Scott L. Pomeroy - US grants

The long-term objective of this project is to determine how glial cells effect changes in synaptic connections. In parasympathetic ganglia, synapses cluster near the nucleus of glia called satellite cells. This relationship is retained as both synapses and satellite cells undergo. rearrangements over time, suggesting that these glia have a role in the maintenance of synapses and that they may participate in synaptic remodeling. The research described here examines this relationship with 3 objectives. The first objective is to determine how satellite cells are added postnatally to neurons. The addition of glia will be measured using quantitative light microscopy. Bromodeoxyuridine immunohistochemistry will be used to explore whether mitosis accounts the addition of glia. To determine if they arise from cells associated with neurons, the fate of individual satellite cells labeled with intracellular dextran-- rhodamine will be monitored by repeated examination with in vivo video microscopy. The second objective is to establish the function of platelet-growth factor (PDGF) in regulating the proliferation of satellite cells after axotomy of their associated neurons. PDGF is present in many neurons and mitogenic to glia. Following axotomy of the superior cervical ganglion, changes of gene expression for PDGF and its receptor will be monitored using the polymerase chain reaction. It will then be determined whether the proliferative response is blocked with antibodies against PDGF and its receptor. The third objective links satellite cell proliferation to synaptic remodeling. It will be determined by direct observation with in vivo microscopy whether synaptic remodeling continues in older mice when satellite cell number no longer increases, and whether synaptic remodeling is induced when satellite cells are stimulated to divide. These experiments are designed to explore how glial cells elicit changes in synaptic connections. They will provide insights as to how the nervous system responds to injury. Furthermore, the understanding of how glial cells bring about synaptic change is directly relevant to the normal development of the nervous system and to disorders arising from abnormal development.

The long-term objective of this project is to determine how glial cells effect changes in synaptic connections. In parasympathetic ganglia, synapses cluster near the nucleus of glia called satellite cells. This relationship is retained as both synapses and satellite cells undergo. rearrangements over time, suggesting that these glia have a role in the maintenance of synapses and that they may participate in synaptic remodeling. The research described here examines this relationship with 3 objectives. The first objective is to determine how satellite cells are added postnatally to neurons. The addition of glia will be measured using quantitative light microscopy. Bromodeoxyuridine immunohistochemistry will be used to explore whether mitosis accounts the addition of glia. To determine if they arise from cells associated with neurons, the fate of individual satellite cells labeled with intracellular dextran-- rhodamine will be monitored by repeated examination with in vivo video microscopy. The second objective is to establish the function of platelet-growth factor (PDGF) in regulating the proliferation of satellite cells after axotomy of their associated neurons. PDGF is present in many neurons and mitogenic to glia. Following axotomy of the superior cervical ganglion, changes of gene expression for PDGF and its receptor will be monitored using the polymerase chain reaction. It will then be determined whether the proliferative response is blocked with antibodies against PDGF and its receptor. The third objective links satellite cell proliferation to synaptic remodeling. It will be determined by direct observation with in vivo microscopy whether synaptic remodeling continues in older mice when satellite cell number no longer increases, and whether synaptic remodeling is induced when satellite cells are stimulated to divide. These experiments are designed to explore how glial cells elicit changes in synaptic connections. They will provide insights as to how the nervous system responds to injury. Furthermore, the understanding of how glial cells bring about synaptic change is directly relevant to the normal development of the nervous system and to disorders arising from abnormal development.

