Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. INVESTIGATING THE ROLE OF HDAC4 SUBCELLULAR DISTRIBUTION IN DROSOPHILA DEVELOPMENT AND MEMORY A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry At Massey University, Manawatū, New Zealand Patrick James Main 2019 “I sometimes feel, in reviewing the evidence on the localization of the memory trace, that the necessary conclusion is that learning is just not possible” (Lashley, 1950) i ABSTRACT The class IIa histone deacetylase HDAC4 has been previously demonstrated to play an essential role in brain development, learning and memory. However, the molecular pathways through which it acts are unknown. HDAC4 undergoes activity-dependent nucleocytoplasmic shuttling, disruption of the balance of nuclear and cytoplasmic HDAC4 has been identified as a factor in developmental and neurodegenerative disorders. This project used Drosophila melanogaster as a model to investigate the effects of altered subcellular distribution of HDAC4 on neural development and memory formation through the overexpression of Drosophila HDAC4 and wild-type human HDAC4 (hHDAC4), as well as nuclear- and cytoplasm-localising mutants of hHDAC4 named 3SA and L175A, respectively. The nuclear or cytoplasmic abundance of HDAC4 was adjusted by expressing the mutants during development or in adult flies. It was established that increased nuclear abundance of hHDAC4 in the brain impaired long-term memory and development, whereas increasing the cytoplasmic abundance did not. Further investigation showed that, contrary to vertebrate models, HDAC4 does not appear to repress memory in Drosophila through inactivation of MEF2 or CREB. Investigation of the transcriptomic changes induced by nuclear and cytoplasmic HDAC4 via RNASeq on RNA isolated from fly heads showed that L175A unexpectedly up-regulates the expression of genes in transcription and DNA synthesis. The relatively low number of transcriptional changes induced by 3SA suggested that it may be acting through largely transcriptionally independent means to impair memory and development in Drosophila. The localisation of HDAC4 to punctate foci in nuclei, potentially forming protein aggregates similar to Marinesco bodies seen in Parkinson’s Disease warrants further investigation. This project has shown that nuclear but not cytoplasmic HDAC4 impairs development and memory in Drosophila. Furthermore, cytoplasmic HDAC4 may play a role in transcriptional regulation of neurons, possibly regulation metabolic activity, suggesting that the activity-dependent nucleocytoplasmic shuttling of HDAC4 may not be primarily to remove HDAC4 from the nucleus and but instead to return HDAC4 to the cytoplasm. ii iii ACKNOWLEDGEMENTS Despite the single name in the authorship it is woefully inaccurate to consider a PhD the work of a single person. I do not believe for a single second I could have achieved this undertaking on my own, the support and input I received from friends, colleagues and family was essential to the completion of this project and I will utilise some space here to thank many of those who were part of this. Firstly, my primary supervisor Dr Helen Fitzsimons without whom none of this could have happened in a very literal sense. The regular meetings and suggestions for interesting articles were vital to my understanding of the field. I am also infinitely grateful for her persistence and patience in nudging me into becoming the scientist I am today. Thank-you. My co-supervisor Dr Tracy Hale, who was invaluable in her assistance with tissue culture and protein work, providing vital feedback and troubleshooting advice for cell-culture experiments in addition to critiquing my immunohistochemistry figures to help me think about communication with a wider audience. The Drosophila neurogenetics group and chromatin research group that constitute our lab space have seen a number of members while I’ve been there, it has been a pleasure working with you all, particular thanks to Sarah Bond, Silvia Schwartz and Patrick Freymuth for teaching me many of the basics when I was new to the lab and Ana Claasen for being an excellent sounding board for ideas and bravely reading through my thesis. I would also like to thank the Manawatū Microscopy and Imaging Centre for training and assistance with microscopy techniques, the technicians and administration team in the School of Fundamental sciences for their assistance in training and help, as well as the maintaining of equipment that is so vital to all of this. Furthermore, I have been fortunate to receive the Massey Doctoral Scholarship to fund me through my research, in addition to the SFS Travel Fund for partially iv funding my trip to the Drosophila Neurobiology conference in Krakow which was an unforgettable experience. Last, and absolutely not least, my family and friends. My family for their unconditional support, my friends for being there when I needed distracting. My loving wife, Ami, for her tolerance, patience and love and my son Wilfred, who changed my life in ways I never considered. v ABBREVIATIONS °C Degrees Celsius 3SA hHDAC4-3SA Ach Acetylcholine AD Alzheimer’s Disease APL Anterior paired lateral neuron Arc1 Activity-regulated cytoskeleton protein 1 ASD Autism spectrum disorder Aβ Amyloid-beta BDSC Bloomington Drosophila Stock Centre bp Base pair Ca++ Calcium CaMK Calcium/Calmodulin-dependent kinase cAMP Cyclic adenosine monophosphate CI Courtship index CIP Calf intestinal phosphatase CRE cAMP response element CREB CRE binding protein Canton S Canton special CS Conditioned stimulus DAL Dorsal anterolateral neuron DAN Dopaminergic neuron DmHDAC4 Drosophila histone deacetylase 4 DNA Deoxyribonucleic acid DPM Dorsal paired medial neurons EDTA Ethylenediaminetetraacetic acid Elav Embryonic lethal abnormal vision FASII Fasciclin II GABA γ-amino butyric acid GFP Green fluorescent protein GMR Glass multimer reporter HAT Histone acetyltransferase vi HDAC Histone deacetylase HDACI Histone deacetylase inhibitor hHDAC4 Human histone deacetylase 4 KC Kenyon cell KCl Potassium chloride KD Knockdown kDa Kilodalton L Litre L175A hHDAC4-L175A LTM Long-term memory M Molar mA Milliampere MAPK Mitogen-activated protein kinase MB Mushroom body MBON Mushroom body output neurons MEF2 Myocyte enhancing factor 2 MgCl2 Magnesium chloride MI Memory index MRE MEF2-response element mRNA Messenger RNA NaB Sodium butyrate NEB New England Biolabs NES Nuclear export signal NLS Nuclear localisation signal NMDARs N-methyl-D-aspartic acid receptors OAN Octopaminergic neurons OE Overexpression PAM Protocerebral anteromedial neurons PBS Phosphate buffered saline PCR Polymerase chain reaction PD Parkinson’s disease PKA Protein kinase A PKC Protein kinase C vii PPL Protocerebral posterior lateral neurons RIPA Radioimmunoprecipitation assay buffer RFP Red fluorescent protein RNAi RNA interference Rpm Rotations per minute RT Room temperature SAHA Suberoylanilide hydroxamic acid SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis STM Short-term memory SUMO Small ubiquitin like modifier TARGET Temporal and regional gene expression targeting TBS Tris-buffered saline TRP Transient receptor potential ts Temperature sensitive UAS Upstream activating sequence V Volts VDRC Vienna Drosophila Resource Centre WB Western blotting wt wild-type viii TABLE OF CONTENTS Abstract ........................................................................................................................ i Acknowledgements ................................................................................................. iii Abbreviations ............................................................................................................. v Table of Figures .......................................................................................................xiii Table of Tables ...................................................................................................... xviii 1 Introduction ........................................................................................................ 