Zebrafish Disease Models to Study the Pathogenesis of Inherited Manganese Transporter Defects and Provide A

Zebrafish disease models to study the pathogenesis of inherited manganese transporter defects and provide a route for drug discovery Dr Karin Tuschl University College London PhD Supervisors: Dr Philippa Mills & Prof Stephen Wilson A thesis submitted for the degree of Doctor of Philosophy University College London August 2016 Declaration I, Karin Tuschl, 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. Part of the work of this thesis has been published in the following articles for which copyright clearance has been obtained (see Appendix): <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">Tuschl K, et al. Manganese and the brain. Int Rev Neurobiol. 2013. 110:277- </li></ul>312. <ul style="display: flex;"><li style="flex:1">-</li><li style="flex:1">Tuschl K, et al. Mutations in SLC39A14 disrupt manganese homeostasis and </li></ul>cause childhood-onset parkinsonism-dystonia. Nat Comms. 2016. 7:11601. I confirm that these publications were written by me and may therefore partly overlap with my thesis. 2Abstract Although manganese is required as an essential trace element excessive amounts are neurotoxic and lead to manganism, an extrapyramidal movement disorder associated with deposition of manganese in the basal ganglia. Recently, we have identified the first inborn error of manganese metabolism caused by mutations in SLC30A10, encoding a manganese transporter facilitating biliary manganese excretion. Treatment is limited to chelation therapy with intravenous disodium calcium edetate which is burdensome due to its route of administration and associated with high socioeconomic costs. Whole exome sequencing in patients with inherited hypermanganesaemia and earlyonset parkinsonism-dystonia but absent SLC30A10 mutations identified SLC39A14 as a novel disease gene associated with manganese dyshomeostasis. Zebrafish loss-of- function mutants for slc30a10 (slc30a10U800) and slc39a14 (slc39a14U801) were generated using TALEN and CRISPR/Cas9 genome editing technologies in order to model these Mn transporter defects in vivo. Both mutants demonstrate prominent manganese accumulation during larval development. Adult slc39a14U801 mutants show significantly increased brain manganese levels similar to the human phenotype. During larval stages slc39a14U801 mutants display increased sensitivity to manganese toxicity, reduced locomotor activity and visual impairment upon manganese exposure. This phenotype is accompanied by a reduction of tyrosine hydroxylase positive cells in the ventral diencephalon suggesting an involvement of dopaminergic circuits. RNA sequencing further identified genes involved in neurotransmitter release and signalling, phototransduction, circadian clock, and hypoxia inducible factor (HIF) signalling to be affected by manganese dyshomeostasis. In summary, slc30a10U800 and slc39A14U801 zebrafish mutants provide disease models of inherited manganese transporter defects that allow the study of disease mechanisms to identify novel therapeutic targets with the view to improve clinical treatment strategies. 3Acknowledgements I would like to express my deep gratitude to my primary supervisor and long term mentor Dr Philippa Mills who has guided and taught me for over 10 years and has always been there for me both as an excellent academic supervisor and as a close friend. Furthermore, I would like to thank my secondary supervisor Prof Steve Wilson, who has been extremely supportive and uplifting whenever my moral seemed to plummet, for giving me the unique opportunity to learn in his laboratory. Eternal thanks go to Dr Leonardo Valdivia who has spent a huge amount of time teaching me everything about zebrafish and helping with experiments, and has tolerated my neverending interruptions of his work with senseless questions. I would like to thank Prof Peter Clayton who, for many years, has been an extremely motivating and unceasingly supportive mentor. I would also like to thank Prof Paul Gissen who has sold zebrafish to me so well that I am now hooked on this stripy animal. A big thank you goes to my colleagues in both the Translational Omics Group at the Institute of Child Health and the Department of Cell and Developmental Biology for their readiness to help and making each day in the lab a fun day. I would like to thank Prof Olaf Bodamer and Dr Christina Hung at the University of Miami for their generous collaboration. I would also like to thank Dr John Spencer, Dr Rupert Purchase and Dr Alaa Abdul-Sada for their interest in my work and their valuable guidance regarding metal chemistry. Thanks also go to Dr Kling Chong for his assistance with the interpretation of radiology images and Dr Tom Jacques for histopathological analysis. I would like to thank Dr Manju Kurian and Dr Esther Meyer for their efforts to publish our joint work. I am indebted to the patients and their families who have taken part in this research and made this work possible. I am sincerely grateful to Action Medical Research for providing the funding for this project and for giving me this unique opportunity. Finally, eternal thanks go to my family for their neverending support and belief in me, particularly to Tomi for enduring our fruitful research discussions and to Tobias for every minute of sleep that allowed me to write this thesis. 4Table of Contents Abstract......................................................................................................................... 3 Acknowledgements...................................................................................................... 4 Table of Contents ......................................................................................................... 5 Table of figures............................................................................................................. 9 List of tables ............................................................................................................... 14 Abbreviations.............................................................................................................. 16 <ul style="display: flex;"><li style="flex:1">Chapter 1. </li><li style="flex:1">Introduction........................................................................................ 22 </li></ul>1.1 Manganese and its role in disease processes ............................................. 