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Towards Therapy for Batten Disease Towards therapy for Batten disease Mariana Catanho da Silva Vieira MRC Laboratory for Molecular Cell Biology University College London PhD Supervisor: Dr Sara E Mole A thesis submitted for the degree of Doctor of Philosophy University College London September 2014 Declaration I, Mariana Catanho da Silva Vieira, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The gene underlying the classic neurodegenerative lysosomal storage disorder (LSD) juvenile neuronal ceroid lipofuscinosis (JNCL) in humans, CLN3, encodes a polytopic membrane spanning protein of unknown function. Several studies using simpler models have been performed in order to further understand this protein and its pathological mechanism. Schizosaccharomyces pombe provides an ideal model organism for the study of CLN3 function, due to its simplicity, genetic tractability and the presence of a single orthologue of CLN3 (Btn1p), which exhibits a functional profile comparable to its human counterpart. In this study, this model was used to explore the effect of different mutations in btn1 as well as phenotypes arising from complete deletion of the gene. Different btn1 mutations have different effects on the protein function, underlining different phenotypes and affecting the levels of expression of Btn1p. So far, there is no cure for JNCL and therefore it is of great importance to identify novel lead compounds that can be developed for disease therapy. To identify these compounds, a drug screen with btn1Δ cells based on their sensitivity to cyclosporine A, was developed. Positive hits from the screen were validated and tested for their ability to rescue other specific phenotypes also associated with the loss of btn1. The same hits were also tested in JNCL patient fibroblasts and in a zebrafish model of the disease. Promising results were obtained for three compounds: alloxazine, prochlorperazine dimaleate and E-64, with the latest being the one with the most potential for developing therapeutic tools. Yeast models for other LSDs (Chédiak Higashi Syndrome, Niemann-Pick disease type C2 and congenital CLN10 disease) were also characterised in terms of cellular phenotypes and the compounds described above were also tested in these models. Overlapping phenotypes were observed on all the yeast models, suggesting at least one common pathway between these LSDs. 3 Acknowledgement Firstly, I would like to thank my supervisor Dr Sara Mole for her continuous support and encouragement during this project. Secondly, I am very grateful for all the help other members of the lab gave me throughout: Dr Mike Bond, Davide Marotta, Rachel Brown, Sophia kleine-Holthaus and Varun Warrier; for all their moral support, coffee breaks, and post-work time. I would also like to thank my PhD committee for their guidance: Julie Pitcher, Jürg Bähler and Robin Ketteler. I am very grateful to all the work that was done by Dr Claire Russell and Dr Jamie Freeman, that was crucial to my project; all the support from the UCL Yeast Club, specially Rob de Bruin’s and Jürg Bähler’s lab, from the NCL community; and all the work of the support staff at the Laboratory for Molecular Cell Biology (LMCB), from the reception girls to the light microscopy unit. I am very grateful to the entities that helped by funding my project: the NCL- Stiftung foundation (specially Frank Stehr, who was always very helpful), the BDFA association and the Medical Research Council. I am incredibly grateful to my parents Ricardo and Carmo, my sisters Francisca and Mafalda, and the rest of my family (grandparents, uncles, aunts and cousins) for all their encouraging words and warm embraces. My friends have been very supporting and present through the good and bad moments, and for that I thank them. Finally, I want to thank my other half, Filipe, for that without him this journey would never be possible! 4 Table of contents Abstract ................................................................................................................. 3 Acknowledgement ................................................................................................ 4 Table of contents .................................................................................................. 5 Table of figures ..................................................................................................... 8 List of tables ........................................................................................................ 10 1 Introduction ..................................................................................................... 14 1.1 Lysosomal storage disorders ............................................................................. 15 1.1.1 Neuronal ceroid lipofuscinosis ...................................................................... 16 1.1.2 Juvenile CLN3 disease .................................................................................. 25 1.1.3 Chédiak-Higashi syndrome and Niemann-Pick type C disease .................... 42 1.2 Fission yeast: a model for juvenile CLN3 disease ........................................... 45 1.2.1 Function of yeast vacuoles ............................................................................ 47 1.2.2 NCL genes in S. pombe ................................................................................. 49 1.2.3 Btn1p, the orthologue of CLN3 .................................................................... 50 1.3 Project aims ........................................................................................................ 56 2 Materials & Methods ....................................................................................... 58 2.1 List of reagents ................................................................................................... 59 2.2 Table of yeast strains ......................................................................................... 62 2.3 Fission yeast methods ......................................................................................... 63 2.3.1 Media............................................................................................................. 63 2.3.2 Growth of yeast cells ..................................................................................... 67 2.3.3 Transformation of yeast cells ........................................................................ 68 2.3.4 Labelling vacuoles with FM4-64 and LysoSensor ....................................... 69 2.3.5 Calcofluor staining ........................................................................................ 69 2.3.6 Viability assays ............................................................................................. 70 2.3.7 Spot assays .................................................................................................... 70 2.4 Molecular biology ............................................................................................... 71 2.4.1 Site-directed mutagenesis.............................................................................. 71 2.4.2 Polymerase chain reaction (PCR) ................................................................. 72 2.4.3 Cloning .......................................................................................................... 74 2.4.4 Sequencing .................................................................................................... 76 2.4.5 Electrophoresis in agarose gels ..................................................................... 76 2.4.6 Table of primers ............................................................................................ 77 2.5 Work with mammalian models ......................................................................... 79 2.5.1 Mammalian cell culture and assays............................................................... 79 2.5.2 Zebrafish generation and assays ................................................................... 80 2.6 Microscopy .......................................................................................................... 81 2.6.1 Fluorescence microscopy .............................................................................. 81 2.6.2 Confocal microscopy .................................................................................... 81 2.7 Drug screen ......................................................................................................... 82 2.8 Data analysis and statistics ................................................................................ 83 3 Results 1. Btn1p mutations affect the protein function in different manners ............................................................................................................... 84 3.1 Generating S. pombe genomic integrants of GFP-tagged Btn1p ................... 85 3.1.1 Making the constructs ................................................................................... 85 5 3.1.2 Protein expression levels and localisation..................................................... 86 3.2 Generating S. pombe genomic btn1 mutants .................................................... 90 3.2.1 Generating the mutants ................................................................................. 90 3.2.2 Localisation of integrated mutants ................................................................ 91 3.3 Characterising phenotypes of integrated
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