent, able to differentiate into all germ layers and were devoid of karyotypic abnormalities. MEA study revealed CLN3 neurons to have lower levels of spike and burst activity compared to CLN3-Cor neurons for most part of the culture period. Network burst activity which indicates the formation of functional circuits in the neuronal network was also lower in CLN3 neurons than in CLN3-Cor neurons. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at both time points in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis of LAMP1 expression revealed a new finding where LAMP1 was hyperglycosylated in CLN3 neurons at all time points, which were not apparent in CLN3-Cor neurons. DQ-BSA Green assay indicated a minor reduction in endocytosis in CLN3 neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Subunit C protein levels as examined by western blot analysis were not statistically different between the isogenic neurons at all time points. Conclusions: iPSC-derived neurons in CLN3 mutations can recapitulate certain features of the disease, including storage material accumulation and lysosomal alterations. This work also provides a new finding on the occurrence of LAMP1 hyperglycosylation in CLN3 mutation, which could serve as a potential target for therapy