Research Awardees: 2001
Research Awards
Tony Charman, Ph.D.
Institute of Child Health, London, UK
"A Detailed Genotype-Phenotype Correlation Study in Rett Syndrome"
2-Year Award: $99,437
Research Sponsor: Family Friends of Jessie Lebson
Final Report (November 2004)
Lay Summary
Research into Rett Syndrome, a disorder that profoundly affects brain development in females, moved into a new era with the recent discovery that up to 80% of cases are caused by mutations in a specific gene (MECP2). The extent to which the presence or absence of a mutation in this gene, or the precise nature of the mutation, affect the natural history of the clinical disorder is not yet clear. We aim to address this question by carrying out mutation detection screens in 3 large groups of Rett Syndrome females in whom the clinical presentation has been detailed with greater precision than has been the case in previous studies. We hope that the findings will contribute to our understanding of the pathology that underlies Rett Syndrome (holding out the possibility for gene therapeutic strategies in the future). However they will also inform clinical decision making by improving diagnostic accuracy and advice on prognosis and likely outcome as well as choosing the most efficacious strategies for each individual patient.
Abstract
MECP2. This protein binds to methylated CpG dinucleotides in DNA and probably functions in the control of transcription competence of numerous chromosomal loci. The effect of mutations in MECP2 on the function of the protein are beginning to emerge, but the extent to which the presence or absence of an MECP2 mutation, or the precise nature of the mutations, affect the natural history of the clinical disorder is not yet clear. Previous studies that have attempted to identify genotype-phenotype correlations have been limited by low sample sizes and poor characterization of the physical, developmental and behavioral phenotype. Few consistent associations have thus far emerged. We aim to address this question by carrying out mutation detection screen in MECP2 and measuring X-inactivation ratios, in 3 large cohorts of Rett Syndrome patients. Using a combination of detailed systematic clinical assessment information and parental questionnaire information (including information from a newly-developed questionnaire with proven ability to discriminate between typical Rett Syndrome behaviors and those found in other individuals with severe and profound developmental disability) we will be able to characterize the phenotype more accurately than has been previously achieved. In addition the size of the cohorts available (N=100 with clinical assessment information, N=300 with questionnaire information) and the number of classic (N=220) and atypical/variant (N=80) cases will allow for a stronger test of genotype-phenotype associations than in previous studies. The identification of phenotypic differences between patients with different mutations will aid in the development of models of the neuropathological processes that occur in the disorder (holding out the possibility for gene therapeutic strategies in the future). It will also aid clinicians now to improve diagnostic and prognostic accuracy, and decide on the best therapeutic strategy for each individual patient.
Jeffrey C. Hansen, Ph.D.
University of Texas Health Science Center at San Antonio
"Solution-state Characterization of Native and Mutant MeCP2 Interactions with Chromatin and Sin3p"
2-Year Award: $99,000
Research Sponsor: Gardner Family Foundation
Final Report (November 2004)
Lay Summary
The cells in our body contain a protein, called MeCP2, that when mutated leads to the development of Rett Syndrome in affected individuals. Now that this molecular link to Rett Syndrome has been discovered, it is important to determine what molecular functions of MeCP2 are altered in the disease state. With today's technology, it is possible to produce and isolate normal MeCP2 and Rett Syndrome-associated MeCP2 mutants in bacteria. These studies will exploit this technology to determine how normal and mutant purified MeCP2s interacts with the chromatin fiber, and with the regulatory protein Sin3p. Chromatin is the genetic material of higher organisms, and consists of an assemblage of DNA and histone proteins. Although MeCP2 is thought to be a chromatin-associated protein, no direct studies of MeCP2-chromatin interactions have been performed. A second important function of MeCP2 is to interact with the regulatory protein Sin3p. We will examine the binding of normal and mutant MeCP2s to Sin3p. This information will allow us to better understand the role of MeCp2-Sin3p, and HDAC. The latter is a chromatin modifying enzyme that is associated with repression of gene expression. In summary, the proposed studies will fill gaps in our knowledge of two key functions of MeCP2 in healthy individuals, and how these functions are affected by Rett Syndrome-associated MeCP2 mutations. Together, the results of the proposed research will significantly improve our understanding of the molecular basis of Rett Syndrome.
