Luận án Tiến sĩ: Rối loạn biểu hiện gen ở não bệnh nhân Rett và chuột Mecp2-null

School ofMedicine Oregon Health & Science University CERTIFICATE OF APPROVAL This is certify that the Ph. dissertation of Vivianne Wei Deng has been approved Mentor/Advtsor <.! ember Member Deregulated Gene Expression in the Brain of Rett Syndrome Patients and Mecp2-null Mice by Vivianne Wei @eng A DISSERTATION Presented to the Department of Cell and Developmental Biology and the Oregon Health & Science University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2005 TABLE OF CONTENTS Acknowledgements. vi CHAPTER ONE Introduction Rett Syndrome and Epigenetic Gene Regulation Mediated by Methyl-CpG Binding Protein 2 (MeCP2). 1 Overview ofRett Syndrome.

2 DNA Methylation and MeCP2-mediated Epigenetic Gene Regulation. 6 Toward Understanding the Role ofMeCP2 in the Brain and the Neuropathology ofRTT .11 FXYD 1 and the FXYD Gene Family. 16 General Hypothesis and Specific Aims. 21 CHAPTER TWO FXYDJ is a MeCP2-target Gene Overexpressed in the Brain ofRett Syndrome Patients and Mecp2-null Mice.

28 Materials and Methods. 49 Figures and Tables. 55 CHAPTER THREE Conclusions and Future Directions. 106 Appendix Chapter 1: FXYDJ and FXYD7 Gene Expression Are Developmentally and Differentially Regulated in the Cortex and Hypothalamus of Rodents.

107 Materials and Methods. 112 1 Appendix Chapter II: Abnormal Expression of Genes Involved in Neurogenesis (SOX2 and WIFJ) in the Brain ofRett Syndrome Patients. 120 Materials and Methods. 128 11 ACKNOWLEDGEMENTS I love science.

Following my passion for science, I crossed the Pacific Ocean from Hong Kong and came to OHSU to pursue my Ph. Over the past six and a half years of my graduate career here at OHSU, there have been many ups and downs, challenges as well as unexpected turning points. Throughout all of these, there have been many people who have been uplifting and have contributed to both my academic and personal growth in their own unique ways. I feel blessed and grateful for having these wonderful people and experiences in this chapter of my life.

My first thanks goes to my advisor, Dr. Sergio Ojeda, for giving me the great opportunity to work on this project and for supporting me through these years of work on this project. I am deeply indebted to every member on my thesis advisory committee (TAC), Drs. Richard Maurer, Charles Roberts and Klaus Freuh, for their valuable scientific advice, productive discussion, time, effort and cooperation, especially when commuting between the two OHSU campuses to every TAC meeting was challenging.

Without their support, I couldn't have gone this far to get where I am standing now. I'd also like to extend my special thanks to our collaborator, Dr. Matthew Frerking, for his sage advice, inputs, and stimulating discussion, and also for the tremendous amount of time and effort as well as the excellent expertise that he contributed to the electrophysiological study of this project. I'd like to thank Dr.

Sudge Budden for bringing this 111 wonderful project onto the lab bench and for sharing her passion for research on Rett syndrome. I'd also like to thank Northwest Rett Syndrome Foundation for allowing me the chance to attend their annual meetings and to interact with many families and girls affected with RTT. It was those experiences that ultimately evoked strong passion and drive within me that gave this project a meaning much deeper than that of a Ph. I'm grateful to have worked with every member of the Ojeda lab and thank you for your friendship and support over these past five years.

My never-ending appreciation goes to my parents, who, as compassionate physicians, imparted to me and fostered my strong interest in life science and human diseases during my early childhood; and whose unconditional love nurtured a deep sense of security within me that has always empowered me to explore and to pursue my dreams. Thank you, my dear mother and father, for your endless love, support, understanding and sacrifice! I'd also like to thank my dearest older sister for her love, patience, courage and sharing of perspectives in every aspect of life, even though we are apart from each other for all these years. Finally I'd like to thank all those people who have left special and beautiful footprints in my life while I was working on this Ph. Thank you, Bin and Dennis, for sharing those special experiences that have deepened my perception and eventually let me grow stronger as a whole person.

