PRION PATHOLOGY IN THE BRAINSTEM: CLINICAL TARGET AREAS IN PRION DISEASE A thesis submitted in partial fulfilment for the degree of Doctor of Philosophy to the University College London by Ilaria Mirabile MRC Prion Unit Institute of Neurology University College London 1 Declaration I, Ilaria Mirabile, 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 List of contributions All the procedures described in this thesis were performed by the candidate, with the following exceptions: In vivo procedures Mice breeding, colony maintenance, ear biopsies, prion inoculation, prion symptoms monitoring, mice culling and brain sampling were performed by designated staff at the Prion Unit animal house facility. Prion inocula were prepared by Dr Jonathan Wadsworth. Immunohistochemistry Paraffin embedding and microtome slicing were performed by designated staff in the MRC Prion Unit histology support group. Molecular biology DNA sequencing was performed by Gary Adamson. Cell culture Flow cytometry was performed by Dr Annika Alexopoulou, Dr Sara Monteiro, and Melania Tangari. 3 Acknowledgments I am extremely grateful to all the members of the MRC Prion Unit for their intellectual, practical and moral support. Firstly, I would like to thank my supervisors, Prof. Parmjit Jat, Prof. John Collinge and Prof. Sebastian Brandner for their guidance. I am particularly grateful to Jackie Linehan, Catherine O‘Malley, Caroline Powell and Lorrain Spence in the Histology Core Facility, and to the Prion Unit Animal Facility, for their tremendous hard work. A big thank goes to the members of Prof Parmjit Jat‘s laboratory who welcome me as a second family, to Prof. Giovanna Mallucci, my supervisor when I first joined the Prion Unit, and to Dr Emanuel Asante for his help on planning experiments once Prof. Mallucci left. I am in debt with the wonderful people at the Education Unit, Dr Caroline Selai and Miss Daniela Warr Schori for their support through these years at the Institue of Neurology. A special thank goes to Michael, who has always been there for me during this sometime not easy journey, and all the other friends I met in the Unit who lifted my mood up, and put things in prospective: Nathalie, Inma, Kat, Savroop, Pela, Hazel, Pedro, Andy, Olivia, Aarti, Jeremy, Kevin, Sabeena, Melania, Sara, Sarah, Steph, Andrew, Annika, Emilie, Samira, Jess, Gary, Chris C, Adrian, Chris M, Mar, Julie, Mike B, Aaron …I am really grateful I could count on your support throughout these years! Enormous thanks to past and present flat mates, Lucy, Sunny, Oy, Kim, Aaren, Kate for tolerating my mood swings and thesis-writing drama. Huge thank you to my friends back home, Alessandra, Maria, Feliciana, and Simona, for their unconditional love and support. My major thanks to my wonderful family, mum, dad and Manuela. Thanks for being my source of inspiration, my rock and my home. Without you I would have probably given up when things got ―messy‖… Dulcis in fundo, an immense thank you to Salim, for his enthusiasm, positive thinking, and faith in me… and, of course, for introducing me to the ―Pomodoro technique‖! 4 To my family 5 Abstract Prion diseases are fatal transmissible neurodegenerative disorders characterized by spongiform changes, neuronal loss, reactive astrocytosis, and deposition of disease associated prion protein (PrP). Our aim was to investigate ―clinical target areas‖ for prion disease, responsible for disease onset, progression, and the clinical phenotype, using PrP overexpressing MloxP and PrP depleted NFH-Cre/MloxP transgenic mouse lines. Upon infection with different prion strains NFH-Cre/MloxP mice have significantly longer survival than MloxP mice (first set of experiments: Me7, ~29 weeks vs. ~17 weeks; Mouse-adapted BSE , ~33 weeks vs. ~20 weeks; second set of experiments: RML, ~35 weeks vs.12 weeks; Me7 ~29 weeks, vs. ~17 weeks; MRC2 ~31 weeks vs. ~22 week. As we found that the first pathological changes in the brains of Me7 and Mouse– adapted BSE infected mice are localized in the brainstem, and clinical signs of prion disease point to brainstem failure, we quantitatively scored spongiosis, abnormal PrP accumulation and astrogliosis at early and late stage of disease in specific brainstem nuclei of RML and Me7 infected MloxP and NFH-Cre/MloxP mice. The first target areas showing abnormal PrP accumulation and gliosis in both prion infections are the locus coeruleus (LC), the nucleus of the solitary tract (NTS) and the pre-Bötzinger complex (PBC). We then studied the pathology progression, scoring prion pathology in these and other brainstem nuclei of infected MloxP and NFH-Cre/MloxP mice in the course of the disease. We show that neural degeneration in the LC, NTS, and PBC correlate