Abstract
MeCP2 is a methyl CpG binding protein implicated in Rett Syndrome (RTT). It consists of two key domains, termed the methyl CpG binding domain (MBD) and the transcriptional repression domain (TRD). The first objective of this proposal is to determine the interactions of normal and RTT-associated MeCP2 mutants with the chromatin fiber. The second objective is to quantitatively analyze native and mutant MeCP2-Sin3p interactions. Both objectives test whether Rett Syndrome associated MeCP2 mutants exhibit altered interactions with functionally important macromolecules that bind to native MeCP2. The information obtained from the proposed studies will test the hypotheses that Rett Syndrome mutations in the MeCP2 MBD will disrupt interactions with chromatin, while MeCP2 mutations in the TRD will abolish interactions with Sin3p. These studies will provide insight into the molecular effects of Rett Syndrome-associated MeCP2 mutations, which in turn will allow for a better understanding of the molecular pathogenesis of Rett Syndrome.
Aharon Razin, Ph.D.
The Hebrew University of Jerusalem-Hadassah Medical School, Israel
"MeCP2 and Rett Syndrome"
1-Year Award: $50,000
Research Sponsor: Rett Syndrome Association of Illinois / Adam (Buddha) Lavey
Final Report (November 2003)
Lay Summary
Rett Syndrome is in most cases caused by a mutation in the MECP2 gene that is located on chromosome X. This gene encodes an important protein that participates in a major regulatory mechanism that is responsible for silencing of a group of genes. We have studied the production of this protein in early mouse development and found that the protein is not present in embryos before implantation to the mother's womb. We propose to study the onset of the production of this protein and discover when in development of the embryo the protein is required. The second part of the proposed research will be devoted to examine the possibility of a rescue of the disease phenotype by introducing a normal MECP2 gene into MeCP2-deficient mice.
Abstract
MECP2, the affected gene in Rett Syndrome patients, will be studied in mouse early embryo development to determine the onset of its synthesis and assembly of the MeCP2-Sin3A-HDAC multiprotein corepressor complex. Introduction of a MeCP2 expression vector by transfection into MeCP2 deficient cells in culture will provide a tool to study the possibility of complementation of the MeCP2 mutation. The repressor activity will be studied on a suitable target gene that will be selected from genes discovered in comparative microarray assays. The results of the transfection experiments will be used to set the stage for more sophisticated experiments in vivo. In these experiments MeCP2 transgenic lines will be established on a WT and a MeCP2 -/- mouse background. These mice will be used to examine whether the mutated phenotype can be rescued and will allow us to answer the question whether Rett Syndrome results from MeCP2 loss of activity, accumulation of a MeCP2 aberrant protein or both.
Bryan M. Turner, Ph.D.
University of Birmingham Medical School
"Histone Deacetylase Complexes Assembled on Wild-Type and Mutant MeCP2: Functional Properties and Genomic Distribution"
1-Year Award: $49,940
Final Report (November 2003)
Final Report (November, 2003):
The DNA-binding protein MeCP2 has a role in controlling the expression of many different genes. It is believed that the mutations in MeCP2 that cause Rett Syndrome, exert their effects by altering patterns of gene expression and thereby disrupting normal pathways of brain development. Mutations in MeCP2 are commonly found in either the region of the protein that binds to DNA, or in a region (called the TRD) that is believed to associate with other specific proteins to assemble a multi-protein gene regulatory complex. In our project we used molecular genetic techniques to make MeCP2 proteins that had mutations the same as those found in Rett patients. We were able to demonstrate that MeCP2 with mutations in the protein-binding domain can assemble a gene regulatory complex with the same constituents as that assembled by normal MeCP2, but does so less efficiently. We used microscopical analyses to show that these mutant complexes are targeted to the same DNA regions as normal MeCP2 complexes. In contrast, MeCP2 mutated in the DNA-binding domain, was not properly targeted. Our results suggest that mutations in the protein-binding region of MeCP2 may lead to the symptoms of Rett syndrome by rather subtle, quantitative effects.
Dean F. Wong, M.D., Ph.D.