DESCRIPTION: Recent discoveries have identified two different molecular signals that appear to be critical regulators of medulloblastoma growth. Mutations of the Patched (Ptc) gene in Gorlin's syndrome, an inherited disorder associated with medulloblastoma, and in about 20 percent of sporadic tumors has implicated the Sonic hedgehog (Shh)/Ptc pathway as a proliferative stimulus. Favorable prognosis has been tightly linked to the expression of the neurotrophin-3 (NT-3) receptor TrkC. TrkC appears to be more than a passive marker for favorable prognosis, for medulloblastomas undergo apoptosis when grown in the presence of nT-3 in vitro and are growth inhibited in nude mice when the receptor is overexpressed in tumor xenografts. These findings support a model of medulloblastoma oncogenesis in which the Shh/Ptc and neurotrophin signaling pathways collaborate to promote and modulate the growth of these tumors that arise from cerebellar granule cell precursors. In this model, they hypothesize that deregulated signaling of the Shh/Ptc pathway, either by gain of Shh function or by inactivating mutations of ptc, promotes tumor proliferation. By either mechanism, loss of Ptc transcription suppression promotes expression of molecules downstream of Ptc that induce excessive granule cell proliferation. They hypothesize that NT-3 inhibits the growth of medulloblastomas either by inducing apoptosis or differentiation of the tumor cells. This model predicts that highly malignant medulloblastomas have a constitutive Shh/Ptc proliferative signal with little or no Nt-3 induced cell death or differentiation, and that the progression of tumors induced by Shh/Ptc signaling will be inhibited by NT-3 especially when there is a high level of endogenous trkC expression. The experiments of this proposal test this model. In Specific Aim 1, they ask whether deregulation of the Shh/Ptc signaling pathway is a general feature of medulloblastomas. The experiments of Specific Aim 2 determine whether excessive Shh signaling is sufficient to induce the growth of medulloblastomas and whether the biological effects of Shh overexpression can be modified by TrkC activation. In Specific Aim 3, they determine whether TrkC activation inhibits the growth of medulloblastomas that arise in Ptc +/- mice, by breeding compound mutant mice deficient in both Ptc and TrkC signaling. In Specific Aim 4, they will determine in a nude mouse/xenograft model whether exogenous NT-3 can induce the regression of medulloblastomas in vivo. By testing whether Shh/Ptc and NT-3/TrkC collaborate to regulate the growth of the tumors, these experiments ultimately aim to define critical determinants of medulloblastoma growth, which have until now been poorly understood.

Medulloblastoma is the most common malignant brain tumor of childhood. It is thought to originate from the progenitors of granule cell neurons cell neurons in the developing cerebellum and it is classified as a primitive neuroectodermal tumor. About half the children and young adults with this disease survive longer than 10 years following surgery and adjuvant therapy. The others relapse and die in the face of identical treatment. Several years ago, we and our colleagues discovered a prognostic indicator for medulloblastoma. We showed that "standard risk" medulloblastomas express high levels of mRNA encoding the neurotrophin-3 receptor protein TrkC. The "high risk" medulloblastomas express low levels of trkC. The experiments we describe here are based upon this observation. We have three specific aims. The first aim is to establish the cellular basis for trkC over-expression in standard risk versus high risk medulloblastomas. Do standard risk tumors contain a greater percentage of cells that express trkC mRNA, or do the cells within these tumor express more trkC mRNA per cell? Quantitative analysis using in situ hybridization and immunohistochemical assays will be used to address this question. This will elucidate whether high and low trk C tumors represent two distinct types of medulloblastoma or sequential stages in the evolution of a common medulloblastoma progenitor. The second specific aim is to determine whether a prognostic correlation can be made with TrkC protein per se or with TrkC signaling functions. We will use phosphotyrosine directed antibodies to determine whether TrkC signaling functions are active in standard risk tumors in vivo. This will be an important step in learning whether activation of TrkC inhibits tumor growth. The third specific aim is to determine which other genes are coordinately up-regulated along with trk C in standard risk versus high risk medulloblastomas. Towards this end we will use a large cohort of standard and high risk medulloblastomas to screen the clones isolated by Dr. Stiles based on their differential expression in a high risk and standard risk tumor. Taken together the studies outlined here will provide a basis for understanding molecular attributes that enable some patients with medulloblastoma to be cured of their disease.

