I , I l¡ \-lN I ìo Molecular Basis for Intellectual Disability and Epilepsy: Role of the Human Homeobox Gene ARX A thesis submitted for the degree of Doctor of Philosophy to the University of Adelaide by Desiree Cloosterman BSc (Hons) school of Medicine, Department of Paediatrics, university of Adelaide November 2005 ll ,rlf the human brain were so simple that we coultl understønd it, we woukl be so simple thøt we couldn't" Emerson M. Pugh lll CONTENTS Summary IV Statement and Declaration v vl Acknowledgements..--- ------ --- List of Abbreviations vlll CHAPTER I Introduction-----------.-- 1 CHAPTER 2 Materials and Methods- 43 CHAPTER 3 Conservation of ARX. _.__-_._.1 10 CHAPTER 4 Yeast Two-Hybrid Screening __-_--_ I 33 CHAPTER 5 Confirmation of Y2H Interactions 176 CHAPTER 6 ZebrafishKnockdown Model------.- 203 CHAPTER 7 Conclusions 237 References lv SUMMARY Mental retardation (MR) is estimated to affect 2-3Yo of the population and is caused by both environmental and genetic factors. Mutations in Ihe Aristaless-related homeobox gene (AR$ have been found in numerous families with X-linked MR with and without other clinical features including infantile spasms, dystonia, lissencephaly, autism and dysarthria. The aim of this study was to investigate the normal function of the ARX protein within a cellular environment and in development. Discernment of ARX function will improve our understanding about the molecular pathology of intellectual disability and epilepsy, as well as improve our knowledge of the genes and mechanisms required for normal brain development. The first part of the thesis addressed the conservation of ARX domains and polyalanine regions by the identification and analysis of characterized and novel ARX orthologs. It was found that the ARX homeodomain and aristaless are highly conserved, but the octapeptide domain, exon 2 NLS, fourth polyalanine tract and to a lesser extent the acidic domain are only conserved in vertebrates and the first three polyalanine tracts are only conserved in mammals. The second part of this thesis involved the identification of IPO13, PICKl, PKM2 and AlUp as interacting with ARX by yeast two-hybrid screening and speculated on their functional roles with respect to ARX. The interactions between ARX and the proteins IPOl3 and PICKI were confirmed by coimmunoprecipitation and colocalization studies. Furthermore, it was found that expansion of the first polyalanine tract in ARX, as seen in human patients, resulted in a marked increase of cytoplasmic aggregation, where IPO13 colocalized to these ARX aggregates. This suggests that nuclear import of ARX may be hindered due to expansion of the first polyalanine tract. Finally, a zebrafish knockdown model was generated by the microinjection of antisense arx morpholinos. Microinjection of arx morpholinos resulted in reduced forebrain expression of emx3, dlx2a and dlx5a indicating that arx plays a significant role in forebrain and GABAergic interneuron development. VI ACKNOWLEDGMENTS I would like to take this opportunity to thank the Cytogenetics unit of the Department of Genetics, Women's and Children's Hospital for their financial and moral support and for use of equipment during my PhD. In particular I would like to thank my supervisors Associate professor Jozef Gecz and Professor Grant Sutherland for giving me the opportunity to work in such an exciting field and for providing guidance and helpful advice when needed- I would also like to acknowledge the MS Mcleod Research Foundation who provided financial support in the form of the MS Mcleod Research Fund Postgraduate Study Award. I would like to thank my colleagues within the Neurogenetics and Molecular Genetics laboratories that have made this experience an enjoyable one. Thank you to Marie Shaw' Dr Cheryl Shoubridge, Shirley Richardson, Joanna Crawford, Merran Finnis, Tod Fullston, Olivia McKenzie, Dr Mark Corbett, Rachael Bennett, Linda Burrows, Lynne Hobson, Dr Marie Mangelsdorf, Dr Kathie Friend and last but not least Lucianne Vandeleur. I would also like to thank those that contributed to the work presented within this thesis. Thank you goes to Joanna Crawford, Dr Karen Lower, Michaella Richards and Dr Anne Davy for their help in setting up the yeast two-hybrid systems. I gratefully thank Dr Michael Lardelli and members of his lab (Ben Tucker, Simon Wells, Svanhild Nornes) who collaborated on the zebrafish work including the maintenance, microinjection and fixation of zebrafish embryos. I would like to say thank you to Dr Marie Mangelsdorf for sequence the start of the zebrafish arx gene. A sincere thank you also goes to Dr Igor Dawid and Dr Marc Ekker for the kind provision of plasmids used in zebrafish whole mount in situ hybridization analysis (detailed in materials and methods). Thank you also to Professor Dirk Görlich for the kind provision of the anti-IPO13 antibody. A special thank you goes to Dr Cheryl Shoubridge and to Olivia McKenzie for their contributions towards my work on ARX. Dr Shoubridge kindly collaborated on the work involving the colocalization of ARX with IPOI3 and PICKI