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Functional Consequences of Setd1a Haploinsufficiency: from Gestation to Behaviour

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Functional Consequences of Setd1a Haploinsufficiency: from Gestation to Behaviour Functional consequences of Setd1a haploinsufficiency: from gestation to behaviour by Matthew Lee Bosworth A thesis submitted for the degree of Doctor of Philosophy September 2019 Declarations Statement 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy. ____________________________ (Matthew Bosworth) Date 24/09/2019 Statement 2 This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is it being submitted concurrently for any other degree or award (outside of any formal collaboration agreement between the University and a partner organisation) ____________________________ (Matthew Bosworth) Date 24/09/2019 Statement 3 I hereby give consent for my thesis, if accepted, to be available in the University’s Open Access repository (or, where approved, to be available in the University's library and for inter-library loan), and for the title and summary to be made available to outside organisations, subject to the expiry of a University-approved bar on access if applicable. ____________________________ (Matthew Bosworth) Date 24/09/2019 Declaration This thesis is the result of my own independent work, except where otherwise stated, and the views expressed are my own. Other sources are acknowledged by explicit references. The thesis has not been edited by a third party beyond what is permitted by Cardiff University's Use of Third Party Editors by Research Degree Students Procedure. ____________________________ (Matthew Bosworth) Date 24/09/2019 i ii Summary Advances in psychiatric genetics are providing opportunities to investigate underlying pathogenic mechanisms. Rare loss of function (LoF) variants that have large effects on risk are of particular interest because they unequivocally implicate LoF of a single gene and are expected to have prominent phenotypic effects. The first LoF variants to be identified for schizophrenia were in the SETD1A gene. SETD1A catalyses methylation of lysine residue 4 on histone 3 (H3K4) and its pathogenic role is consistent with convergent evidence implicating disrupted H3K4 methylation in schizophrenia and other neurodevelopmental disorders. However, understanding of the biological mechanisms underlying the association between SETD1A LoF and psychopathology is lacking. This thesis investigated the functional consequences of Setd1a haploinsufficiency using a mouse model. Setd1a haploinsufficiency resulted in modest transcriptomic changes in the developing mouse brain that were enriched for mitochondrial annotations (Chapter 3). However, there was no enrichment for schizophrenia common variant association. Additionally, placental weight was reduced in Setd1a+/- mice and sexually dimorphic changes in placental gene expression and postnatal growth were observed in Setd1a+/- males but not females (Chapter 4). Behavioural phenotyping revealed that constitutive Setd1a haploinsufficiency caused heightened emotional reactivity and aberrant sensorimotor gating (Chapter 5). The effects on sensorimotor gating were robust and could not be rescued by established antipsychotics (haloperidol and risperidone) (Chapter 6). However, male (but not female) Setd1a+/- mice showed an insensitivity to the startle-inhibiting effects of risperidone, potentially indicating 5-HT2A receptor dysfunction. Finally, behavioural phenotyping of a conditional knockout with Setd1a haploinsufficiency constrained to the nervous system revealed evidence for a degree of convergence with the constitutive model (Chapter 7). In conclusion, this thesis provides novel insights into the diverse effects of Setd1a haploinsufficiency. These findings warrant further investigation to establish the pathophysiological relevance of effects on the developing brain, placenta, and adult brain function. iii iv List of Contents Section Page Chapter 1: General Introduction 1 1.1. Unmet clinical need of schizophrenia 1 1.2. Neurobiology of schizophrenia 2 1.2.1. Neuroanatomical abnormalities 2 1.2.2. Functional brain alterations 3 1.3. Schizophrenia aetiology 7 1.3.1. Environmental risk factors 7 1.3.2. Genetic architecture 9 1.3.2.1. Common variants 9 1.3.2.2. Rare variants 11 1.3.2.2.1. Copy number variants 11 1.3.2.2.2. Loss of function variants 12 1.3.2.2. Summary of genetic findings 14 1.4. SETD1A: utility for disease modelling 14 1.4.1. Histone modifications 14 1.4.2. H3K4 methylation 15 1.4.3. H3K4 methylation in the brain 15 1.4.4. SETD1A biochemical function 17 1.4.5. Setd1a and embryonic development 17 1.4.6. Setd1a