Many fundamental insights into signal transduction mechanisms that control cell growth and development have come via analysis of genetically simple, pediatric tumors. Via the joint rain tumor clinical at DFCI/Children's Hospitals, our program has exceptional access to rare pediatric tumors of neural ectodermal origin. This primitive neural ectodermal tumors can be conceptualized as multi-potent neural progenitor cells are developmentally stalled. Some of the larger pediatric hospitals and pediatric brain tumor consortiums have established PNET banks. However the tissue samples in these banks are, in general of no use to molecular biologists because no effort has been made to preserve information mRNAs and phosphoproteins. Under the direction of Dr. Scott Pomeroy, we have begun to bank PNETs and other rare solid tumors of childhood in a format that is of use to molecular biology researchers. Tumors are snap frozen in liquid nitrogen in the operating room so as to preserve mRNA to tyrosine phosphoprotein. Patient blood samples are also collected, together with family histories. It would be irresponsible to ignore this important clinical resource as a route to discovery-especially when the studies we propose could have practical overtones for the pediatric patients at DFCI/Children's Hospitals. According we request support for this facility as a core resource. Over time, as the research progresses, Drs. Whitman and Greenberg will almost certainly want to explore the function of FAST and BAD homologues in these "informative" pediatric tissue samples. Synthetic phosphopeptides can be used to raise antibodies that report the phosphorylation state of tyrosine, serine or threonine at defined positions within a specific growth factor receptor or signaling generating protein. Phosphopeptide-directed antibodies can be targeted to virtually any growth factor receptor or signaling generating protein which is regulated by phosphorylation. These antibodies are considerably more selective than conventional antibodies. Thus they lend themselves to studies on receptor activation in complex animal tissues such as brain. Some will need of such antibodies targeted to proteins such as SMADs, BADs, PDGF receptors and Trks. Economies of scale can be achieved with a core facility.

DESCRIPTION: The Imaging Core provides a variety of state-of-the-art digital imaging techniques to MRRC investigators, including three-dimensional volume rendering from optical, electron microscopic, or confocal microscopic images, multi-label fluorescence imaging for localizing cellular molecules, high quality digital light microscopic imaging, high temporal resolution time-lapse confocal imaging, quantitative confocal calcium imaging, and output as high quality print, slides, or digital images. Dr. Scott Pomeroy, formerly the Associate Director, replaced Dr. Kristen Harris, who moved to Boston University in 1999. Dr. Jon Kornhauser has been the Core?s System Manager since 1999. In addition, work-study students assist in the daily administration of the Core facility. The Core occupies 300 square feet of space on two floors of the Enders Building and is supported administratively by Ms. Amy Weinberg.

DESCRIPTION (provided by applicant): Medulloblastomas are the most common malignant brain tumors in children. More than one third of children with these tumors do not survive and essentially 100% of survivors have life changing neurocognitive sequelae. New approaches to treatment with reduced toxicity are needed. In the first five years of this project, the largest medulloblastoma genomic dataset created to date was established. Using integrated analysis of molecular, genomic and clinical data, medulloblastomas were discovered to consist of multiple molecular subtypes, each with a unique molecular signature reflecting underlying mechanisms of tumorigenesis and correlated with clinical outcome. These data were used to develop the most accurate risk-stratification schema developed to date, which proved to be generalizable to a fully independent dataset. The goals of the next funding period will be to define molecular subtypes at a deeper level, to identify molecular markers for targeted therapies, and to complete the development of our risk stratification model with a goal of translating to a test with that will be incorporated into the next generation of medulloblastoma clinical trials. These goals will be accomplished through the following Specific Aims: 1) Refine and validate medulloblastoma subtyping and outcome prediction in large-scale multi-institutional prospective clinical trials; 2) Implement a real time test for risk stratification and molecular subtyping of medulloblastoma patients; and 3) Develop an interactive website, or portal, which will provide a single, web-based, publicly available gateway to deliver genomic data, genesets, and computational methodology to the general clinical and scientific community.