with clinical signs characteristic of terminally ill mice. We therefore propose that these areas are potential clinical target areas of prion disease. We also studied the spatial and temporal characteristics of Cre-mediated recombination. With immunohistochemistry in reporter mice, we estimated that in the LC, NTS, and PBC, Cre-mediated recombination is 60% or lower, and this can explain why mice proceed to terminal stage of the disease. In NFH-Cre/MloxP mice we found 6 that recombination is a progressive event and in the hippocampus it is complete by 5 weeks post-natally, differently from previous data. Finally, we produced anti PrP RNAi –encoding lentivirus which could be used as focal therapy in the clinical target areas we propose. 7 Table of contents Title 1 Declaration 2 List of contributions 3 Acknowledgments 4 Abstract 6 Table of contents 8 List of figures and table 17 Abbreviations 21 1 INTRODUCTION 24 1.1 Prions and prion disease 24 1.1.1 Prion diseases in animals 24 1.1.1.1 Scrapie 24 1.1.1.2 Chronic wasting disease 25 1.1.1.3 Transmissible mink encephalopathy 25 1.1.1.4 BSE 25 1.1.2 Human prion diseases 26 1.1.2.1 Sporadic CJD 27 1.1.2.2 Inherited prion diseases 28 1.1.2.3 Acquired prion diseases 28 1.1.2.3.1 Kuru 28 1.1.2.3.2 Iatrogenic CJD 29 1.1.2.3.3 Variant CJD 30 1.1.3 Nature of the infective agent and protein-only hypothesis 31 1.1.3.1 Prion protein gene 32 8 1.1.3.2 Structural characteristics of PrPC and PrPSc 33 1.1.3.3 Models of prion conversion and replication 33 1.1.3.4 Prion strains and transmission barriers 36 1.1.4 Cellular prion protein 40 1.1.4.1 Prion protein structure 40 1.1.4.2 PrPC localization and trafficking 42 1.1.4.3 PrP expression during development in the nervous system 43 1.1.4.4 PrPC physiological function 43 1.1.5 Prion mediated neurotoxicity 46 1.1.5.1 The role of PrPC in prion disease 46 1.1.5.2 PrPSc neurotoxicity 47 1.1.5.3 Lessons from adult PrP knockout mice 48 1.1.5.4 The concept of the ―toxic species‖ 51 1.1.5.5 Mechanism for prion-mediated neurodegeneration 53 1.1.5.6 The concept of clinical target areas 56 1.1.6 Therapeutic strategies in prion disease 58 1.1.6.1 RNA interference 60 1.1.6.2 RNAi in the treatment of neurodegenerative diseases 62 1.2 The brainstem and its main functions 63 1.2.1.1 The brainstem in prion disease 69 1.3 Thesis hypotheses 70 1.4 Aims of the thesis 71 1.5 Outline of the thesis 72 9 2 MATERIALS AND METHODS 74 2.1 Mice 74 2.1.1 Genotyping 74 2.1.2 Prion inoculation 74 2.1.2.1 Prion inoculum preparation and titration 74 2.1.3 Diagnosis of scrapie symptoms 76 2.1.4 Removal of brains and embryos 76 2.2 Immunohistochemistry 76 2.2.1 β-galactosidase staining assay 76 2.2.2 Preparation of paraffin blocks 77 2.2.3 Pre-treatment prior to immunostaining: Re-hydration and de-hydration of wax embedded sections 77 2.2.4 Immunostaining of paraffin-embedded sections 77 2.2.4.1 Haematoxylin and Eosin staining 78 2.2.4.2 Immunostaining with the PrP-specific antibody ICSM35 78 2.2.4.3 Immunostaining for the astroglial marker GFAP 79 2.2.4.4 Immunostaining for NK1 -receptor 79 2.2.4.5 Immunostaining for β- galactosidase 79 2.2.4.6 Immunostaining for tyrosine hydroxylase 79 2.2.5 Neuropathological analysis 80 2.3 Techniques involving nucleic acids 80 2.3.1 Extraction of DNA from tails, ear biopsies, brain samples, and embryos. 80 2.3.2 Polymerase chain reaction 81 10 2.3.3 Real time polymerase chain reaction (qPCR) 83 2.3.4 Plasmid DNA minipreps 83 2.3.5 Maxipreps of plasmid DNA 84 2.3.6 Spectroscopic measurement of DNA 85 2.3.7 Restriction enzyme digestion 85 2.3.8 Ligation of DNA 86 2.3.9 Transformation of DNA into E.coli 86 2.3.10 Agarose gel electrophoresis 87 2.3.11 Extraction of DNA from agarose gel 87 2.3.12 DNA Sequencing 88 2.4 Cell culture 88 2.4.1 Propagation of N2A cells 88 2.4.2 Propagation of HEK293 cells 89 2.4.3 Cryopreservation of cells 90 2.5 Lentiviral procedure 90 2.5.1 Design and preparation of shRNA insert oligonucleotides 90 2.5.2 Annealing of oligonucleotides 91 2.5.3 Recombinant lentivirus production 91 2.5.4 Transduction of HEK293 cells 92 2.5.5 Determination of lentiviral titre 92 2.5.6 Measurement of PrP knockdown 93 3 EFFECT OF CRE-MEDIATED RECOMBINATION IN ME7 AND MOUSE- ADAPTED BSE PRION INFECTED MICE 94 11 3.1 Background 94 3.2 Aims 94 3.3 Experimental setup 94 3.4 Results 95 3.4.1 Extended survival of prion infected NFH-Cre/MloxP mice 95 3.4.2 Time course of prion pathology in mice infected with Me7 prion strains 96 3.4.3 Time course of prion pathology in mice infected with Mouse-adapted BSE prion strain 101 3.5 Discussion 104 3.6 Summary 106 4 PRION PATHOLOGY IN THE BRAINSTEM OF RML AND ME7 MLOXP AND NFH-CRE/MLOXP INFECTED MICE 107 4.1 Introduction 107 4.2 Aims 108