Generating and Characterising Knockout and Transgenic Mouse Models of Frontotemporal Dementia Caused by CHMP2B Mutation This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to University College London By Shabnam Ghazi-Noori MRC Prion Unit and Department of Neurodegenerative Disease Institute of Neurology University College London Declaration I hereby declare that this thesis is my work and effort, and that it has not been submitted anywhere for any award. Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to the literature, and acknowledgement of collaborative research and discussions. 2 Abstract A mutation in the charged multivesicular body protein 2B (CHMP2B) gene, identified in a kindred from the Jutland region of Denmark, segregates with affected family members with clinical presentations of frontotemporal dementia (FTD) and is absent in control populations (Gydesen et al., 1987; Gydesen et al., 2002; Skibinski et al., 2005). The mutation is a G>C transition in the splice acceptor site of exon 6 resulting in two novel splice variants CHMP2BInt5 and CHMP2B10 leading to C-terminal truncation of the CHMP2B protein (Skibinski et al., 2005). Chmp2b knockout (Chmp2b-/-) mice and transgenic mice expressing either wild-type or C-terminally truncated mutant CHMP2B splice variants CHMP2BInt5 and CHMP2B10 were generated with the aims of examining the normal function of Chmp2b and the effect of mutant CHMP2B species in vivo, as well as providing insight into a potential common FTD mechanism of disease. Quantification of Chmp2b protein in Chmp2b-/- mice demonstrates a significant (85%) depletion of endogenous Chmp2b in the mouse brain. No pathology is identified in the CNS or muscle tissue of these mice however, they do demonstrate significant motor and behavioural abnormalities. CHMP2BInt5 transgenic mice demonstrate neurodegenerative changes including progressive gliosis, accumulation of CHMP2B, p62 and ubiquitin inclusions which are negative for TDP-43 and FUS proteins, consistent with the inclusion pathology observed in patients with CHMP2B mutation. Furthermore, these mice have reduced survival and develop progressive axonopathy characterized by axonal swellings and accumulation of mitochondria and vesicles likely from the endosome- lysosome and autophagy pathway, implicating altered axonal function in disease pathogenesis. This thesis describes the first mouse models of FTD-3 caused by CHMP2B mutation and presents evidence consistent with a gain-of-function effect unique to the CHMP2BInt5 isoform and provides new insights into the mechanisms of CHMP2B- induced neurodegeneration. 3 Table of contents Declaration ----------------------------------------------------------------------------------------------------------------------- 2 Abstract --------------------------------------------------------------------------------------------------------------------------- 3 List of tables-------------------------------------------------------------------------------------------------------------------- 10 List of figures ------------------------------------------------------------------------------------------------------------------ 11 List of film clips ---------------------------------------------------------------------------------------------------------------- 15 Abbreviations ------------------------------------------------------------------------------------------------------------------ 16 Antigen retrieval abbreviations ------------------------------------------------------------------------------------------ 18 Researcher initials ------------------------------------------------------------------------------------------------------------ 18 Dedication ---------------------------------------------------------------------------------------------------------------------- 19 Acknowledgments ----------------------------------------------------------------------------------------------------------- 20 1 Introduction ------------------------------------------------------------------------------------------------------------ 21 1.1 Project Overview -------------------------------------------------------------------------------------------------- 21 1.1.1 A brief history of clinical understanding of FTD -------------------------------------------------- 21 1.1.2 Frontotemporal dementia linked to chromosome 3 in a Danish family- Historical perspectives ------------------------------------------------------------------------------------------------------------ 24 1.2 Clinical Presentation and Classification --------------------------------------------------------------------- 27 1.2.1 Behavioural variant frontotemporal dementia --------------------------------------------------- 29 1.2.2 Semantic dementia --------------------------------------------------------------------------------------- 30 1.2.3 Primary non-fluent aphasia ---------------------------------------------------------------------------- 31 1.3 FTD Clinical Variation -------------------------------------------------------------------------------------------- 32 1.3.1 FTDP-17 ------------------------------------------------------------------------------------------------------ 32 1.3.2 FTD-3 --------------------------------------------------------------------------------------------------------- 32 1.3.3 FTD-MND ---------------------------------------------------------------------------------------------------- 34 1.4 Epidemiology ------------------------------------------------------------------------------------------------------- 35 1.5 Genetics ------------------------------------------------------------------------------------------------------------- 38 4 1.5.1 CHMP2B ----------------------------------------------------------------------------------------------------- 38 1.6 Neuropathology --------------------------------------------------------------------------------------------------- 44 1.6.1 FTLD-tau ----------------------------------------------------------------------------------------------------- 46 1.6.2 FTLD-TDP ---------------------------------------------------------------------------------------------------- 47 1.6.2.1 FTLD-TDP Clinical and Genetic Associations ----------------------------------------------- 48 1.6.3 FTLD-FUS ---------------------------------------------------------------------------------------------------- 49 1.6.4 FTLD-Other ------------------------------------------------------------------------------------------------- 50 1.6.5 FTD-3 Neuropathology ---------------------------------------------------------------------------------- 50 1.7 Endosomal Sorting Complex Required for Transport Proteins and Multivesicular Bodies ---- 53 1.7.1 Human homologues of yeast ESCRT III proteins -------------------------------------------------- 54 1.7.2 Mutant CHMP2B Causes dendritic spine and late endosome pathology ------------------ 56 1.7.3 Mutant CHMP2B impairs the endosome-lysosome pathway --------------------------------- 58 1.7.4 Mutant CHMP2B impairs autophagy ---------------------------------------------------------------- 59 1.8 Mouse Models of FTD -------------------------------------------------------------------------------------------- 61 1.8.1 ESCRT subunit mouse models ------------------------------------------------------------------------- 62 1.8.2 CHMP2B mouse models--------------------------------------------------------------------------------- 64 1.9 Project Aims -------------------------------------------------------------------------------------------------------- 64 2 MATERIALS AND METHODS --------------------------------------------------------------------------------------- 65 2.1 Equipment and Reagents --------------------------------------------------------------------------------------- 65 2.1.1 Equipment -------------------------------------------------------------------------------------------------- 65 2.1.2 Plastic and glassware ------------------------------------------------------------------------------------ 66 2.1.3 Commercial kits ------------------------------------------------------------------------------------------- 66 2.1.4 Antibodies -------------------------------------------------------------------------------------------------- 67 2.1.5 Reagents----------------------------------------------------------------------------------------------------- 71 2.1.6 Prepared solutions---------------------------------------------------------------------------------------- 73 2.2 Mouse Strains ------------------------------------------------------------------------------------------------------ 75 2.2.1 CHMP2B transgenic mouse colony ------------------------------------------------------------------- 75 2.2.2 Chmp2b knockout mouse colony --------------------------------------------------------------------- 75 2.3 Animal Husbandry and Ethical Approval ------------------------------------------------------------------- 75 2.4 Molecular Methods ----------------------------------------------------------------------------------------------- 76 2.4.1 Primer design ---------------------------------------------------------------------------------------------- 76 2.4.1.1 Primer sequences --------------------------------------------------------------------------------- 76 5 2.4.2 Tissue digestion and DNA extraction ---------------------------------------------------------------- 77 2.4.3 RNA extraction and reverse