1 1.1 Neurodevelopmental disorders & neurodegeneration ............................ 1 1.2 The study of learning and memory ............................................................ 4 1.2.1 Short and long-term memory .............................................................. 8 1.2.2 The physiology of memory .................................................................. 9 1.3 The Drosophila mushroom body ............................................................... 11 1.3.1 Intrinsic neurons of the mushroom body ......................................... 11 1.3.2 Genetic tools for Drosophila memory research .................................. 14 1.3.3 Extrinsic neurons of the mushroom body ........................................ 18 1.4 Epigenetic regulation of memory formation ........................................... 23 1.4.1 Histone acetyltransferases (HATs) and Histone deacetylases (HDACs) ............................................................................................................ 24 1.4.2 Histone acetylation and memory ...................................................... 24 1.4.3 The HDAC family members .............................................................. 25 1.4.4 Histone deacetylases in memory ....................................................... 27 1.5 Histone deacetylase 4 (HDAC4) in the brain ........................................... 30 1.5.1 Domain structure and catalytic activity of HDAC4 ......................... 30 1.5.2 Expression of HDAC4 in the brain .................................................... 32 1.5.3 HDAC4 and neuronal function ......................................................... 33 1.6 Downstream targets of HDAC4 ................................................................ 37 1.6.1 MEF2 and Arc in brain development and memory ......................... 38 1.6.2 CREB in memory and learning .......................................................... 40 1.6.3 HDAC4 and SUMOylation ................................................................ 40 ix 1.7 Drosophila melanogaster as a model to study memory ............................. 42 1.7.1 Behavioural assays to evaluate learning and memory .................... 42 1.7.2 Investigating developmental pathways in neurons......................... 46 1.8 Aims of this project .................................................................................... 48 2 Methods and Materials .................................................................................... 50 2.1 Fly strains ................................................................................................... 50 2.1.1 Fly strain maintenance ....................................................................... 50 2.1.2 Genetic crosses .................................................................................... 50 2.2 Eye phenotype analysis ............................................................................. 51 2.2.1 Light microscopy ................................................................................ 51 2.2.2 Scanning electron microscopy (SEM) ................................................ 51 2.3 Drosophila brain isolation ........................................................................... 52 2.3.1 Immunohistochemistry on whole fly brains .................................... 53 2.4 Protein extraction from flies ...................................................................... 53 2.4.1 Fly head isolation ................................................................................ 53 2.4.2 Protein isolation .................................................................................. 54 2.4.3 RNA extraction from flies .................................................................. 55 2.5 Plasmid subcloning .................................................................................... 56 2.5.1 Generation of competent cells ........................................................... 56 2.5.2 plasmid preparation from Escherichia coli ......................................... 57 2.5.3 Quantification of DNA ....................................................................... 57 2.5.4 Restriction endonuclease digestion of DNA .................................... 57 2.5.5 Agarose gel electrophoresis ............................................................... 57 2.5.6 DNA purification from agarose gels ................................................. 58 2.5.7 Plasmid ligation .................................................................................. 58 2.5.8 Transformation of competent cells .................................................... 58 2.6 SDS-PAGE and Western Blots .................................................................. 59 2.7 Sequencing of DNA ................................................................................... 60 x 2.8 Drosophila transgenesis .............................................................................. 60 2.8.1 Crosses to generate stable fly lines .................................................... 62 2.9 Transient transfection of human cell lines ............................................... 63 2.10 Total protein extraction of human cell lines ......................................... 63 2.11 Quantification of protein ....................................................................... 63 2.12 Luciferase assay ...................................................................................... 64 2.12.1 Tissue preparation for flies................................................................. 64 2.12.2 Tissue preparation for human cell culture ........................................ 64 2.12.3 Luciferase assay .................................................................................. 64 2.13 Transcriptome analysis .......................................................................... 65 2.14 Courtship suppression assay ................................................................. 65 2.14.1 Statistical analysis of courtship data ................................................. 67 2.15 Electrical stimulation of whole flies ...................................................... 67 2.16 TrpA1 activation ..................................................................................... 68 3 The subcellular distribution of HDAC4 variants ........................................... 