and provided yet to be published results regarding aggregate formation of ARX in mammalian cells. I wish to thank Olivia McKenzie for the information and figures regarding the unpublished work of ARX transcriptional repression studies and the yeast two-hybrid work involving ARX polyalanine tracts and AlUp. I would also like to thank Cathy Derwas for cell establishment and maintenance of cell lines. vil I would like to thank my family for their support during my studies. To my parents Robert and Christine, who lead by example in both working hard and achieving what I aim for. To Nana who shows me every day what courage is and who I am very proud of. To my brother Shannon who provided unconditional encouragement and who shares a love of a good cup of coffee. To my sister Serenity who showed me that persistence pays off and who understands exactly what I mean whether it be work or life. To Dion, my future brother in law, thank you for making my sister happy and welcome to the family. Finally to Rascal (AKA Evil Kitty dam cute' rd ), you are expensive to keep and I may loose a limb one day, but you are so vlll LIST OF ABBREVIATIONS 3AT 3 -Amino- 1, 2,4 -T riazole aa amino acid AC acidic domain ACC agenesis ofthe corpus callosum AD activation domain ADP adenosine diphosphate al aristaless gene ARX Ar is t ale s s -related homeobox gene ATP adenosine triphosphate bp base pair CNS central nervous sYstem DBD DNA binding domain ECL enhanced chemiluminescent detection EST expressed sequence tag GAP GTPase activating Protein GDI guanine nucleotide disassociation inhibitor GEF nucleotide exchange factor GFP green fluorescent protein HD homeodomain hpf hours post fertilization HRP horseradish peroxidase HYD/AG hydranencephaly with ambiguous genitalia IPOl3 Importin l3 IQ intelligence quotient ISSX X-linked Infantile Spasms/)(-linked West syndrome ORF open reading frame kb kilo base pairs KD kilo Daltons MR mental retardation NE nuclear envelope NJ Neighbour Joining NLS nuclear localization sequence IX NPC nuclear pore complex NS-XLMR non-syndromic X-linked mental retardation OAR aristaless domain OMIM Online Mendelian Inheritance in Man OP octapeptide domain PBS phosphate buffer solution PICKl Protein Interacting with C Kinase I PK pyruvate kinase PKM2 Pyruvate Kinase M2 polyA polyalanine PRTS Partington syndrome SBH subcortical band heterotopia SC synthetic complete medium SD standard deviation SHH sonic hedgehog S-XLMR syndromic X-linked mental retardation TCS tonic-clonic seizures UPGMA unweighted pair group method using arithmetic averages UTR untranslated region XLAG X-linked lissencephaly with abnormal genitalia XLMR X-linked mental retardation XMESID X-linked myoclonic epilepsy with genetalized spasticity and intellectual disability Y2H Yeast two-hybrid YPD yeast extract peptone dextrose medium CHAPTER 1 Introduction 1.1.1 AErloI-ocy oF MENTAL RETARDATION ............'...." """""""'4 I3 l. 2. I . I Neur ons, Cytoskeleton Remodelling and Dendrite s 1.2. 1.2 Synapse Signalling... 17 I .2. l. 3 Trans cription Re gulation...... 1.3.1 PAIRED-CLASS HOMEODOMAIN PROTEINS AND ARX .......''..20 1.3.2 GENOMIc CrnRactsruzArIoN AND EXPRESSION oF ARX '......'.'... """"""'22 1.3.3 DEVELOPMENTAL ROLE oF ARX """"""""""25 1.3.4 ARX MurRtIoNS: PHENOTYPE-GENOTYPE CORRELATIoN .."""""""""""'26 l. 3.4. I XJinked West syndrome.. -.......... 28 ].3.4.2 XMESID l. 3.4. 3 Partington Syndrome ........30 L3 4.4 Proud Syndrome....-......... 3I ..31 l. 3. 4. 5 Lissencephaly and XL4G......... 1.3.5 INren,cND INTRAFAMTLTAL PHENorypE VARIATIoN lN PATIENTS wtrs ARX 428-45lour(24nr) 1.3.6 PoLyALAnmr TRacr EXPANSION DISORDERS.... ..".""""""'37 1.4 AIMS CHAPTER 1 - 2 1.1 MENTAL RETARDATION A person's success in society is often determined or influenced by their level of intelligence' Though there is no consensus on the dehnition of intelligence, most people have a general impression of the extreme limits to intelligence such as higher than average intelligence or a lower than averuge intelligence. Intelligence is most commonly determined through an individual's performance in standardized IQ (intelligence quotient) tests. Observations have shown that the distribution of IQ score in the general population follows a bell curve (Figure 1.1), with the major proportion of the general population (95%) within 2 standard deviations (SD) above or below the mean score of 100 (Dingman and Tarjan, 1960; Moser and Wolf, teTr). -t +3rd 400 0 o ôa E 22 55 t¡13 IO ¡cofr¡ Figure 1.1: Distribution of intelligence across the general population (modihed from Mackintosh, 1998). The proportion of the population who possess an IQ of less than 70 (2 SD below the mean where 1 SD is 15 IQ points) are classified as being mentally retarded (World Health Organization (WHO), 1993). Though not perfect, a recent definition of mental retardation CHAPTER I - 3 (MR) provided by the American Association on Mental Retardation (AAMR, 2002) characterizes MR by 1) IQ score; 2) limitations in adaptive behaviour in conceptual, social and practical skills; and 3) the conditions manifesting before 18 years of age.