Johns Hopkins University School of Medicine
"Dysfunction of High-Affinity a4/b2 Nicotinic Acetylcholine Neuronal Receptors in Rett Syndrome"
1-Year Award: $50,000
Research Sponsor:Family and Friends of Chelsea Coenraads
Lay Summary
Several lines of evidence suggest that abnormalities of the high-affinity a4/b2 neuronal nicotinic acetylcholine receptors (nAChRs) in the brain play an important role in Rett Syndrome (RTT). The objective of this study is to identify the binding of nAChRs in specific brain regions of ten participants with RTT and ten age and sex-matched normal control subjects. The binding of nAChRs will be determined by the utilization of single photon emission computed tomography (SPECT) scans after the administration of the novel nAChR radioligand (S)-5[123I] iodo-3-(2-azetidinylmethoxy)pyridine (51A), which localizes high-affinity a4/b2 nAChRs in the brain. Determination of binding on nAChRs in the brain of people with RTT may provide the basis for the development of safe and effective supportive treatments including nicotine patches and sprays. Additionally this technique may provide the basis to determine the effectiveness of other interventions for RTT.
Abstract
The central methodology of this study will be the examination of the distribution of high-affinity a4/b2 neuronal nicotinic acetylcholine receptors (nAChRs) in the brains, the ascertainment of plasma, salivary, and urinary levels of nicotine and cotinine, and the abnormal involuntary movements, and the psychological functioning of people with RTT and control groups. Two groups of neuroleptic-na.ve subjects, ten patients with RTT who were never exposed to nicotine and ten age and sex-matched normal control subjects without RTT who never smoked cigarettes and never had other nicotine exposure, will be assessed utilizing a protocol consisting of (A) single photon emission computed tomography (SPECT) utilizing the nAChR radioligand (S)-5[123I] iodo-3-(2-azetidinylmethoxy)pyridine (51A), which localizes high-affinity a4/b2 nAChRs (Musachio, et al., 1999, 2001), (B) determination of plasma, salivary, and urinary levels of nicotine and cotinine, and (C) structured assessment of psychiatric and movement disorders. Because the parameters measured by the instruments may not be normally distributed, nonparametric tests of significance including the Mann-Whitney test (Conover, 1999) will be used to analyze the results. Data will be analyzed utilizing statistical procedures appropriate for the small sample sizes. If the data are from normally distributed populations, then paired t tests will be used to analyze the results.
RSRF Post-Doctoral Fellowship Awards
Carolyn Ellaway, Ph.D.
The Children's Hospital at Westmead, Sydney, Australia
"Assessment of Central Autonomic Nervous System Function in Rett Syndrome"
Mentor: John Christodoulou, Ph.D.
2-Year Award: $88,000
Lay Progress Report (August 2002)
Lay Summary
The future of Rett Syndrome research will be directed towards understanding the underlying pathogenesis of the disorder and the development of specific treatments, ideally prior to the onset of symptoms or early in the evolution of the disorder.Ê It is therefore vitally important to establish objective methods for assessment of the clinical status of patients with Rett Syndrome, prior to commencement of clinical trials and to be able to accurately monitor changes over time.Ê The central autonomic nervous system, which controls the activity of the nerves controlling the function of the heart and lungs, has been shown to function abnormally in Rett Syndrome subjects.Ê The abnormalities include irregular breathing patterns, extreme agitation with large pupils and flushed face, vacant spells, abnormal brainwave activity, seizures and poor circulation. A relatively new instrument for evaluating the central autonomic nervous system, the Neuroscope TM can measure and monitor the activity of the central autonomic nervous system.Ê Information derived from the assessment of the autonomic nervous system will provide a valuable tool for monitoring Rett Syndrome patients during clinical treatment studies.Ê In addition an individual treatment plan can potentially be developed with the ultimate aim of improving patient care and quality of life.Ê It has been suggested that abnormal function of the autonomic nervous system could be the cause of abnormal heart beat rhythms and sudden death.Ê Since at least 25% of deaths in Rett Syndrome are sudden and unexpected, it would be reasonable to establish objective measures of disordered breathing in Rett Syndrome.Ê Since the discovery of mutations in the MECP2 gene as a cause of Rett Syndrome, numerous studies have attempted to correlate the type of mutation (gene error) in the MECP2 gene (genotype) with clinical characteristics (phenotype).Ê The Neuroscope TM, a reliable clinical method of characterizing autonomic nervous system function, may assist these studies in Rett Syndrome.