69 3.1 Investigating the effects of nuclear and cytoplasmic HDAC4 on neuronal development ......................................................................................................... 69 3.2 Characterisation of the subcellular distribution of wild-type and mutant HDAC4 in the Drosophila brain ........................................................................... 71 3.2.1 A note on colour schemes .................................................................. 71 3.2.2 Generation of N-terminal GFP-HDAC4 fusion ................................ 71 3.3 Effects of altered subcellular distribution on neuronal development. .. 81 3.3.1 Characterising the effects of altered HDAC4 distribution on Drosophila mushroom body development ...................................................... 81 3.3.2 Effects of altered subcellular distribution on Drosophila eye development ..................................................................................................... 87 3.4 Discussion ................................................................................................... 90 4 Investigating the role of altered HDAC4 subcellular distribution on long- term memory ............................................................................................................ 92 4.1 Courtship suppression assay .................................................................... 92 4.2 Drosophila and human HDAC4 in long-term memory ........................... 94 4.3 Nuclear and cytoplasmic hHDAC4 in long-term memory .................... 96 xi 4.4 Discussion ................................................................................................... 98 5 HDAC4 and transcriptional regulators ........................................................ 100 5.1 Co-localisation of HDAC4 and MEF2 .................................................... 100 5.1.1 A second note on colour schemes ................................................... 100 5.2 Co-localisation of HDAC4 and MEF2 in the brain................................ 102 5.3 Effects of altered abundance of MEF2 on Drosophila mushroom body development ....................................................................................................... 104 5.4 Investigating the role of MEF2 in memory ............................................ 107 5.5 Investigating a potential HDAC4-MEF2 interaction ............................ 109 5.6 Characterising CREB activity in cultured human cells ........................ 119 5.7 Co-localisation of HDAC4 and CREB .................................................... 121 5.8 Discussion ................................................................................................. 123 6 Investigating the effects of altered HDAC4 subcellular distribution on transcription in Drosophila ..................................................................................... 126 6.1 Introduction .............................................................................................. 126 6.2 Preparation and QC of samples .............................................................. 127 6.3 Quality control and read verification ..................................................... 130 6.4 Analysis of differential expression between treatment groups ........... 134 6.5 Transcriptional changes induced by L175A .......................................... 135 6.6 Transcriptional changes induced by 3SA .............................................. 142 6.7 Differential expression between the L175A and 3SA groups .............. 147 6.8 GO-TERM analysis .................................................................................. 152 6.9 Comparisons to previous sequencing data............................................ 159 6.10 Candidate genes from RNASeq analysis............................................ 165 6.11 Screening candidate genes for interactions with HDAC4 ................ 168 6.12 Discussion ............................................................................................. 176 7 Summary and future directions .................................................................... 183 7.1 Increased nuclear but not cytoplasmic HDAC4 significantly impairs memory and development in Drosophila. ......................................................... 183 7.2 HDAC4 does not appear to act through MEF2 or CREB in the repression of Drosophila memory. ........................................................................................ 186 7.3 Transcriptional effects of increased nuclear or cytoplasmic HDAC4 in the Drosophila brain. ................................................................................................. 189 7.4 Conclusion ................................................................................................ 192 8 Bibliography ................................................................................................... 193 9 Appendix 1 Fly stocks .................................................................................... 220 xii 10 Appendix 2 Details on GFP::HDAC4v cloning ............................................ 223 11 Appendix 3 Primers, plasmids and sequences ............................................ 229 11.1 PCR Primers .......................................................................................... 229 11.2 Plasmids ................................................................................................ 230 11.3 Antibodies ............................................................................................. 231 11.4 HDAC4 variant sequences ................................................................... 232 12 Appendix 4 hsp70MRE and hsp70ΔMRE development.............................. 235 13 Appendix 5 RNASeq quality control ............................................................ 236 14 Appendix 6 Most significantly differentially expressed RNASeq genes identified in differential expression heatmaps .................................................... 248 xiii TABLE OF FIGURES Figure 1.1 Schematic of an example synaptic junction. .......................................... 4 Figure 1.2 Schematic showing the gill withdrawal reflex in Aplysia californica. ... 5 Figure 1.3 Molecular similarities between Aplysia and mammalian neurons. ..... 7 Figure 1.4 Lateral view of a single mushroom body and surrounding structure. ................................................................................................................................... 12 Figure 1.5 Simplified schematic of the MB from the anterior of the fly. ............. 13 Figure 1.6 Schematic showing an example of the UAS-GAL4 system. ............... 15 Figure 1.7 Whole mount confocal microscopy images showing the structure of the mushroom body in the adult brain. ................................................................. 15 Figure 1.8 Schematic of the mechanism behind the TARGET system. ................ 17 Figure 1.9 Schematic of the nucleosomes and their structure. ............................. 23 Figure 1.10 Simplified diagram comparing the 11 Histone Deacetylases in humans...................................................................................................................... 