Thank you, all my dear friends-- See-yan and Luk, Jasper, Wei, Jenny, Matthew and many others, for lV your love and encouragement, for your precious friendship, and for your continuing support. Thank you, Anuji, my fellow graduate student and dear friend, for your valuable inputs, critiques as well as kind help and support, especially during the final phase of the dissertation preparation on those winter days when the weather was relentlessly cold and snowy and you had to walk home on foot. Once again, I thank everyone who has impacted and helped shape my life greatly. Without you, I could not have become who I am now.

v ABSTRACT Found almost exclusively in young females, Rett syndrome (RTT) is a progressive X chromosome-linked neurodevelopmental disorder characterized by loss of acquired psychomotor abilities, decelerated head growth, mental retardation, stereotyped hand movements, autism, and seizure. Girls affected with RTT usually present a dynamic age-dependent clinical course with an abrupt onset of symptomatic manifestation after 6-18 months of seemingly normal development. Mutations in the MECP2 gene, encoding methyi-CpG-binding protein 2 (MeCP2), are responsible for a majority of RTT cases. MeCP2 acts as a transcriptional repressor that interprets DNA methylation signals in the mammalian genome by binding to methylated DNA, and subsequently induces gene silencing via chromatin remodeling by recruitment of Sin3A-histone deacetylases (HDAC1/2) corepressor complex.

Gene silencing, mediated by DNA methylation, represents an important epigenetic mechanism and is essential for many biological processes during normal development in vertebrates. The MeCP2 protein is most abundantly expressed in mature neurons of the mammalian central nervous system (CNS) and is thought to play a role in maintenance and modulation of neuronal maturity and plasticity. The precise Vl function of MeCP2 in the brain and the mechanism by which MeCP2 mutations cause RTT currently remain elusive. Given the molecular nature of MeCP2, it is predicted that loss of MeCP2 function may lead to widespread aberrant gene expression in the CNS.

Identification of the neuronal targets of MeCP2 will not only advance our current knowledge about the role of MeCP2 in neuronal functioning, but will also shed light on the neuropathology of RTT. In this thesis study, using Affymetrix GeneChip oligonucleotide arrays, we compared gene expression profiles of postmortem brain tissues from RTT patients with their age- matched control subjects. We found that the gene expression level of FXYD1 was abnormally elevated in the frontal cortex of RTT patients. We further detected that the FXYD1 gene was also selectively overexpressed in the frontal cortex of Mecp2-null mice, a mouse model of RTT.

The FXYD1 gene encodes a small, single-span membrane protein, namely FXYD domain-containing ion transport regulator 1 (FXYD1) or phospholemman (PLM), which regulates cell excitability via modulation of tissue- specific Na+,K+-ATPase activity. Here, we demonstrated that the FXDY1 gene harbored multiple methylated CpG sites in its promoter regions, and that MeCP2 was physically associated with the native FXYD1 promoters in both human cell culture and mouse brain tissues. These results suggested that the FXYD1 gene was subjected to DNA methylation-dependent epigenetic transcription repression mediated by MeCP2. We further evaluated functional consequences of FXYD1 overexpression by electrophysiological studies.

The data revealed that Na+,K+- Vll ATPase-dependent currents were reduced in cortical neurons of Mecp2-null mice, possibly attributable to FXYD1 overexpression. Taken together, results presented in this thesis have suggested for the first time that FXYD1 is a bona fide MeCP2-target gene, whose selective derepression in the frontal cortex due to MeCP2 deficiency may contribute to the neuropathogenesis of RTT by altering neuronal Na+, K+ ATPase activity. This finding will potentially provide a new line of investigation for designing therapeutic strategies and agents to treat this debilitating childhood disorder. Keywords: Rett syndrome (RTT), DNA methylation, MeCP2, FXYD1, Gene expression, Transcription repression, Affymetrix GeneChip arrays, Frontal cortex, Na+, K+-ATPase, Mecp2-null mouse.