The goal of the Bioinformatics Core (BC) will be to provide web-accessible annotation, cataloging facilities and state-of-the-art bioinformatics analyses. This will enable researchers from all three of the projects to maximally utilize the gene expression, polymorphism and proteomic data sets to determine functional dependencies among the known genes and Expressed Sequence Tags (ESTs) and direct further biological validation of these putative dependencies (the expression vector core addresses this critical biological validation step). The BC will connect data from in situ hybridization studies in project three to microarray centers using Affymetrix GeneChip arrays of human and murine expressed sequences. It will also integrate proteomic data and genotype data from the projects.

4.a.2. Overall Objective The overall objective of the Imaging Core is to provide MRDDRC investigators access to state-of-theart microscopic imaging and the capacity for three-dimensional reconstructions of neural preparations. Over the past five years, new imaging systems have been added to the already substantial capabilities of the Core. The three-dimensional image reconstruction system that has been the center of the Core for two decades now is based on affordable personal computer platforms, allowing individual laboratories to import and export images to their own desktops as well as to work on the computers within the Imaging Core. The confocal microscopes and digital imaging microscopes add powerful image acquisition and analysis capabilities. Moreover, image files generated by these microscopes can be easily formatted to be compatible with such standard image programs as Adobe Photoshop running on desktop PC or Macintosh computers. By adopting standard image formats and "point-and-click" Windows/Maclntosh/Xwindows computer interfaces, the Core has been designed for ease of use and maximum flexibility. Moreover and importantly, the Core Director and System Manager are available for individualized instruction in the use of the Core, and training workshops have been conducted as new imaging systems have been added. This training objective is a critical aspect of the Core's function and the MRDDRC mission.

DESCRIPTION (Provided by applicant): This Center grant requests funding for seven Core facilities to support a broadly-based research program in the study of mental retardation and developmental disabilities. The seven Core facilities are: Administrative, Proteomics, Cellular Neuroscience, Imaging, Gene Manipulation, Molecular Genetics, and Cell Sorter. The cores have undergone major development and support 123 research projects and 73 investigators. The research is in two programmatic areas (Neuroscience and Genetics). In Neuroscience, there are two major programs (Basic Neuroscience and Clinical/Translational Neuroscience). The Basic Neuroscience Program, directed by the Center Director, Dr. Michael Greenberg, consists of 47 investigators whose research efforts span a broad spectrum of interdigitated research in molecular neuroscience and systems neurobiology. A research project in Basic Neuroscience, proposed in this grant as a "New Program," addresses " the function of the GTPase regulatory enzyme, ephexin5, in synapse formation and dendritic spine development" (Mustafa Sahin, M.D., Ph.D.). A second major program in Neuroscience, i.e., Clinical/Translational Neuroscience, directed by Dr. Scott Pomeroy, includes 15 investigators whose research efforts include such areas as neuro-oncology, brain injury, brain behavior and brain development in neonates, human visual development, HIV, heart disease, surgery, and the developing brain, in vivo studies of brain development, neurocognitive development, developmental neuropsychology and learning disabilities, and endogenous and exogenous pxins and brain development. These areas of clinical research interdigitate with the basic research in the MRDDRC. In Genetics, directed by Dr. Louis Kunkel, 11 investigators are studying the molecular genetics of neuromuscular disease and human cerebral cortical development, the molecular basis of genetic disorders, retinal development and degenerative disorders, signal transduction, muscle stem cells, the genetics of complex traits and inherited musculoskeletal disorders. The multidisciplinary approaches to the research of this MRDDRC include the various basic science disciplines within Genetics and Neuroscience, and the clinical science fields of Neurology, Pediatrics, Neonatology, Infectious Disease, Endocrinology, Metabolism, Genetics, Cardiology, Neuropathology, Ophthalmology, Psychiatry, and Psychology. The research programs are housed in over 142,000 sq. ft. in two research buildings at Children's Hospital and several other facilities within the Harvard-Longwood Medical Area.

