Marotta, D; (2015) Defining the role of the Golgi apparatus in juvenile NCL (Batten disease). Doctoral thesis , UCL (University College London).

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Abstract

The neuronal ceroid lipofuscinoses (NCLs) are a group of severe neurodegenerative lysosomal storage disorders characterised by accumulation of autofluorescent ceroid lipopigments in most cells. NCLs are caused by mutations in at least fourteen recessively inherited human genes. The NCL genes encode both soluble and transmembrane proteins localised to the endoplasmic reticulum, Golgi apparatus or endosomal/lysosomal organelles. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinoses (JNCL, Batten disease). JNCL represents the worldwide most common form of NCL. Currently more than 40 mutations have been characterised in the CLN3 gene. However, the most common mutation causes a 1-kb deletion. CLN3 encode a multi-pass type III transmembrane protein, which is conserved in single-celled eukaryotes such as the fission yeast Schizosaccharomyces pombe, suggesting a fundamental role for this protein in eukaryotic cells. CLN3 has been functionally linked to many different cellular processes, including lysosomal homeostasis, autophagy, lipid synthesis or modification, cytoskeleton organisation and trafficking. Despite these endeavours, the function of CLN3 remains unclear. The main goal of this project was to investigate the role of the Golgi apparatus in the pathogenesis of juvenile CLN3 disease. The role of CLN3 at the Golgi apparatus was studied in mammalian cells and in fission yeast model. The morphology of the Golgi complex was studied in fibroblast cell lines from patients and in HeLa cells depleted for CLN3 using RNAi. The observed changes in morphology were accompanied by manganese dyshomeostasis within the Golgi complex, ER stress and apoptosis. The morphology of the Golgi complex was studied in S. pombe using electron microscopy in order to confirm the changes observed in mammalian cells. Finally, drugs shown to ameliorate aspects of the yeast model of CLN3 disease were tested for their efficacy in mammalian cells as an early step in therapeutic development. In this study I have shown that both morphology and size of the Golgi apparatus result to be affected by the loss/depletion of CLN3. Moreover, the changes in Golgi complex morphology and size are accompanied by manganese dyshomeostasis within the Golgi complex with activation of ER stress and activation of the proapoptotic protein caspase 2. Together, these data suggest that the loss/depletion of CLN3 activates secretory stress pathways and cell death. A dysfunctional Golgi apparatus may be the key to uncover the role of CLN3 and find new targets for therapeutic development.

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