The Effect of CLN3 Disease on Organellar Characteristics

CLN3 disease, or juvenile dementia, is a lysosomal storage disorder with largely neurological symptoms. Towards the end of their lives, individuals with CLN3 disease develop a cardiac phenotype, which has not yet been systematically studied. On a cellular level, mutations in CLN3 have been shown to disrupt the endo-lysosomal system. However, the function of the CLN3 protein, which is localised on the lysosomal membrane, is still unknown. Positioning of lysosomes is affected by the physiological state of a cell and can be changed in diseases. This study developed a powerful assay for the automated analysis of lysosomal movement and positioning. This assay was then applied to study different cell lines with mutations in their CLN3 gene, specifically a CLN3 knockout HeLa cell line and patient-derived fibroblasts with two different CLN3 mutations. Lysosomal movement was found to be reduced in CLN3 patient fibroblasts. Additionally, the calcium signalling, mitochondrial membrane potential and responses to oxidative stress were characterised in the cells. These were found to be not affected by the mutations in CLN3. Another part of this study aimed to characterise changes in the structure and function of cardiac myocytes in CLN3 disease. This was approached in two ways: Firstly, changes in the structure of ventricular cardiac myocytes were investigated in hearts from mice with a common mutation causing CLN3 disease. Hearts from animals of three different ages were analysed, and changes in the mitochondria were found. Surprisingly, only little of the storage material, characteristic for lysosomal storage disorders, was found. Secondly, iPSC-derived cardiac myocytes were to be used to study changes in the cell’s function