26 Figure 1.11 Comparison of human HDAC4 and Drosophila HDAC4. ................. 31 Figure 1.12 Subcellular distribution of HDAC4 in the Drosophila mushroom body. ................................................................................................................................... 33 Figure 1.13 Schematic outlining the T-maze test used in olfactory conditioning. ................................................................................................................................... 43 Figure 1.14 Characteristic courtship behaviours of a male Drosophila. ............... 45 Figure 1.15 Schematic showing the courtship suppression assay. ...................... 45 Figure 1.16 Schematic outlining the rough eye phenotype screen. ..................... 47 Figure 3.1 Confocal microscopy images showing the mushroom body. ............ 71 Figure 3.2 Visual summary of the cloning strategy. ............................................. 73 Figure 3.3 Restriction digests of pUAS-attB GFP::HDACv plasmid maxi-preps. ................................................................................................................................... 74 Figure 3.4 Schematic of Drosophila genetic cross. .................................................. 76 Figure 3.5 Western blot probed with anti-GFP antibody. .................................... 77 Figure 3.6 DAPI staining of a whole Drosophila brain from the posterior. .......... 77 Figure 3.7 Subcellular distribution of HDACv in Drosophila KC bodies. ............ 78 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164432 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164432 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164433 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164433 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164434 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164434 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file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164453 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164453 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164454 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164454 xiv Figure 3.8 Single layer images to visualise HDACv subcellular distribution. .... 79 Figure 3.9 Averaged phenotype scores of the elav x HDACv F1 progeny. ........ 83 Figure 3.10 Typical phenotypes observed following overexpression of HDAC4v in mushroom bodies. ............................................................................................... 86 Figure 3.11 Light and scanning electron microscopy images of Drosophila eyes. ................................................................................................................................... 89 Figure 4.1 Courtship indices of flies tested in behaviour analysis. ...................... 95 Figure 4.2 Memory indices of flies expressing hHDAC4 and DmHDAC4. ........ 95 Figure 4.3 Courtship indices of flies expressing hHDACv. .................................. 96 Figure 4.4 Memory indices of flies expressing hHDACv. .................................... 97 Figure 5.1 Confocal microscopy images with a single slice of the Kenyon cell bodies of the mushroom body. ............................................................................. 101 Figure 5.2 Confocal images of hHDAC4 variants and MEF2. ............................ 102 Figure 5.3 Confocal images of hHDAC4 variants and MEF2. ............................ 103 Figure 5.4 Phenotype scores from flies with altered MEF2 activity................... 105 Figure 5.5 Representative phenotypes from altered MEF2 activity in mushroom bodies. ..................................................................................................................... 106 Figure 5.6 Courtship indices of flies with altered MEF2 activity. ...................... 107 Figure 5.7 Memory indices of MEF2 KD flies and controls. ............................... 108 Figure 5.8 Memory indices of MEF2 OE flies and controls. ............................... 108 Figure 5.9 Sequences of the 3xMRE and 3xΔMRE DNA. .................................... 109 Figure 5.10 Initial testing of MRE-luciferase construct in flies. .......................... 110 Figure 5.11 Luciferase activity in Drosophila heads expressing MEF2-related constructs. ............................................................................................................... 111 Figure 5.12 Initial testing of MRE-luciferase in human MCF7 cells. .................. 112 Figure 5.13 Comparing the original MRE-luciferase to the hspMRE-luciferase in MCF7 cells. .............................................................................................................. 113 Figure 5.14 Activation of the MRE-luciferase in MCF7 cells. ............................. 113 Figure 5.15 MEF2-VP16 activation of hsp70MRE-luciferase in Drosophila. ...... 114 Figure 5.16 Activation of MRE-luciferase by DmMEF2 in Drosophila. ............. 115 Figure 5.17 Activation of the MRE-luciferase in HeLa cells. .............................. 115 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164455 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164455 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164456 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file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164477 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164478 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164478 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164479 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164479 xv Figure 5.18 Luciferase activity 60 minutes post-electrical stimulation. ............. 116 Figure 5.19 Luciferase activity of TrpA1 expressing flies. ................................. 117 Figure 5.20 Fold-activation of luciferase by CRE in mammalian tissue culture. ................................................................................................................................. 120 Figure 5.21 Anterior images of Drosophila brains expressing CREB and HDAC. ................................................................................................................................. 121 Figure 5.22 Drosophila calyces expressing CREB and HDAC4 ........................... 122 Figure 5.23 Drosophila Kenyon cell bodies expressing HDAC4 and CREB. ...... 122 Figure 6.1 Trace from Labchip analysis of RNA integrity. ................................. 128 Figure 6.2 Agarose gel electrophoresis of RNA samples. ................................... 129 Figure 6.3 Sample of quality checking data from fastqc 0.6.0. ............................ 131 Figure 6.4 Pair-wise principal component analysis of the samples. .................. 132 Figure 6.5 Variant sequences from read data aligned with human chromosome 2. ................................................................................................................................. 133 Figure 6.6 PCA of L175A treatment and control groups. ................................... 135 Figure 6.7 Volcano plot of identified genes in L175A vs control sample. ......... 