DESCRIPTION (provided by applicant): Barriers and challenges to the conduct of clinical trials are obstacles to the improvement of scientific knowledge and the advancement of diagnosis and treatments for neurologic disorders. The innovative aspects of the NEXT model are aimed at overcoming these barriers. The Department of Neurology at Children's Hospital Boston has the resources, personnel, patient population, institutional support, research expertise, and dedication that make it the logical choice for a NEXT Clinical Research Site (CRS) focused on pediatric neurology. The Specific Aims of this proposal are: (1) to develop the Children's Hospital Boston NEXT CRS as a shared infrastructure platform and assign roles for CRS leadership and staff; (2) to identify and recruit eligible patients for NEXT and NINDS trials and establish the extent of available disease-specific patient populations; (3) to submit trial applications for Network review, be named lead CRS on a future NEXT study, and demonstrate experience and expertise in the conduct of clinical research; (4) to collaborate with the NEXT Data Coordinating Center and demonstrate expertise in publication and dissemination of research results; (5) to abide by standardized Network policies and implement and optimize parameters of institutional, divisional, departmental, and individual collaborative agreements; and (6) to train new clinical investigators in skills required for an independent career in clinica research and identify means by which the CRS will maximize co-investigator use of local clinical research educational and support services.

A1. OBJECTIVE The overall objective of the Administrative Core is to facilitate the mission of this IDDRC. Thus, the goals of the Administrative Core are to ensure that 1) research in this Center is focused on problems of relevance and importance to intellectual and developmental disabilities, 2) the research is organized around clearly-defined themes, 3) the research is of the highest scientific quality, 4) there is facilitation of multidisciplinary research and research training, 5) translation of basic research findings to clinical application has high priority, and 6) there is fostering of centerness, that leads to synergistic, collaborative interactions between IDDRC investigators. We believe that the activities outlined throughout this grant (see Progress Report, Core Descriptions, and Research Projects Proposed for Core Usage) demonstrate that we are achieving these goals, and it is our intention to build upon our previous successes during the next funding period.

DESCRIPTION (provided by applicant): This Center grant requests funding for seven Core facilities and one Subcore;Administrative with Translational Neuroscience Subcore, Proteomics, Cellular Imaging, Neurodevelopmental Behavioral, Mouse Gene Manipulation, Molecular Genetics and Stem Cell, to support a broadly-based research program in intellectual and developmental disabilities at Children's Hospital Boston and Harvard Medical School. Our specific aims are to: 1) Maintain and introduce new "state of the art" core facilities that can be shared by Intellectual and Developmental Disabilities Research Center (IDDRC) investigators, providing core research services not possible in a single laboratory;2) Use the framework of the core laboratories to enhance and encourage collaboration between IDDRC investigators;3) Provide core services to aid in the training of young investigators and trainees and 4) Use the core services provided by the IDDRC program to leverage collaboration outside the institution, particularly with other IDDRCs, to speed the development of effective clinical interventions in intellectual and developmental disabilities. The Center supports 124 research projects and 77 investigators who receive approximately $75M in external funding, $57M of which is from the NIH. Research in the Center occurs in over 142,000 sq.ft. of space in research buildings at Children's Hospital Boston and Harvard Medical School affiliated institutions in the Harvard Longwood Medical Area. Our research focuses on three programmatic areas- Basic Neuroscience, Clinical / Translational Neuroscience and Genetics- and our primary goal is to identify the causes of and develop therapies for children with intellectual and developmental disabilities. The research of this IDDRC encompasses laboratory research on fundamental processes of normal and abnormal neurodevelopment and plasticity, as well as clinical and behavioral studies directed at disorders including, but not limited to, autistic spectrum disorders brain injury, neuro-oncology, learning and cognitive disabilities, the effects of surgery and environmental toxins on neural development, and multiple neurogenetic disorders, including those that affect neural formation, migration, specification and synaptic connectivity, as well as muscular dystrophy.