Abstract
The future of Rett Syndrome (RTT) research will be directed towards understanding the underlying pathogenesis of the disorder and the development of targeted therapeutic interventions, ideally in the pre-symptomatic stage or early in the evolution of the disorder.Ê It is therefore vitally important to establish objective methods for assessment of the clinical status of patients with RTT, prior to commencement of clinical trials and to be able to accurately monitor clinical changes over time.Ê A relatively new neurophysiological approach to the evaluation of brainstem function has been developed.Ê The Neuroscope TM can measure and monitor the activity of the central autonomic nervous system, in particular brainstem function, cardiac vagal tone and respiratory patterns.Ê Information derived from the assessment of the autonomic nervous system will be valuable for monitoring RTT patients during clinical intervention trials.Ê In addition the characterization of autonomic dysfunction will enable a patient outcome oriented treatment plan to be developed, with the ultimate aim of improving patient care and their quality of life.Ê Since the discovery of mutations in the MECP2 gene as a cause of RTT, numerous studies have attempted to identify phenotype-genotype correlations.Ê It has become apparent that the clinical method of characterizing the phenotype in RTT is difficult and unreliable.Ê The study of phenotype-genotype correlations in RTT may be assisted by the Neuroscope TM, a reliable clinical method of characterizing the phenotype.
Joel M. Harp, Ph.D.
The University of Tennessee - Oak Ridge National Laboratory
"X-ray Crystallographic Studies of MeCP2 on a Chromatin Substrate"
Mentor: Gerard J. Bunick
2-Year Award: $100,000
Final Report (November 2003)
Lay Summary
Final Report (November, 2003):
The MeCP2 protein recognizes a chemical signal on DNA and binds to it. Portions of the MeCP2 protein then recruit other proteins to change the structure of chromatin and silence gene expression. Chromatin is the form in which our DNA is packaged and consists of repeating units called nucleosomes. Questions of how MeCP2 binds to DNA on nucleosomes and then modifies the nucleosomes are very difficult to answer. While graduate student at the University of Tennessee and the Oak Ridge National Laboratory, I worked on the structure of the nucleosome core particle using the techniques of macromolecular crystallography. I received a fellowship from RSRF while still at Tennessee with hopes of solving the structural basis of how MeCP2 recognizes methylated DNA on the nucleosome and how it then affects the structure of chromatin. While this work is still ongoing, the RSRF Fellowship has allowed me to move twice. Once to the University of Virginia where I worked with Drs. David Allis, Sepideh Khorasanizadeh, and Fraydoon Rastinejad. There I had an opportunity to work on proteins involved in the histone code as well as work on my primary interest in MeCP2. The second move has been to Vanderbilt University where I am now Director of the Macromolecular Crystallography Facility. This position provides opportunities to continue the studies begun during the term of the RSRF Fellowship and, hopefully, to begin new collaborations with Rett Syndrome researchers.
Noriyuki Kishi, M.D., Ph.D.
Children's Hospital, Harvard Medical School
"Molecular Controls Over Differentiation of Cortical Projection Neurons from Neural Precursors: Normal and MECP2 -/-"
Mentor:ÊJeffrey Macklis, M.D., D.HST
2-Year Award:$110,000
Research Sponsor: Festival of Food and Wine
Final Report (November 2004)
Lay Summary
Rett syndrome is a disease of brain development that causes mental retardation and autistic behavior in girls.Ê Recent experiments have revealed that a defect in a gene called the methyl-CpG-binding protein 2 (MECP2) causes Rett syndrome.Ê There is evidence from human autopsies that neurons in the cerebral cortex that make long-distance connections between the two hemispheres of the brain express MECP2 at high levels during brain development.Ê In addition, recent work using MECP2 mutant mice has shown that mutation of the MeCP2 protein in neurons is sufficient to cause neuronal dysfunction in various parts of the brain including the cerebral cortex; these animals manifest symptoms that are similar to those of Rett syndrome.Ê However, the specific neuronal abnormalities have not been elucidated.Ê The cerebral cortex is the most complex structure of the brain and is responsible for high functions unique to humans; even a subtle abnormality of connections of this complex structure can cause diseases affecting behavior and cognition, such as Rett syndrome.Ê The development of cortical connection neurons is critical to cortical function; the role of MECP2 mutation in this process may be central to understanding the neurobiological basis of Rett syndrome.Ê The molecular mechanisms underlying development of cortical connection neurons, and the specific role of MECP2 in these processes, remain largely unclear.Ê Therefore, I propose to investigate directly the molecular mechanisms of cortical connection neuron development and effects of MECP2 mutation on this process.Ê I propose to: 1) investigate the effects of MECP2 on development of connections between the two hemispheres of the cerebral cortex using MECP2 mutant mice and analysis of MeCP2 production in select populations of cortical connection neurons during normal development; and 2) investigate developmental events that follow MeCP2 production and function, using precursor cells and purified cortical connection neurons in cell culture experiments.Ê This knowledge will not only add to our understanding of the neurobiological basis of Rett syndrome and the mechanisms underlying connection neuron development in the cerebral cortex, but also may contribute to the potential for molecular and therapies for neurological disorders involving neuronal compromise and loss, including Rett syndrome.
