NUCLEAR LAMINA AND AGEING

NUCLEAR LAMINA AND AGEING RAFIDAH BTE ABDUL MUTALIF (B.Sci(Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2015 Acknowledgements Firstly, I would like to express my gratitude to my PhD supervisor, Professor Colin Stewart for providing me the opportunity to work on these interesting and exciting projects, while working full-time as a Research Officer in his lab. I am grateful for all the guidance, advice, discussions and support on various research aspects. In IMB, I am thankful to all past and present members of CS and BB labs for being very helpful and for creating a wonderful working environment. In particular, I would like to thank Dr Esther Wong for all the guidance in mouse husbandry and experiments, to Dr Alexandre Chojnowski for guiding me with the virus work, and various techniques. I would also like to thank Dr Gracy Rosario and Dr Hendrikje Werner for teaching me immunofluorescence and histology techniques, to Dr Nardev Ramanathan on the guidance on metabolic studies, to Dr Henning Horn and Dr Loo Tsui Han for all the interesting discussions on various research topics. I am grateful to all the Research Officers in the lab who have been supportive and doing their part to ensure that the lab runs smoothly. I am also thankful to Dr Bina Rai for her help with the MicroCT bone scans. In IMCB, I am thankful to Dr Christine Cheung who guided me with the directed differentiation of stem cells into the specific lineages of SMC. In NUS, I am thankful to Dr Angeli Veronique and Angeline Tan who guided me on how to extract arteries from mice. From SBIC, I would like to thank Dr Han Weiping and Dr Li Hongyu for allowing me to utilize their metabolic cages. From SMART, I am grateful to Dr Sebastian Bayer for the opportunity to re-create blood vessels with the cells that we generated using his fluidics system. A special thanks to my parents, my brother and my partner for their understanding, motivation, support and unrelenting love, especially through the challenging times. Lastly, I would like to dedicate this thesis to my late sister, Nurulhuda (1987 to 2010), for always believing in me. ii Table of Contents Declaration of Originality i Acknowledgements ii Summary ix List of Tables xi List of Figures and Illustrations xii List of Abbreviations xvii Chapter 1: General Introduction 1.1 Ageing Populations 1.2 Ageing Theories 1.3 Ageing Research 1.4 Premature Ageing Diseases in Humans 1.5 The Nuclear Lamina 10 1.5.1 The LINC Complex 13 1.5.2 KASH-SUN Domain Interaction 14 1.5.3 KASH-Domain Proteins 15 1.5.4 SUN-Domain Proteins 17 1.6 Laminopathies 23 1.6.1 Laminopathies Affecting Striated Muscle 23 1.6.2 Fat Based Laminopathies 25 1.6.3 Premature Ageing 26 1.7 Nuclear Lamina and Ageing 28 1.8 Project Outline 31 iii Chapter 2: Materials and Methods 2.1 In Vivo Murine Studies 34 2.1.1 Generation of Smooth Muscle Specific Lmna9 Mouse 34 2.1.2 Generation of Lmna9/9:SUN1-/- Mouse Model 35 2.1.3 35 2.1.4 2.2 2.3 Generation of E145K Mouse Model Genotyping of Mice 36 2.1.4.1 Tail Digest 36 2.4.1.2 PCR Genotyping 37 2.1.5 Weight Monitoring and Death Curve 37 2.1.6 Mouse Necropsy Analysis 37 2.1.7 MicroCT Scan 38 2.1.8 Body Composition Analysis using EchoMRI 38 2.1.9 Metabolic Analysis 39 Cell Culture 39 2.2.1 Primary Mouse Embryonic Fibroblast (MEF) Derivation 40 2.2.2 Primary Mouse Adult Fibroblast (MAF) Derivation 41 2.2.3 Primary Stromovascular Fraction (SVF) Cell Derivation 41 2.2.4 Lentiviral Transfection Experiments 42 2.2.5 Growth curves and proliferation assays 43 2.2.6 Oil Red O Staining 43 Molecular Biology 44 2.3.1 44 Quantitative PCR (qPCR) iv 2.4 2.3.2 Protein Preparation from Cells 45 2.3.3 Protein Prepartion from Tissues (Whole Tissue Lysates) 46 2.3.4 Immunoblotting 46 2.3.5 Immunofluorescence (IF) 47 2.3.6 Chromatin Immunoprecipitation Assay (ChIP) 49 Statistical Analysis 49 Chapter 3: Tissue Specific Lmna9 Mouse Model 3.1 Introduction 51 3.1.1 Lmna9 Mouse Model 51 3.1.2 Vascular Smooth Muscle Cells and ECM Production 58 3.1.3 Adult Smooth Muscle Specific Cre Recombinase Mouse Control Experiment to Check Specificity of SM22α-CreKI 63 3.1.4 3.2 60 Results 65 3.2.1 Expression of Cre recombinase is