Overall Objective The overall objective of the Molecular Genetics Core is to provide a central location where IDDRC investigators can have access to high quality, low cost genomic technology services and expertise in a timely, affordable manner. These services can be broken down into six main functional components: Sequencing; Microsatellite Genotyping; High-Throughput SNP Genotyping and qPCR; Microarray Analysis; Next-Generation Sequencing; and Sample Management. By utilizing automated instrumentation in conjunction with the most advanced genetic equipment, the Core strives to rapidly generate consistent and reliable data at a low cost to investigators for each of these services. Through strong bioinformatics support and harboring a full complement of technologies in one centralized unit, the Core aims to serve as a preeminent unique resource of genetic analysis knowledge for investigators pursuing a broader understanding of the genetic basis of developmental disabilities. Specific Objectives The specific objectives are as follows: -High-throughput, low cost DNA sequencing including support with primer design, purification techniques, and software analysis tools. -Microsatellite genotyping including support. -High-throughput quantitative and digital PCR services including support with assay design and software analysis tools. -High-throughput and microarray SNP genotyping services. -Microarray gene expression analysis (including splice-variant analysis) and gene discovery. -Genomic deep sequencing capabilities and analysis using next-generation sequencing technology including analysis software support. -DNA and RNA sample extractions from blood and saliva including sample storage and preparation for downstream applications. -Project design and bioinformatics support for all services offered.

B1. OBJECTIVE The Proteomics Core aims to provide all investigators at the Children's/Harvard IDDRC with access to the state-of-the-art mass spectrometry-based proteomics required to advance studies in the cell and molecular biology of neurodevelopmental disabilities. This core provides instruction, consultation and service for the analysis of proteins by mass spectrometry, including protein identification, characterization of post-translational modifications, and quantification of proteins in samples with a wide range of complexities. Overview. Mass spectrometry-based proteomics is currently the most sensitive, quantitative and comprehensive technology available for the characterization of proteins. A major goal of Core B is to use proteomics to assist IDDRC investigators in the identification of protein complexes relevant to neuronal function, to measure the quantitative changes in protein composition that occur in the nervous system during development and under conditions that lead to developmental disabilities, and to identify functional posttranslational modifications of neuronal proteins. Proteomics is a rapidly progressing field and the Core is committed to introducing and implementing new methodology and applications as they become available. Dr. Steen, an Assistant Professor in the Neurobiology Program at Children's Hospital, and an expert in protein biochemistry and mass spectrometry, was recruited to setup and now act as director of the IDDRC Proteomics Core located within the Kirby Neurobiology Center at Children's. The Steen Laboratory is actively involved in proteomics research and has a computational/bioinformatics subgroup, which develops proteomics related statistical and data analysis tools and provides access to this statistical and computational expertise via the Core to all members of the IDDRC. The Core provides advice and the technology for protein quantification and comparative proteomics to IDDRC members. This Core has been designed to provide qualitative and quantitative mass spectrometry-based proteomics technologies. Since the inception of the Proteomics Core in 2005, these powerful proteomic technologies have been successfully used in many IDDRC laboratories for the proteomic analysis of cells and tissues to study intellectual and developmental diseases. The application of proteomic techniques to the study of the developing brain is poised to accomplish i) characterization of the full constellation of proteins in defined cells and tissues, ii) an assessment of changes in protein content or modifications as a function of disease or change in cell state, and iii) characterization of protein function. These investigations involve both large scale and directed proteomic experiments. Large scale studies are often quantitative, involving isotope tagging methods to find differentially expressed or modified proteins; whereas directed studies examine the structure of a single protein including its co-translational and post-translational modifications with respect to the protein's function. The proteomics Core provides services which encompass both these areas. Genome sequencing has provided the opportunity to address questions regarding brain development, disease and dysfunction using approaches previously impossible. To understand the molecular mechanisms of intellectual development and pathophysiology, IDDRC investigators can now directly interrogate the proteome of cells or tissues. This Core will provide a critical interactive center for IDDRC investigators by providing protein identification services, post-translational modification analyses and protein quantification.