136 Figure 6.8 Heatmap of the 50 most significantly differentially expressed genes in L175A vs. control sample. ..................................................................................... 138 Figure 6.9 PCA of the 3SA and control samples. ................................................. 142 Figure 6.10 Volcano plot comparing 3SA and control samples. ........................ 143 Figure 6.11 Heatmap of the 50 most significantly differentially expressed genes in 3SA vs. control sample. ..................................................................................... 144 Figure 6.12 PCA of the 3SA and L175A samples. ................................................ 147 Figure 6.13 Volcano plot comparing L175A and 3SA samples. ......................... 148 Figure 6.14 Heatmap of the 50 most significantly differentially expressed genes in L175A vs. 3SA samples. .................................................................................... 149 Figure 6.15 Representative image of GMR-driven HDACv in fly eyes. ............ 169 Figure 6.16 Representative images of ACXD KD in Drosophila eyes. ................ 170 Figure 6.17 Representative images of fu12 KD in Drosophila eyes. .................... 171 Figure 6.18 Representative images of kat80 KD in Drosophila eyes. ................... 172 Figure 6.19 Representative images of mthl8 KD in Drosophila eyes. .................. 173 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164480 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file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164504 xvi Figure 6.20 Representative images of npc2g KD in Drosophila eyes. .................. 174 Figure 10.1 Diagram of the pUAST-attB-GFP plasmid. ...................................... 225 Figure 10.2 Agarose gel electrophoresis of digested vectors and inserts .......... 226 Figure 10.3 pUAST-attB-GFP-3SA plasmid map. ................................................ 227 Figure 10.4 pUAST-attB-GFP-DmHDAC4 plasmid map. .................................. 227 Figure 10.5 pUAST-attB-GFP-L175A plasmid map. ........................................... 228 Figure 12.1 Map of the hsp70MRE-luciferase plasmid. ...................................... 235 Figure 13.1 Per read quality analysis for sample 3SA_1_1 ................................. 236 Figure 13.2 Per read quality analysis for sample 3SA_1_2 ................................. 236 Figure 13.3 Per read quality analysis for sample 3SA_2_1 ................................. 237 Figure 13.4 Per read quality analysis for sample 3SA_2_2 ................................. 237 Figure 13.5 Per read quality analysis for sample 3SA_3_1 ................................. 238 Figure 13.6 Per read quality analysis for sample 3SA_3_2 ................................. 238 Figure 13.7 Per read quality analysis for sample 3SA_4_1 ................................. 239 Figure 13.8 Per read quality analysis for sample 3SA_4_2 ................................. 239 Figure 13.9 Per read quality analysis for sample CS_1_1 ................................... 240 Figure 13.10 Per read quality analysis for sample CS_1_2 ................................. 240 Figure 13.11 Per read quality analysis for sample CS_2_1 ................................. 241 Figure 13.12 Per read quality analysis for sample CS_2_2 ................................. 241 Figure 13.13 Per read quality analysis for sample CS_3_1 ................................. 242 Figure 13.14 Per read quality analysis for sample CS_3_2 ................................. 242 Figure 13.15 Per read quality analysis for sample CS_4_1 ................................. 243 Figure 13.16 Per read quality analysis for sample CS_4_2 ................................. 243 Figure 13.17 Per read quality analysis for sample L175A_1_1 ........................... 244 Figure 13.18 Per read quality analysis for sample L175A_1_2 ........................... 244 Figure 13.19 Per read quality analysis for sample L175A_2_1 ........................... 245 Figure 13.20 Per read quality analysis for sample L175A_2_2 ........................... 245 Figure 13.21 Per read quality analysis for sample L175A_3_1 ........................... 246 Figure 13.22 Per read quality analysis for sample L175A_3_2 ........................... 246 Figure 13.23 Per read quality analysis for sample L175A_4_1 ........................... 247 Figure 13.24 Per read quality analysis for sample L175A_4_2 ........................... 247 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164505 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164505 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164506 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164506 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164507 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164507 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file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164532 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164532 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164533 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164533 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164534 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164534 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164535 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164535 xvii xviii TABLE OF TABLES Table 3.1 Restriction digest predicted lengths for pUAS-GFP::HDACv. ............ 75 Table 3.2 Frequency of the observed mushroom body phenotypes brains......... 82 Table 5.1 Phenotype frequency from altered MEF2 during development. ....... 104 Table 6.1 Qubit analysis of RNA samples. ........................................................... 129 Table 6.2 Overview of each sample comparison showing number of significant changes. ................................................................................................................... 134 Table 6.3 The 30 most significantly differentially expressed genes between the L175A and control groups. .................................................................................... 141 Table 6.4 The 30 most significantly differentially expressed genes between the 3SA and control groups. ........................................................................................ 146 Table 6.5 The 30 most significantly differentially expressed genes between the L175A and 3SA groups. ......................................................................................... 151 Table 6.6 GO-Term analysis from L175A expression. ......................................... 154 Table 6.7 GO-Term analysis from 3SA expression. ............................................. 155 Table 6.8 GO-Term analysis from comparing the 3SA sample to the L175A sample. .................................................................................................................... 155 Table 6.9 DAVID analysis of genes up- or down-regulated by L175A expression. ................................................................................................................................. 156 Table 6.10 DAVID analysis of genes up- or down-regulated by 3SA expression. ................................................................................................................................. 