Specific to Smooth Muscle Cells in SM22α-CreKI Mice 65 3.2.2 Smooth Muscle Specific Lmna9/- Mice Exhibit Similar Pathology to Lmna9/9 Mice 69 3.2.3 Specificity of Cre Recombinase Expression in Lmna9/- SM22αcre+/- Mice 70 3.2.4 Lmna9/9 Mice Expressed Reduced Levels of ECM Components Most Significantly in the Arteries 3.3 Discussion 72 75 v Chapter 4: In Vitro Human Studies 4.1 Introduction 79 4.1.1 Embryonic Development of Vascular SMC 80 4.1.2 Generation of VSMCs from HESCs 82 4.1.3 SMC Disease Modeling Using iPSCs 84 4.1.4 Proximity-dependent biotin identification (BioID) to identify potential differences in the protein interactome between progerin and lamin A expressing VSM 85 Approach to Modeling In-Vitro systems 87 4.1.5 4.2 Results 88 4.2.1 Directed Differentiation of Stem Cells into Different Lineages of VSMCs 88 4.2.2 Characterization of Early Differentiation Markers 89 4.2.3 Characterization of VSMC Markers 90 4.2.4 Characterization of LMNA/C and SUN1 94 4.2.5 3T3 assay of differentiated VSMCs proliferation 97 4.2.6 Identification and characterization of the Lamin A and Progerin interactomes 98 4.3 Discussion 107 Chapter 5: Amelioration of Lmna9 Phenotype 5.1 5.2 Introduction 111 5.1.1 114 Mouse Models of SUN1 Results 5.2.1 117 Loss of Sun1 Ameliorates Lmna9/9 Pathologies vi 117 5.2.2 Validation of Lamin A and SUN1 Expression 119 5.2.3 Tissue Pathologies Are Improved in Lmna9/9: SUN1-/- 123 5.2.4 SUN1 Accummulates at the Nuclear Envelope and the Golgi of Lmna9/9 Mice 124 5.2.5 SUN1 Overaccumulation in HGPS Cells Correlates with Dysfunction 5.3 125 Discussion 127 Chapter 6: Characterization of a Novel Progeric Mouse – E145K Mouse Model 6.1 Introduction 131 6.2 Results 134 6.2.1 E145K Mutant Mouse Missense Mutation 134 6.2.2 E145K Mutant Mouse Phenotype Characterization 136 6.2.3 Necropsy Analysis of LmnaE145K/E145K mouse 136 6.2.4 Computed Tomography (CT) Analysis 144 6.2.5 In-vitro Characterization 146 6.2.6 Metabolic Analysis 151 6.2.6.1 Body Composition Analysis 151 6.2.6.2 Indirect Calorimetry / Metabolic Cage Studies 152 vii 6.3 6.2.6.3 In-Vitro Adipocyte Differentiation of E145K MAFs 155 6.2.6.4 In-Vitro Adipo Differentiation of E145K Pre-Adipocytes 157 Discussion 160 Chapter 7: General Discussion and Conclusion 165 7.1 The Lmna9 Mouse Model 165 7.2 Amelioration of Lmna9 Phenotype by Loss of SUN1 167 7.3 In-Vitro Studies on Human HGPS Cells 168 7.4 Analysis of a Novel Progeric Mouse Model 169 7.5 Conclusion 172 References 174 List of Publications 191 viii Summary Ageing affects every individual and the world’s ageing population is estimated to double by 2050. Research in ageing would provide insights as to how the quality of life of aged individuals can be improved. Ageing research can generally be approached in two ways – the study of extended life models or the study of accelerated aging models. One of the most drastic accelerated ageing model in human is the Hutchinson Gilford Progeria Syndrome (HGPS). Most HGPS cases are caused by a splicing defect in exon 11 of the LMNA gene that results in a truncated form of Lamin A protein that remains farnesylated. In this thesis, ageing research is pursued by looking at mouse models of accelerated ageing and human in-vitro system. The Lmna9 mouse model was previously established and similar to HGPS, it expresses a truncated Lamin A protein that retains the farnesyl group. The Lmna9 mice shows a similar phenotype to HGPS patients and were found to be defective in ECM production. Subsequently it was identified that Wnt signalling was affected with the reduction of LEF-1 levels. 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Developmental Cell, 19, 413–425 191 [...]