The primary focus of the Stem Cell Core is the isolation and characterization of various types of stem cells and the fate tracing of their committed derivatives, although it will also be useful for sorting neuronal and glial cells for many IDDRC projects. This is accomplished using flow cytometry, based on the detection of cell surface markers, fluorescent proteins expressed under the control of specific gene promoters within transfected vectors or staining of nucleic acids with various staining dyes. The core is instrumental in the purification and characterization of adult stem cells isolated from neural and skeletal muscle tissue, purification of progenitor cells from iPS (induced Pluripotent Stem) and ES (Embryonic Stem) ceil lines, and isolation of cells whether cultured or from tissue that are expressing a fluorescent protein under the control of a specific promoter in a transfected vector. The core is proficient and experienced in handling zebrafish, mouse, and human cells. The extensive experience of the core's leadership ensures investigators expert assistance for their experiments and training in the use of successful techniques. Our principle objective is to provide both IDDRC and non-IDDRC researchers with comprehensive stem cell and stem cell derivative isolation and characterization services in a timely, dependable and cost-effective manner.

DESCRIPTION (provided by applicant): The Children's Hospital Boston Neurological Sciences Academic Development Award will be carried out in the Longwood Medical Area, a physically and intellectually unified area composed of Children's Hospital Boston, Harvard Medical School, the Beth Israel Deaconess Medical Center, the Brigham and Women's Hospital and the Harvard School of Public Health. Our long-term goal will be to facilitate and accelerate the development of independent physician scientists by providing research training experiences with outstanding research mentors who are committed to helping them achieve independent research faculty status by the end of their training. We will build on the strong track record of Neurology training in the Longwood area that has fostered the development of academic physician-scientists since the inception of the specialty. We intend to recruit a total of four scholars from an internal applicant pool of more than 50 child neurology and neurodevelopment disabilities residents and subspecialty neurology clinical fellows. Applications will also be accepted from qualified trainees from other institutions. An outstanding group of 54 mentors will offer remarkable depth and breadth to NSADA scholars in molecular, cellular and systems/integrative neurobiology as well as clinical and translational research. Clinical investigators will be linked not only to research mentors but also to a wealth of clinical centers and programs focused on a wide variety of neurological and neurogenetic disorders. Research development programs tailored to individual backgrounds and educational histories will be crafted for each NSADA scholar by an internal Steering Committee, an external Advisory Committee and special Mentoring Committees that will be formed for each resident. All NSADA programs will include major components of 1) advice and guidance; 2) formal course work, including formal training in research ethics and the responsible conduct of research; 3) research experience with one or more strong and committed mentors; 4) career planning assistance and monitoring and evaluation of progress.

Project Summary This competing continuation proposal will provide an opportunity for selected residents in the neurology residency training programs of the Beth Israel Deaconess Medical Center (BIDMC) and Boston Children's Hospital (BCH) to participate for 6 to 36 months in an intensive, mentored, research educational experience during the final year of residency and subsequent fellowship years. This training will be designed to prepare participating residents for successful competition for NIH funded independent mentored research awards, and will facilitate the transition from resident/fellow to clinician-scientist. Each participant will work with one of 57 mentors, who have been recruited from the faculties of BCH, BIDMC, and Harvard Medical School (HMS). All have active NIH funding and a history of training clinician-scientists. The proposed mentors cover all major areas of the clinical and basic neurosciences, and include 30 investigators from BCH, 15 investigators from BIDMC, seven investigators from HMS and five investigators from other Harvard and Harvard Hospital affiliates. Thirty of these investigators are engaged in clinical/translational neuroscience research and forty-two are engaged in basic neuroscience research. Mentors have been drawn not only from the Departments of Neurology/Neurobiology, but also from Divisions/Departments of Anaesthesia, Cell Biology, Genetics, Neurosurgery, Ophthalmology, Pathology, Radiology, Developmental Medicine, and Psychiatry. Selected resident participants will learn state-of-the-art laboratory skills and will acquire the critical expertise necessary for the conduct of responsible research. Data collected and analyzed will serve for publications as well as for future NIH proposals. The program will be governed by a Steering Committee consisting of the PD/PIs, the Department Chairs, and the Residency Directors of the participating residency programs. This Committee will work together to recruit and select trainees, to monitor their progress, and to evaluate the effectiveness of the training experience.