22 1.2 Inherited hypermanganesaemia with dystonia- parkinsonism caused by mutations in SLC30A10.......................................................................................... 24 1.2.1 Clinical phenotype ..................................................................................... 25 1.2.2 Diagnosis .................................................................................................. 28 1.2.3 Treatment.................................................................................................. 33 1.3 Mn transport and homeostasis ..................................................................... 35 1.3.1 Mn uptake ................................................................................................. 36 1.3.2 Mn export .................................................................................................. 38 1.4 Mechanisms of Mn toxicity............................................................................ 43 1.4.1 Mn and its effect on dopamine neurotransmission..................................... 43 1.4.2 Effects of Mn on Glutamine, Glutamate and γ-Aminobutyric acid (GABA) signalling............................................................................................................... 45 1.4.3 Effects of Mn on oxidative stress and mitochondrial dysfunction................ 46 1.4.4 Role of Mn in neuroinflammation ............................................................... 47 1.5 Zebrafish as a disease model........................................................................ 48 1.5.1 Transcription activator-like effector nucleases (TALENs)........................... 49 1.5.2 CRISPR/Cas9 genome editing .................................................................. 50 1.6 Aims and scope of this thesis....................................................................... 52 <ul style="display: flex;"><li style="flex:1">Chapter 2. </li><li style="flex:1">Materials & Methods .......................................................................... 53 </li></ul>2.1 Reagents......................................................................................................... 53 2.2 Subjects.......................................................................................................... 53 2.3 Molecular biology........................................................................................... 53 2.3.1 Genomic DNA extraction ........................................................................... 53 2.3.2 PCR and Sanger sequencing .................................................................... 54 2.3.3 Whole exome sequencing (WES).............................................................. 60 2.3.4 DNA purification ........................................................................................ 60 2.3.5 Restriction enzyme digestion..................................................................... 60 2.3.6 Total RNA isolation, reverse transcription and RT-PCR............................. 61 2.3.7 Quantitative real time PCR (qRT-PCR)...................................................... 64 2.3.8 Conventional molecular cloning................................................................. 65 2.3.9 TOPO TA cloning for sequencing .............................................................. 67 2.3.10 In-Fusion cloning....................................................................................... 67 2.3.11 Transformation of competent E. coli cells .................................................. 69 2.3.12 Colony PCR............................................................................................... 69 2.3.13 Plasmid preparation................................................................................... 70 2.3.14 In vitro transcription of capped RNA .......................................................... 70 2.3.15 Probe synthesis for in situ hybridisation..................................................... 71 2.3.16 5’ and 3’ rapid amplification of cDNA ends (RACE) ................................... 72 2.3.17 TALEN mRNA design and synthesis ......................................................... 76 2.3.18 Generation of CRISPR gRNAs and Cas9 nuclease mRNA........................ 79 2.3.19 High resolution melting analysis (HRMA)................................................... 81 52.3.20 KASP genotyping ...................................................................................... 82 2.3.21 Yeast complementation studies................................................................. 84 2.3.22 Vectors and constructs used ..................................................................... 87 2.4 Embryology .................................................................................................... 89 2.4.1 Zebrafish husbandry.................................................................................. 89 2.4.2 Injection procedure.................................................................................... 89 2.4.3 Generation of stable mutant lines using TALEN or CRISPR genome editing .................................................................................................................. 90 2.4.4 Environmental Mn exposure...................................................................... 92 2.4.5 Whole mount in situ hybridisation .............................................................. 92 2.4.6 Sectioning of whole mount in situ hybridisation samples ........................... 93 2.4.7 Immunofluorescence and confocal imaging............................................... 94 2.4.8 Apoptosis analysis..................................................................................... 95 2.4.9 Locomotor behaviour analysis ................................................................... 96 2.4.10 Optokinetic response (OKR)...................................................................... 98 2.5 Metal determination using inductively coupled plasma – mass spectrometry ICP-MS.............................................................................................. 98 2.5.1 Blood samples........................................................................................... 98 2.5.2 Zebrafish larvae......................................................................................... 98 2.5.3 Zebrafish tissues ....................................................................................... 