Abstract
Rett syndrome is a neurodevelopmental disorder and one of the causes of mental retardation and autistic behavior in girls. Recent work has revealed that a defect in the methyl-CpG-binding protein 2 (MECP2) gene causes Rett syndrome.Ê There is evidence from human autopsy material that cortical projection neurons express MECP2 at high levels during development.Ê In addition to the gene discovery, recent mouse genetic work has demonstrated that, in MECP2 mutant mice, a MeCP2 deficiency in neurons is sufficient to cause neuronal dysfunction in neocortex, hippocampus, and cerebellum, with manifestating symptoms that mimic those of Rett syndrome.Ê However, the specific neuronal abnormalities have not been elucidated.Ê The neocortex is the most complex structure of the brain and gives us unique talent, but even a subtle disruption of cortical connectivity can cause behaviorally and cognitively significant diseases, such as Rett syndrome.Ê The development of cortical projection neurons is critical to cortical connectivity; the role of MECP2 mutation in this process may be central to understanding the neurobiological basis of Rett syndrome.Ê The molecular mechanisms underlying development of cortical projection neurons, and the specific role of MECP2 in these processes, remain largely unclear.Ê Therefore, I propose to investigate directly the molecular mechanisms of callosal projection neuron development and effects of MECP2 targeted gene deletion ("knockouts") on this process.Ê I propose to: 1) investigate the effects of MECP2 on development of interhemispheric connections using MECP2 knockouts and analysis of MeCP2 expression in select populations of projection neurons during normal development; and 2) investigate developmental events downstream of MeCP2 expression, using cultured neural precursors and purified cortical projection neurons.Ê This knowledge will not only add to our understanding of the neurobiological basis of Rett syndrome and the mechanisms underlying projection neuron development in neocortex, but also may contribute to the potential for molecular and cellular therapies for neurological disorders involving neuronal compromise and loss, including Rett syndrome.
Tomoki Yokochi, Ph.D.
National Cancer Institute (NIH)
Recipient of the Alan P. Wolffe Memorial Fellowship
"Chromatin Activities in Neurons: Structural and Functional Analysis of the MeCP2 and HDAC Complexes"
Mentor: Keith D. Robertson, Ph.D.
2-Year Award: $100,000
Final Report (November 2003)
Final Report (November, 2003):
In this research, I have attempted to identify MeCP2 and its interacting partners in the brain. Previous reports suggested that MeCP2 interacts with a group of proteins called histone deacetylases (HDACs) in the tissue culture (tissue cells that were isolated and kept in medium on a culture dish). In the native mammalian brain tissue, I found that there are two protein complexes containing MeCP2. These complexes also contain several proteins other than MeCP2. One of the common components is Sin3A, which is regarded as the structural framework for these protein complexes. Neither HDAC1 nor HDAC2 was identified in these complexes, which is inconsistent with previous reports. Thus, it is assumed that MeCP2 plays a unique role in the brain tissue with a distinct group of proteins. To further understand the structural difference of MeCP2s in tissue culture and in brain tissue, I have identified the MeCP2 complex by an alternative approach, immunoprecipitation. Just recently, it was reported that there are two forms of MeCP2 in cultured nerve cells. One receives modification at particular position with a phosphate group, whereas the other was not modified. In contrast, I found that MeCP2 in native brain tissue appeared to have a different modification status. These results suggest that the structural modification of MeCP2 proteins may impart it with a brain-specific function in native brain tissue.