157 Table 6.11 RNASeq data from DmHDAC4 overexpression. .............................. 161 Table 6.12 Comparison of log2-fold changes in read counts from the overexpression of DmHDAC4 (Schwartz, 2016), L175A, and 3SA compared to controls. ................................................................................................................... 162 Table 9.1 UAS-construct fly lines used in this project, their genotype, and source ................................................................................................................................. 221 Table 9.2 GAL4-driver lines, control lines and balancer fly lines used in this project, their genotype, and source....................................................................... 222 Table 9.3 UAS-RNAi fly lines used in this project, their genotype, and source 222 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164538 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164538 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164539 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164539 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164540 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file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164549 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164549 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164550 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164550 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164551 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164551 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164551 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164551 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164552 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164552 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164552 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164553 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164553 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164553 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164554 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164554 xix Table 10.1 Restriction digest plan for digestion of vector and excision of HDACv inserts ...................................................................................................................... 225 Table 11.1 PCR Primers used in this project, primers are from Sigma Aldrich 229 Table 11.2 Plasmids used in this project............................................................... 230 Table 11.3 Primary antibodies used in immunohistochemistry (IHC) and Western blotting (WB). ......................................................................................................... 231 Table 11.4 Secondary antibodies used in immunohistochemistry (IHC) and Western blotting (WB). .......................................................................................... 231 Table 14.1 50 most significantly differentially expressed genes from CS vs. L175A. ................................................................................................................................. 250 Table 14.2 50 most significantly differentially expressed genes from CS vs. 3SA. ................................................................................................................................. 253 Table 14.3 50 most significantly differentially expressed genes from L175A vs. 3SA. ......................................................................................................................... 256 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164555 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164555 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164555 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164556 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164556 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164557 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164557 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164558 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164558 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164558 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164559 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164559 file:///C:/Users/Patrick/Google%20Drive/PhD%20Project/Thesis/Chapters/emendations_Patrick%20Main_PhD_Thesis%20(AutoRecovered).docx%23_Toc20164559 xx 1 1 INTRODUCTION 1.1 Neurodevelopmental disorders & neurodegeneration Neurodevelopmental disorders are defined as “a group of conditions with onset in the developmental period characterised by developmental deficits that produce impairments of personal, social, academic, or occupational functioning” (American Psychiatric Association, 2013). Disorders such as intellectual disability, autism spectrum disorders, epilepsy, and others are well recognised as developmental disorders that place a significant burden on the health of the individual as well as their families and the healthcare system. Often these disorders are evident in children as they grow up but others, such as attention- deficit hyperactivity disorder and autism-spectrum disorders can be subtle and remain undiagnosed well into adulthood. Neurodegenerative disorders such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are characterised by dysfunction and death of neurons. This breakdown of the nervous system results in many symptoms such as loss of co- ordination or motor function; loss of memory and impaired sensory input (Kumar et al., 2015; Przedborski, 2017). These disorders predominantly affect the elderly and, whilst they share many outward symptoms, are the result of a complex and diverse set of underlying causes, many of which are yet to be identified. In general, neurodegenerative disorders are the result of the body’s repair systems failing. These systems often breakdown with ageing and, as such, the increasing average age of the population is a growing concern. It was recently determined over 45 million people worldwide live with dementia, an increase of over 100% since 1990 (Prince et al., 2015). Parkinson’s disease, Alzheimer’s disease and other dementias more than doubled from 0.7% of all deaths in 2000 to 2.6% in 2015 (World Health Organisation, 2016) , a trend which is expected to continue as life expectancies increase. In developed nations the prevalence of Alzheimer’s in individuals over the age of 65 has been reported to be as high as 2 11% (Hebert et al., 2013). This increase is expected to be more marked in underprivileged populations that, thanks to more widespread healthcare, are undergoing a more rapid increase in average life-expectancy (Prince et al., 2015). While the reduction in overall lifespan caused by neurodegeneration is a major concern, an additional factor is the years of healthy life lost during the progression of the disease, caused by steadily declining cognitive and motor function. The slow creep of these disorders makes them challenging to diagnose early as the symptoms are often confused with signs of stress and aging. A complication often leading to delayed diagnosis that results in worse treatment outcomes (Bradford et al., 2009). Alzheimer’s disease accounts for