99 2.5.4 Cell culture samples .................................................................................. 99 2.5.5 ICP-MS analysis........................................................................................ 99 2.6 RNA sequencing .......................................................................................... 100 2.6.1 Preparation of larvae for RNA and DNA extraction .................................. 100 2.6.2 RNA sequencing ..................................................................................... 100 2.7 Statistical analysis ....................................................................................... 100 <ul style="display: flex;"><li style="flex:1">Chapter 3. </li><li style="flex:1">Clinical phenotype and genetics of SLC30A10 deficiency............ 101 </li></ul>3.1 Introduction.................................................................................................. 101 3.2 Case presentations ...................................................................................... 103 3.3 Novel mutations identified in SLC30A10.................................................... 113 3.4 Discussion.................................................................................................... 119 3.4.1 Further delineation of the clinical characteristics of SLC30A10 deficiency............................................................................................................ 119 3.4.2 Expansion of the genetic spectrum of SLC30A10 deficiency ................... 122 Chapter 4. deficiency Identification of a new Mn transporter defect - SLC39A14 .......................................................................................................... 124 4.1 Introduction.................................................................................................. 124 4.2 Whole exome sequencing identifies SLC39A14 as a novel disease gene ....................................................................................................................... 124 4.3 Properties of SLC39A14............................................................................... 130 4.4 Clinical presentation.................................................................................... 131 4.5 Diagnostic features of SLC39A14 deficiency............................................. 134 4.6 Na2CaEDTA effectively lowers whole blood Mn levels and can lead to improvement of clinical symptoms ..................................................................... 140 4.7 SLC39A14 is a Mn uptake transporter at the cell membrane; isoform 1 and 2 show differences in tissue expression, Mn transport ability and transcriptional regulation.............................................................................. 142 4.7.1 Tissue expression.................................................................................... 142 4.7.2 Mn transport efficacy ............................................................................... 147 4.7.3 Transcriptional regulation ........................................................................ 148 4.8 Discussion.................................................................................................... 150 4.8.1 SLC39A14 deficiency – a novel Mn transportopathy................................ 150 64.8.2 SLC39A14 functions as a Mn uptake transporter and its isoforms play diverse roles in the regulation of Mn homeostasis............................................... 155 <ul style="display: flex;"><li style="flex:1">Chapter 5. </li><li style="flex:1">slc30a10 zebrafish do not recapitulate all phenotypes of human </li></ul>SLC30A10 deficiency................................................................................................ 159 5.1 Introduction.................................................................................................. 159 5.2 Characterisation of the slc30a10 orthologue in zebrafish......................... 159 5.2.1 Temporal expression and sequence verification of zebrafish slc30a10.... 159 5.2.2 Spatial expression of zebrafish slc30a10................................................. 165 5.2.3 Yeast complementation studies to assess the function of zebrafish Slc30a10............................................................................................................. 167 5.3 TALEN and CRISPR genome editing to generate a zebrafish slc30a10 null mutant ............................................................................................................ 169 5.3.1 Generation of a slc30a10 loss-of-function mutant using TALENs targeting exon 1 ................................................................................................................ 169 5.3.2 Generation of a slc30a10 loss-of-function mutant using CRISPRs targeting exon 3 ................................................................................................................ 172 5.4 Acute Mn toxicity in wild-type zebrafish..................................................... 174 5.5 Phenotypic characterisation of slc30a10U800 mutants ............................... 176 5.5.1 slc30a10U800 mutants show differences in Mn levels compared to wild-type larvae depending on their stage of development.................................. 176 5.5.2 slc30a10U800 mutants are more resistant to Mn toxicity during early larval development ....................................................................................................... 179 5.5.3 slc30a10U600 mutants harbouring the p.P240Afs*92 mutation do not show phenotypic differences to slc30a10U800 mutants .................................................. 185 5.6 Discussion.................................................................................................... 186 5.6.1 Zebrafish slc30a10 facilitates Mn export and encodes two transcripts..... 186 5.6.2 Slc30a10 loss-of-function in zebrafish causes an unexpected phenotype 187 Chapter 6. mutant CRISPR genome editing to generate a slc39a14 loss-of-function .......................................................................................................... 189 6.1 Introduction.................................................................................................. 189 6.2 Characterisation of the slc39a14 orthologue in zebrafish......................... 189

Zebrafish Disease Models to Study the Pathogenesis of Inherited Manganese Transporter Defects and Provide A

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