and neurogenesis 18 1.4.7. Summary: current understanding of Setd1a function 19 1.5. Thesis aims 19 Chapter 2: General Methods 21 2.1. Animal lines 21 2.1.1. Knockout strategy 21 2.1.2. Animal husbandry 22 2.2. Genotyping 22 2.2.1. DNA extraction 22 2.2.2. PCR 22 2.2.3. Embryonic sex determination 25 2.3. Behavioural methods 25 2.3.1. Elevated plus maze 26 2.3.2. Open field test 26 v Section Page 2.3.3. Locomotor activity 26 2.3.4. Sensorimotor gating 27 2.3.5. Rotarod performance test 27 2.3.6. Novel object recognition 28 2.4. Molecular methods 29 2.4.1. Timed-matings and dissections 29 2.4.2. RNA extraction 29 2.4.3. cDNA synthesis 29 2.4.4. RT-qPCR 29 2.4.5. Protein extraction 30 2.4.6. Western blotting 30 2.5. Data analysis 31 Chapter 3: Investigating transcriptomic changes in the developing 33 brains of Setd1a+/- mice 3.1. Introduction 33 3.2. Methods 34 3.2.1. Trajectory of Setd1a expression across neurodevelopment 34 3.2.2. Generation of the model 35 3.2.3. Model validation 35 3.2.4. RT-qPCR and Western blot data analysis 36 3.2.5. RNA-seq 36 3.2.5.1. RNA extraction and quality control 36 3.2.5.2. Library preparations and sequencing 36 3.2.5.3. Analysis pipeline 36 3.2.5.4. Gene ontology enrichment analysis 38 3.2.5.5. Schizophrenia common variant gene set enrichment 38 analysis 3.3. Results 38 3.3.1. Trajectory of Setd1a expression across neurodevelopment 38 3.3.2. Model validation 39 3.3.3. RNA-seq 40 3.3.3.1. Differential expression 40 3.3.3.2. Gene ontology enrichment analysis 40 vi Section Page 3.3.3.3. Schizophrenia common variant gene set enrichment 40 analysis 3.4. Discussion 43 Chapter 4: Gestational compromise in Setd1a+/- mice and 47 consequences for growth in the early post-weaning period 4.1. Introduction 47 4.2. Methods 51 4.2.1. Timed-matings, dissections, and assessment of embryonic and 51 placental weights 4.2.2. Placental gene expression RT-qPCR 51 4.2.3. Postnatal growth 52 4.2.4. Data analysis 53 4.3. Results 53 4.3.1. E13.5 foetal and placental weight 53 4.3.2. Placental gene expression 54 4.3.3. General viability 57 4.3.4. Postnatal growth curves 57 4.4. Discussion 59 Chapter 5: Behavioural consequences of Setd1a haploinsufficiency in 63 a constitutive knockout mouse 5.1. Introduction 63 5.1.1. Aims 63 5.1.2. Anxiety 63 5.1.3. Locomotor activity 64 5.1.4. Motoric function abnormalities 65 5.1.5. Sensorimotor gating 66 5.1.6. Learning and memory 67 5.1.7. Summary 67 5.2. Methods 68 5.2.1. Behavioural testing 68 5.2.2. Data analysis 68 5.3. Results 69 5.3.1. Elevated plus maze 69 5.3.2. Open field test 70 vii Section Page 5.3.3. Locomotor activity 71 5.3.4. Sensorimotor gating 72 5.3.5. Rotarod performance test 74 5.3.6. Novel object recognition 76 5.4. Discussion 77 Chapter 6: Pharmacological modulation of sensorimotor gating 81 deficits in Setd1a+/- mice using antipsychotics 6.1. Introduction 81 6.2. Methods 84 6.2.1. Subjects 84 6.2.1.1. Pilot work 84 6.2.1.2. Main experiment 84 6.2.2. Apparatus and materials 84 6.2.2.1. Testing apparatus 84 6.2.2.2. Drugs 85 6.2.3. Design 85 6.2.3.1. Test protocol 85 6.2.3.2. Testing procedure 85 6.2.4. Data analysis 86 6.3. Results 87 6.3.1. Pilot work 87 6.3.1.1. Pilot study 1 87 6.3.1.2. Pilot study 2 88 6.3.2. Main experiment 90 6.3.2.1. Pre-test session 90 6.3.2.2. Drug challenge 91 6.3.2.2.1. Haloperidol 91 6.3.2.2.2. Risperidone 93 6.4. Discussion 95 Chapter 7: Isolating the contribution of Setd1a haploinsufficiency in the nervous system to behavioural phenotypes using a conditional 99 Setd1a KO mouse model 7.1. Introduction 99 7.2. Methods 100 viii Section Page 7.2.1. Generation of the model 100 7.2.2. Animals 100 7.2.3. Data analysis 101 7.3. Results 102 7.3.1. Model validation 102 7.3.2. Elevated plus maze 103 7.3.3. Open field test 104 7.3.4. Locomotor activity 106 7.3.5. Sensorimotor gating 108 7.3.6. Rotarod performance test 109 7.3.7. Novel object recognition 112 7.4. Discussion 113 Chapter 8: General Discussion 117 8.1. Overview 117 8.2. Setd1a haploinsufficiency causes neuropsychiatric endophenotypes 117 that cannot be rescued by antipsychotic treatments 8.3. Potential mechanisms underlying pathogenic effects of Setd1a 121 haploinsufficiency 8.3.1. Disrupted transcriptome of the developing brain 121 8.3.2. Prenatal programming by the placenta: preliminary evidence for 122 sexually dimorphic effects of Setd1a haploinsufficiency 8.3.3. Nervous system specific mechanisms 123 8.3.4. Summary 125 8.4. Limitations 126 8.4. Future directions 127 8.5. Concluding remarks 128 Bibliography 129 Appendix 1. Differentially expressed genes (Benjamini Hochberg padj < .05) 179 in E13.5 Setd1a+/- brain ix x List of Figures Figure Page Figure 1.1.
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