upwards of 60% of the 50 million dementia cases worldwide (Alzheimer’s Association, 2018). Alzheimer’s disease is highly associated with the development of β-amyloid plaques and neurofibrillary tangles in the brain that are thought to lead to neuron death and subsequent degeneration in many regions of the brain (Alonso et al., 1996; Murphy and LeVine III, 2010). As these physiological signs of the disorder develop and spread, they lead to increasing cognitive impairment. This impairment manifests initially as a reduced ability to form long-term memories. A complicating factor in the diagnosis of neurodegenerative disorders is the prevalence of age-associated memory impairment (AAMI) (Crook et al., 1986), the impairment of learning and memory caused by age-related loss of neural plasticity (Tulving and Markowitsch, 1998; Price et al., 2004). As mentioned, the presence of AD is often missed in early diagnoses due to misidentification of the symptoms as “normal” AAMI. Multiple studies have demonstrated that older humans perform poorly in spatial memory tasks and are typically outperformed by their younger counterparts (Uttl and Graf, 1993; Wilkniss et al., 1997; Moffat et al., 2001; Price et al., 2004). It is suggested that this reduction in neural function is due to age-related reductions in synaptic plasticity (the ability of a synapse to change in strength) a process required for learning and formation of new memories (Deupree et al., 1993; Rosenzweig et al., 1997; Vance and Wright, 2009). 3 Current research on AAMI is aimed at elucidating the molecular mechanisms underpinning this condition and determining whether they are related to the pathological effects that cause neurodegenerative diseases. Due to the complex nature of learning and memory, it is often more feasible to study from the ground up, investigating the machinery by playing with individual cogs and levers and observing the effects, usually with a simplified model, an approach that is aided by the conservation of molecular mechanisms in learning and memory across a wide array of organisms. 4 1.2 The study of learning and memory Learning is broadly defined as the acquisition of knowledge. The subsequent recall of this acquired knowledge is referred to as memory. The retention time of this memory can be brief (Short-term memory, STM) or can endure for longer periods of time (Long-term memory, LTM). This allows an individual organism to modify their behaviour to the changing environment by altering behaviour in response to experience. This behavioural plasticity was first observed by Santiago Ramon y Cajal (1894) in a series of studies that form the basis of modern neuroscience, identifying the role of “histological alterations” in the malleability of an individual’s behaviour. The cell-based changes in this case are fluctuations in synaptic strength and number, a mechanism that is critical to learning and memory (Bailey and Kandel, 1993). Synapses are the connection points between neurons, this connection is carried out through the transmittance of neurotransmitters that allow the propagation of an electrical signal across the cleft between individual neurons (Figure 1.1). Figure 1.1 Schematic of an example synaptic junction. A simplified figure emphasising the main components of synaptic transmission, notably the neurotransmitters (white circles) and the neurotransmitter receptors (purple) that are responsible for transmitting the electrical impulse from the axon (top) to the receiving dendrite (bottom) across the synaptic cleft. Figure modified from Osnimf, Wikipedia Commons, public domain. 5 Historically, the study of memory in invertebrates was believed to be futile. However, as the basic neural architecture is preserved across the animal kingdom it was proposed by Eric Kandel that the molecular mechanisms of memory are also conserved. Subsequently, he opted to study this conservation of systems in the sea snail Aplysia californica. This choice of organism seems unusual at first but Aplysia possess a number of large, easily identifiable neurons that permit the repeated testing of physiological responses within the same neuron across a range of individuals (Koester and Kandel, 1977). In a simple but elegant series of experiments it was demonstrated that Aplysia exhibited long-term memory. This was done through exploitation of the gill- withdrawal reflex, a defensive, involuntary reflex in which the snail retracts its gill and siphon when disturbed to prevent damage to these sensitive structures. In these experiments, exposure to a noxious tactile stimulation caused the natural gill-withdrawal reflex (Kandel, 2001), (Figure 1.2). However, when this stimulation was repeated in an identical fashion the reflex response was reduced, a reduction that was shown to be independent of mechanical fatigue or sensory adaptation indicating a memory of the initial stimulus had been formed (Kandel and Tauc, 1965; Pinsker et al., 1970; Frost et al., 1985). Figure 1.2 Schematic showing the gill withdrawal reflex in Aplysia californica. A) Stimulation of the siphon results in rapid retraction of the delicate gill and siphon structures in an involuntary reflex. B) Repeat stimulation triggers a desensitisation characterised by reduced withdrawal of the gill (Kandel, 2001). A) Reproduced with permission from AAAS, B) Copyright (1985) National Academy of Sciences (Frost et al., 1985). 6 Further study showed that this training could be repeated in a fashion that would result in a much longer retention of the withdrawal reflex with a single training session being forgotten within minutes and multiple, time-spaced training sessions resulting in memories that last for days (Hawkins et al., 2006). Investigating further, it was shown that initial exposure to stimulus results in activation of the cyclic adenosine monophosphate (cAMP) kinase signalling cascade that involves activation of the cAMP-dependent protein kinase A (PKA) and subsequent phosphorylation of multiple targets (Abel and Lattal, 2001), which prevent the repolarisation of the neuronal membrane. This increases the release of neurotransmitters triggered by calcium ion influx when the wave of depolarisation reaches the synaptic cleft (Abel and Lattal, 2001). In addition to this, changes to the post-synaptic neuron’s sensitivity to the released neurotransmitters can enhance this sensitisation (Kandel, 2012). These transient changes in neurotransmitter release and response result in short-term memory; a temporary memory in which the information is stored for approximately an hour (Rosenzweig et al., 1993; Izquierdo et al., 2002). Continued activation of the same pathway (for example by repeat exposure to the same stimulus) results in a persistent increase in cAMP and PKA levels. This sustained increase results in the recruitment of a mitogen-activated protein kinase (MAPK) p42 that forms a complex that translocates into the nucleus and phosphorylates transcription factors such as cAMP response element binding protein (CREB). Phosphorylated CREB then binds to response elements in the genome and activates transcription of genes involved in dendrite growth and increasing synaptic density (De Roo et al., 2008; Bourne and Harris, 2011; Chen et al., 2017; Zhu et al., 2018). The depth of study following Kandel’s pioneer studies have shown that many of the mechanisms involved in learning and memory are conserved between vertebrates and invertebrates (Barco et al., 2006), a high level overview of these similarities is shown in Figure 1.3. 