... proteins that form the nuclear lamina (Figure 1.4A) This nuclear lamina may be directly attached to the INM, by the farnesyl residues at the c-termini of the lamins and by interactions with integral membrane (INM) proteins [34] The major components of the nuclear lamina are the nuclear lamins In mammals, nuclear lamins fall into 2 categories – type A and type B Lamins (Figure 1.4B) Lamin-A and Lamin-B are... have given rise to at least 7 other types of disease and are collectively called the Laminopathies 9 1.5 The Nuclear Lamina In cells, nuclei are enclosed by the nuclear envelope (NE), which consists of the inner nuclear membrane (INM) and outer nuclear membrane (ONM) The membranes are connected where they are traversed by the nuclear pore complexes, and together with the endoplasmic reticulum (ER), form... Population aged 60 or Over in 2014 and 2050 These maps show the proportion of population aged 60 or over in 2014 and 2050 and demonstrate the speed at which populations are ageing Source: Global Age Watch Index 2014 (2014, December 22) Population Ageing Maps Retrieved from http://www.helpage.org/global-agewatch/population -ageing- data/population -ageing- map/ The increase in ageing populations will have significant... A/C and SUN1 levels in HES2-VSMC Differentiation 95 4.9 Lamin A/C and SUN1 levels in N1-1-VSMC Differentiation 95 xiii Figure Title Page No 4.10 Lamin A/C and SUN1 levels in PG1-1-VSMC Differentiation 96 4.11 Cumulative 3T3 Assay of Progeria and Control – iPS derived SMCs of Different Lineages 98 4.12 Western Blot was carried out using nuclear and cytoplasmic protein extracted from PASMC 99 4.13 Nuclear. .. organisms which best represent ageing to investigate the molecular causes and effects, and to discover potential interventions Examples of genetic mutations in mice that 6 decrease longevity and appear to accelerate ageing are listed in Table 1.1 The role of these genes highlights the importance of maintaining DNA stability and preventing anti-oxidative stress to regulate aging and longevity [22] Reduced... importance of the conserved terminal loop structure[57] Figure 1.4 Lamins are the Major Components of the Nuclear Lamina (A) Native nuclear lamina of Xenopus oocytes Freeze-dried/metal shadowed nuclear envelope extracted with Triton X-100 revealing the nulear lamina meshwork partially covered with arrays of nuclear pore complexes Adapted from Aebi et al Nature 1986 (B) Structures of the mammalian lamins consisting... [8], Hutchinson Gilford Progeria Syndrome (HGPS) and restrictive dermopathy [9] have defective DDR and genomic instability resulting in a premature ageing phenotype [4] It was only when telomeric structures and telomerase were discovered that the disposable soma theory was validated At the same time, this provided a potential link between senescence and ageing Telomeres, which cap the ends of chromosomes,... and Dyekratosis Congenita [1, 13, 14] On the other hand, immortalized cells typically contain telomerase which maintains telomere length Telomerase is a ribonucleoprotein consisting of a reverse transcriptase (TERT) and its RNA moiety (TERC) Examples of telomerase containing cells include germ cells, stem cell and 85% of cancers[1] 4 1.3 Ageing Research There are generally two broad approaches in ageing. .. Lamin C2 [39-42] Both Lamin A and C usually appear to be incorporated into the nuclear lamina in equal amounts [43] although Lamin A levels are reduced (relative to Lamin C levels) in neurons of the central nervous system[44] On the other hand, Lamin AΔ10 and Lamin C2 are minor products with Lamin C2 being specific to the male germline [45] Individual lamins self-associate and form coiled- 10 coil homodimers... somatic and germ cell lines have different fates during organismal life which was later known as the wear -and- tear theory of ageing The disposable soma theory was proposed by Thomas Kirkwood in 1977 This theory proposed that higher organisms develop differential kinetic 2 proofreading and accuracy-promoting mechanisms in somatic and germ lines In somatic cells, reduced accuracy allows energy saving and . 1.6.2 Fat Based Laminopathies 25 1.6.3 Premature Ageing 26 1.7 Nuclear Lamina and Ageing 28 1.8 Project Outline 31 iv Chapter 2: Materials and Methods 2.1 In Vivo Murine Studies 34 2.1.1. of Figures and Illustrations xii List of Abbreviations xvii Chapter 1: General Introduction 1.1 Ageing Populations 1 1.2 Ageing Theories 2 1.3 Ageing Research 5 1.4 Premature Ageing Diseases. guidance in mouse husbandry and experiments, to Dr Alexandre Chojnowski for guiding me with the virus work, and various techniques. I would also like to thank Dr Gracy Rosario and Dr Hendrikje Werner