Investigating the brain in mouse models of Duchenne muscular dystrophy Emine Bagdatlioglu A thesis submitted for the degree of Doctor of Philosophy Newcastle University Faculty of Medical Sciences Institute of Genetic Medicine September 2017 i i Abstract Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disease caused by mutations in the DMD gene, which encodes the large cytoskeletal protein dystrophin. Alongside severe muscle pathology, one-third of DMD patients exhibit cognitive problems ranging from reduced verbal intelligence to severe autism. There is conclusive evidence that the muscle pathology exhibited by DMD patients is progressive, yet it remains unknown whether the cognitive impairments in DMD are also progressive. Previous studies have highlighted a cognitive impairment in the mdx mouse model of DMD, but no studies have investigated if this cognitive impairment worsens with age. We assessed the consequences of dystrophin deficiency on brain morphology and cognitive function in two dystrophin-deficient mouse models (mdx and Cmah-/-mdx mice). The overall project aim was to identify outcome measures to monitor central nervous system (CNS) pathology non-invasively in DMD mice. Magnetic resonance imaging (MRI) identified a total brain volume increase in DMD mice, alongside morphological changes in brain ventricles. Behavioural testing revealed a deficit in hippocampal spatial learning and memory, particularly long-term memory, in mdx mice, which appears to progressively worsen with age. Immunoblotting identified a progressive reduction of aquaporin-4 (AQP4) expression, the major water channel of the CNS, in DMD mice. Moreover, contrast enhancing MRI and Evans blue extravasation demonstrated a progressive impairment in blood-brain barrier (BBB) integrity in mdx mice. Proteomic profiling of the mdx cerebellum identified changes in expression of mitochondrial subunit complexes, suggestive of changes in mitochondrial function. Additionally, elevated levels of inflammatory markers were identified and confirmed in the mdx cerebellum. Our studies suggest that dystrophin deficiency causes a progressive cognitive impairment in mdx mice. We also present evidence showing that changes in osmotic equilibrium may be involved in the pathogenesis of DMD, with reductions in AQP4 expression and BBB disruptions. We speculate that some of the changes in the mdx cerebellar proteome, in comparison to wild type mice, ii serve as compensatory mechanisms whilst others may contribute directly to cognitive dysfunction in DMD. These results support a role for dystrophin in normal brain morphology and cognitive function. iii iv Dedication This thesis is dedicated to patients with Duchenne muscular dystrophy and their families. v Acknowledgements I would like to thank my supervisors Professor Volker Straub and Professor Andrew Blamire for their help, support, and guidance throughout this project and for also providing me with the opportunity to undertake this research. I am grateful for the opportunities and support that I have received from the John Walton Muscular Dystrophy Research Centre. I would also like to thank Dr Andreas Roos and Dr Steven Laval for their advice with practical aspects of this project. Thanks to my PhD assessors, Professor Helen Arthur and Professor David Elliott, for their insightful knowledge. The animal care staff: Brigid Griffin, Steve Smith, Lynne Todd, and other members of the FGU have been instrumental in helping me with mouse work, without which this research would not have been possible. I would like to thank Dr Chris Blau and Ella Dennis for their help with the X-ray of mice. Thank you to Dr Alison Blain and Elizabeth Greally for your time, support, and friendship over the past 4 years, you really helped me to settle into the team, made me feel welcome, and our discussions regarding the project have been incredibly beneficial. Many thanks to Dr Paola Porcari and Dr Dara O’Hogain for setting up the MRI scan protocols. Thank you to Dr Ross Laws, Tracey Davey and Professor Jochaim Weis for their work on electron microscopy. I would also like to thank Vietxuan Phan for her work on cerebellar proteomic profiling. A special thanks to my family: my parents, brothers, and sister for their continuous support and for always encouraging me to strive further, I am proud to share this with you. Thank you to my friends at the IGM and outside, Yasmin Issop and Lauren Phillips, I have really enjoyed working together over the past 4 years and your constant encouragement, kindness, and help has been invaluable. Lastly, I want to thank Tom Sage, for your never-ending patience, unwavering belief in my ability to complete this thesis, and immeasurable support. This work was funded by the Medical Research Council UK. vi Table of Contents Chapter 1. Introduction ............................................................................................ 27 1.1 Duchenne muscular dystrophy ...................................................................... 27 1.1.1 Clinical phenotype ...................................................................................... 28 1.1.2 Current care strategies for DMD ................................................................. 29 1.2 The dystrophin-glycoprotein complex (DGC) ............................................... 30 1.2.1 DGC in muscle ........................................................................................... 30 1.2.2 The DGC in brain ....................................................................................... 32 1.3 The DMD gene and dystrophin....................................................................... 33 1.4 Cognitive functioning in DMD ........................................................................ 35 1.4.1 Variable protein expression in DMD ........................................................... 38 1.5 Potential role of dystrophin in the CNS ......................................................... 40 1.6 The Cerebellum and dystrophin ..................................................................... 44 1.7 The hippocampus and dystrophin ................................................................. 49 1.8 Fluid movement within the brain ................................................................... 51 1.8.1 The blood-brain barrier (BBB) .................................................................... 51 1.8.2 The blood-cerebral spinal fluid barrier (BCSFB) ......................................... 53 1.9 Alteration of brain structure in DMD patients ............................................... 54 1.10 The use of corticosteroids in DMD .............................................................. 55 1.11 Mouse models for DMD ................................................................................ 56 1.11.1 The mdx mouse model of DMD ................................................................ 56 1.11.2 The Cmah-/-mdx mouse model of DMD ................................................... 57 1.12 Statement of aims ......................................................................................... 61 1.12.1 Overall study aims .................................................................................... 61 1.12.2 Hypotheses to be tested ........................................................................... 62 1.12.3 Specific chapter aims ............................................................................... 63 Chapter 2. Materials and Methods ........................................................................... 64 2.1 Buffers and solutions ..................................................................................... 64 2.2 Standard molecular biology techniques ....................................................... 68 2.2.1 DNA extractions ......................................................................................... 68 2.2.2 Measuring DNA concentrations .................................................................. 68 2.2.3 Genotyping ................................................................................................. 68 2.2.4 Genotyping mdx mice ................................................................................. 68 2.2.5 Genotyping Cmah mice .............................................................................. 69 2.2.6 Agarose gel electrophoresis ....................................................................... 70 2.2.7 DNA purification by gel extraction............................................................... 70 2.2.8 DNA sequencing and alignments ............................................................... 71 2.3 Animal work .................................................................................................... 71 vii 2.3.1 Animal care and husbandry ........................................................................ 71 2.3.2 Transgenic mice used in this study ............................................................. 71 2.4 Behavioural testing ......................................................................................... 73 2.4.1 Barnes maze testing ................................................................................... 73 2.4.2 Novel Object Recognition ........................................................................... 75 2.5 Histological studies ........................................................................................ 76 2.5.1 Sample Preparation ...................................................................................