7 Figure 1.3 Molecular similarities between Aplysia and mammalian neurons. Key factors in Aplysia neurons (a) and mammalian hippocampal neuron (b) signalling. 1) Release of neurotransmitters and short-term strengthening of synaptic connections; 2) maintained equilibrium between synaptic kinase and phosphatase activities; 3) transport of components from the synapse to the nucleus; 4) nuclear transcription regulation; 5) depolarisation induced gene expression; 6) epigenetic shifts in gene expression; 7) transport of newly transcribed products to synapse; 8) protein synthesis at synapses; 9) growth and development of new synapses; 10) activation of silent synapses; 11) memory persistence based on recurring mechanisms. These events move from the synapse (1-2), to the nucleus (3-6) and then back to the synapse (7-11). Figure from Barco et al., 2006, reproduced with permission. 8 The cAMP pathway is also conserved in the fruit fly Drosophila melanogaster. Two of the first memory related mutants characterised in Drosophila were the cAMP associated genes dunce and rutabaga (Quinn et al., 1974; Livingstone et al., 1984). dunce was first identified in an olfactory learning assay and has since been shown to be a large, complex locus that encodes at least 10 differentially spliced variants of a cAMP phosphodiesterase, the enzyme responsible for the degradation of cAMP (Conti and Beavo, 2007). rutabaga plays a complimentary role, encoding a Ca2+/Calmodulin-stimulated adenylate cyclase, an enzyme that makes cAMP and was first identified through learning deficiencies in the same assay as dunce (Quinn et al., 1974). This data supports the original hypothesis proposed by Eric Kandel in that the short- and long-term memory systems rely on a cAMP-dependent signalling cascade that is highly conserved between vertebrates and invertebrates allowing for the study of vertebrate learning and memory through invertebrate models. 1.2.1 SHORT AND LONG-TERM MEMORY Although short and long-term memory are often referred to as a continuum of the same process, the two systems are independent from one another. This has been exemplified by the disruption of STM without affecting LTM and vice versa (Izquierdo et al., 1999, 2000). This has been shown through pharmacological inhibition of memory formation by infusing rat brains with a range of treatments from receptor antagonists/agonists to enzyme inhibitors and stimulants and testing short- and long-term memory in the treated rats using an aversive memory assay. In this study it was observed that 11 treatments, including CNQX (a glutamate AMPA receptor blocker) and muscomil (a GABAA receptor agonist) inhibited the formation of short-term memory without altering LTM performance in the same animals (Izquierdo et al., 2002). Paralleling these rat studies, it has been shown that treatment of Aplysia with serotonin during training was shown to disrupt the formation of STM without affecting LTM development (Emptage and Carew, 1993; Izquierdo et al., 1998) once more 9 reinforcing the conservation of the molecular mechanisms involved in learning and memory. 1.2.2 THE PHYSIOLOGY OF MEMORY Historically, it was believed that memory was stored in specific “memory neurons” that existed to facilitate information storage and recall. However, the potential for memory storage is built into the basic neural architecture (Kandel, 2012) and the formation of memory relies on the strengthening of connections in the existing neural pathway. This relies on increasing the sensitivity to stimulus, increasing the output from a single stimulation, or increasing the number of connections between neurons, all of which result in a greater neuronal output for a single stimulation (Hawkins et al., 2006; De Roo et al., 2008). In both mammal and insect models the formation and storage of memories occurs in specific structures within the brain. In Drosophila melanogaster the electrical impulses and gene expression associated with memory have been shown to be localised largely within the mushroom body (MB) (Davis, 2005; Plaçais et al., 2012). These traces are modulated and acted upon by the extrinsic neurons that allow for the mushroom body to communicate with other brain regions (Aso et al., 2014a; Masek and Keene, 2016). In mammalian systems it is harder to pinpoint a single, specific structure in the brain associated with memory. This difficulty arises due to the complex interplay between structures of the mammalian brain and the tendency of individual stimulus associated impulses (traces) to pass through multiple structures (Shu et al., 2003). In mammals, the hippocampus is particularly involved in converting short- to long-term memory as well as spatial memory (Berger et al., 1976; Ergorul and Eichenbaum, 2004), the amygdala has been indicated as highly associated with emotional memories (McGaugh et al., 1996; LaBar and Cabeza, 2006), the entorhinal cortex acts as a conduit from the hippocampus to the rest of the neo- cortex, as well as acting to retrieve memory (Segal, 1973; Takehara-Nishiuchi, 2014) and the cerebellum is highly associated with motor memories, physical responses to conditioned stimuli (Mishkin and Appenzeller, 1987; Attwell et al., 10 2002). These four regions barely scratch the surface of the various regions in the mammalian brain involved in learning and memory. The complexity and diversity of the role these structures play can be shown by comparing the role of a single protein across the different structures. In a subset of neurons in the hippocampus, knocking out protein kinase C results in normal STM with defective LTM whereas in the entorhinal cortex PKC is required for both STM and LTM (Izquierdo et al., 1999). It has also been shown that protein kinase A is involved in both forms of memory in the hippocampus, acting to generate STM in the first 90 minutes post-training and acting to generate LTM during training and approximately 180 minutes post-training (Vianna et al., 1999). These studies show that the complexity of the mammalian system make determining the role of a protein challenging adding to the benefits of using the Drosophila model. Using a simplified model allows the characterisation of proteins with a key role in memory as a whole, without being concerned about the influence of that protein’s role in different substructures. 11 1.3 The Drosophila mushroom body The mushroom body is a bilateral neuropil structure initially identified by Félix Dujardin in 1850, who initially compared it to the cerebral cortex of vertebrates and considered it to be the major processing centre of the insect brain (Dujardin, 1850; Heisenberg, 1998). The mushroom body is part of the olfactory system which is a major pathway in courtship and olfactory memory (Section 1.7.1) It has subsequently been shown to be the main memory centre in Drosophila and is a structure present in other insects such as locusts and honeybees (Verlinden, 2018). 1.3.1 INTRINSIC NEURONS OF THE MUSHROOM BO