Expression and function of Npas4 during early development THOMAS STEPHEN KLARIC A thesis submitted for the degree of Doctor of Philosophy DECEMBER 2011 Discipline of Genetics School of Molecular and Biomedical Science The University of Adelaide Table of contents Table of contents Table of contents ............................................................................................. i List of figures ............................................................................................... viii List of tables .................................................................................................. ix Declaration ..................................................................................................... x Acknowledgements ....................................................................................... xi Abbreviations ............................................................................................... xii Notes .......................................................................................................... xviii Abstract ........................................................................................................ xix 1 - Introduction............................................................................................... 1 1.1 Overview ................................................................................................................... 1 1.2 An introduction to Npas4 ........................................................................................... 1 1.2.1 Npas4 is a member of the bHLH PAS transcription factor family ..................................... 1 1.2.1.1 Transcription factors ........................................................................................................ 1 1.2.1.2 The bHLH PAS transcription factor family ........................................................................ 2 1.2.1.3 The newest member of the bHLH PAS family: Npas4 ...................................................... 6 1.2.2 Genomic organisation of the mouse Npas4 gene ............................................................ 7 1.2.3 Domain structure and sequence conservation within the Npas4 protein ......................... 7 1.2.4 Interaction with other bHLH PAS factors ......................................................................... 9 1.2.5 DNA binding specificity of Npas4 transcriptional complexes ........................................... 9 1.3 Expression of Npas4 ................................................................................................. 10 1.3.1 Tissue distribution of the Npas4 transcript in the adult organism .................................. 10 1.3.2 Npas4 expression in the adult brain ............................................................................... 11 1.3.3 Cellular distribution of Npas4 expression ....................................................................... 12 1.3.4 Subcellular localisation .................................................................................................. 12 1.3.5 Npas4 expression during development .......................................................................... 13 1.4 Regulation of Npas4 expression ............................................................................... 13 1.4.1 Npas4 is a stimulus-response transcription factor .......................................................... 13 1.4.1.1 Immediate-early genes .................................................................................................. 14 1.4.2 Regulation of Npas4 expression by neural activity ......................................................... 15 1.4.2.1 Neural activity and NMDAR-mediated Ca2+ signalling ................................................... 15 1.4.2.1.1 Neural activity ....................................................................................................................... 15 1.4.2.1.2 Neurotransmitters and their receptors ................................................................................. 15 i Table of contents 1.4.2.1.3 Excitatory synaptic transmission: glutamate and glutamate receptors ............................... 16 1.4.2.1.4 Glutamate receptor signalling and cation flux ...................................................................... 16 1.4.2.1.5 Ca2+ signalling ........................................................................................................................ 18 1.4.2.1.6 Creb: an example of a Ca2+-sensitive transcription factor .................................................... 20 1.4.2.2 Activity-dependent regulation of Npas4 expression in neurons ................................... 20 1.4.2.2.1 Neuronal depolarisation ....................................................................................................... 21 1.4.2.2.2 Nuclear Ca2+ signalling .......................................................................................................... 21 1.4.2.3 Excitotoxic brain injury and Npas4 expression .............................................................. 22 1.4.2.3.1 Seizure................................................................................................................................... 22 1.4.2.3.2 Cerebral ischaemia................................................................................................................ 22 1.4.2.4 Learning and synaptic plasticity ..................................................................................... 24 1.4.2.4.1 Long-term potentiation and long-term depression .............................................................. 24 1.4.2.4.2 Regulation of Npas4 by Chem-LTP and Chem-LTD ............................................................... 27 1.4.2.4.3 Npas4 expression in the context of learning and memory ................................................... 27 1.4.3 Regulation of Npas4 expression by neurotrophin signalling .......................................... 28 1.4.3.1 Neurotrophins ............................................................................................................... 28 1.4.3.2 Neurotrophin receptors ................................................................................................. 28 1.4.3.3 Neurotrophin-dependent Npas4 expression ................................................................. 30 1.4.3.4 Other growth factors ..................................................................................................... 31 1.4.4 Post-translational regulation of Npas4 expression and activity ..................................... 33 1.4.5 Npas4 expression in response to psychological stress ................................................... 34 1.4.5.1 Psychological stress ....................................................................................................... 34 1.4.5.2 Acute psychological stress ............................................................................................. 35 1.4.5.2.1 IEG expression in response to acute psychological stress .................................................... 35 1.4.5.3 Chronic psychological stress .......................................................................................... 35 1.4.5.3.1 Repression of IEGs in response to chronic psychological stress ........................................... 36 1.4.5.4 The effect of psychological stress on Npas4 expression ............................................... 37 1.4.6 Npas4 expression in response to cellular stress ............................................................. 38 1.4.6.1 Oxidative stress .............................................................................................................. 38 1.4.6.2 Osmotic stress ............................................................................................................... 39 1.4.7 The effect of drugs of abuse on Npas4 expression ......................................................... 40 1.4.7.1 Psychostimulants: cocaine and methamphetamine ..................................................... 40 1.4.7.2 Opioids: morphine and heroin ....................................................................................... 40 1.4.7.3 Other drugs of abuse: nicotine and ethanol .................................................................. 41 1.4.8 Circadian regulation of Npas4 expression...................................................................... 41 1.4.8.1 Circadian rhythm ........................................................................................................... 42 1.4.8.1.1 The biological clock of the suprachiasmatic nuclei ............................................................... 42 1.4.8.1.2 Clock output and circadian control of melatonin release ..................................................... 43 ii Table of contents 1.4.8.2 Melatonin and melatonin receptor signalling ................................................................ 43 1.4.8.2.1 Melatonin receptors .............................................................................................................. 44 1.4.8.2.2 Melatonin-dependent circadian regulation of gene expression in the pars tuberalis .......... 44 1.4.8.3 Circadian expression of Npas4 in the pars tuberalis ...................................................... 46 1.4.8.3.1 Possible mechanisms involved in circadian expression of Npas4 ......................................... 46 1.4.9 Post-transcriptional