Vlll Vivianne Deng CHAPTER ONE INTRODUCTION Rett Syndrome and Epigenetic Gene Regulation Mediated by Methyi-CpG Binding Protein 2 (MeCP2) Vivianne W. Deng 1 Vivianne Deng Overview of Rett Syndrome Clinical Characteristics: Rett syndrome (RTT, OMIM #312750) is a severe X-linked neurodevelopmental disorder that predominantly affects young females with a prevalence of about 1:10,000-15,000 worldwide 1-3. It is characterized by an abrupt arrest and deterioration of brain development that becomes symptomatically evident after the first 6-18 months of seemingly normal postnatal growth 4. The disease subsequently leads to a variety of neurological abnormalities, including "acquired" microcephaly, impaired language development, loss of voluntary movement, stereotypical hand wringing, mental 4 retardation, irregular breathing, autism, hypotonia, ataxia, apraxia, and seizure · 5.

Born apparently normal, affected individuals soon lose their ability to stand, walk, talk and use their hands. Surpassed only by Down syndrome, RTT is the second leading cause of mental retardation in females 6. The course of the disease is irreversible, and the disease is incurable at the present time. Although young girls with RTT can generally live into their adulthood, the mortality rate is 1.2% per year, with about 25% of these deaths reported as sudden and unexplained 7.

It has been speculated that breathing dysfunction and cardiac abnormalities may account for these incidents of sudden death 8. Neuropathology: Due to the neurological regressive features presented in RTT, the syndrome was initially thought to be a neurodegenerative disease. It is not until 2 Vivianne Deng recently that evidence from neuropathologic studies has supported RTT as being a disorder of arrested neuronal development 9. Although the gross brain anatomy reveals only subtle and nonspecific abnormalities, the brain size of individual with RTT is about 30% smaller than normal at any given age 9"11.

Further studies using neuroimaging and postmortem brain weight have indicated that the 10 12 caudate nucleus and frontal cortex display the largest reduction in volume · , 11 suggesting a selective decrease in the size of certain brain areas. Impaired neuronal development has also been implicated by the observation that the dendrites of pyramidal neurons in the frontal and motor cortex were markedly 13 14 shortened and had much less complexity •. In fact, the results from three- dimensional confocal microscopy studies confirmed these findings and further detected a reduction in the number of dendritic spines 15. Finally, while neuronal migration in RTT brains seemed normal, neurons in the areas of cerebral cortex, basal ganglia, thalamus, hippocampus, amygdala, and substantia nigra showed smaller soma and were more densely packed than in control subjects 16.

The mechanisms underlying the neuropathology of RTT are currently unknown, however, the dendritic alterations demonstrated in the above studies suggest that a disrupted synaptic development is involved, and may be associated with the reduced levels of a number of molecules, such as microtubule-associated protein 2 (MAP2, an important cytoskeletal component of neuronal dendrites), substance P (a neuropeptide that stimulates neurite extension in cultured neuroblastoma cells), and prostaglandin endoperoxide H synthase-2 (PGHS-2, the rate-limiting enzyme in prostanoid biosynthesis that localizes to dendritic spines and marks 3 Vivianne Deng mature neurons) 17-19. And yet, whether or not there is a causative relationship between changes in synaptic connectivity and the levels of these molecules remains to be determined by in-depth studies in the future. MECP2 as a RTT-responsible Gene and Mecp2-null Mouse as a Rodent Model forRTT: Even though the almost exclusive involvement of females is best explained by X-linked dominant inheritance with lethality in the hemizygous males 4 , the molecular genetics underlying the pathology of RTT had been an enigma for several decades since the first description of the syndrome in1966 by Andreas Rett 20 , a Viennese physician.