DESCRIPTION (provided by applicant): We propose an innovative, systems biology approach to uncover new therapeutic strategies for childhood embryonal tumors. Our project is a collaboration between labs in two separate Integrative Cancer Biology Program (ICBP) centers and a leading hospital-based translational research lab that is not within the ICBP network. Embryonal tumors are the most common central nervous system malignancies in childhood, and there is a pressing need for better therapies. Current survival rates range from 30 - 80%, and nearly all survivors have impaired neurological and neurocognitive function. Extensive genomic analysis of medulloblastomas, the most common embryonal tumors, failed to identify driver genes that could explain the origin of most tumors or suggest new strategies. Nevertheless, these tumors can be grouped into a small number of subtypes that share transcriptional patterns and clinical outcomes. We believe that it is time for a fundamentally new approach that seeks oncogenic driver pathways rather than driver genes. As many different genomic changes can all affect the same driver pathway, such pathways cannot be uncovered by looking for recurring genomic changes. Rather, we will use a systems biology approach to identify these oncogenic driver pathways. We will collect comprehensive datasets in human medulloblastoma tumors and cell lines by measuring mutations, copy number variations, mRNA expression, miRNA expression and epigenomic data. We will then construct network models identifying shared pathways altered across many patients within a subtype. Finally, we will functionally test driver pathways nominated from the network modeling. By merging these diverse genomic and transcriptional data collected from tumors of individual patients, we will have an unprecedented ability to uncover the root causes of cancer, providing new therapeutic strategies. The collective expertise of our collaboration provides a unique environment for solving this critical barrier in cancer, by combining strengths in analyzing genomic data, modeling signaling pathways and transcriptional regulatory networks and clinical expertise in embryonal brain tumors. Together, we will generate and merge all types of transcriptional, genomic and epigenomic data, extract biologically-relevant network models and experimentally validate novel drug targets.

? DESCRIPTION (provided by applicant): This competing continuation proposal will provide an opportunity for selected residents in the neurology residency training programs of the Beth Israel Deaconess Medical Center (BIDMC) and the Children's Hospital Boston (CHB) to participate for 6 to 24 months in an intensive, mentored, research educational experience during the third year of residency and subsequent fellowship years. This training will be designed to prepare participating residents for successful competition for independent mentored research awards, and will facilitate the transition from resident/fellow to clinician-scientist. Each participant will work with one of 52 mentors, who have been recruited from the faculties of CHB, BIDMC, and Harvard Medical School (HMS). All have active NIH funding and a history of training clinician-scientists. The proposed mentors cover all major areas of the clinical and basic neurosciences, and include 30 investigators from CHB, 12 investigators from BIDMC, four investigators from HMS and six investigators from other Harvard and Harvard Hospital affiliates. Thirty-three of these investigators are engaged in clinical/translational neuroscience research and nineteen conduct basic neuroscience research. Mentors have been drawn not only from the Departments of Neurology/Neurobiology, but also from Divisions/Departments of Cell Biology, Genetics, Cardiology, Neuropathology, Neuroradiology, Neurosurgery, Developmental Medicine, and Psychiatry. Selected resident participants will learn state-of-the- art laboratory skills and will acquire the critical expertise necessary for the conduct of responsible research. Data collected and analyzed will serve for publications as well as for future NIH proposals. The program will be governed by a Steering Committee consisting of the PD/PIs, the Department Chairs, and the Residency Directors of the participating residency programs. This Committee will work together to recruit and select trainees, to monitor their progress, and to evaluate the effectiveness of the training experience.

ABSTRACT This Center grant requests funding for six core facilities; Administrative, Clinical/Translational, Cellular Imaging, Mouse Neurodevelopmental Behavior, Mouse Gene Manipulation and Molecular Genetics, to support a broadly-based research program in intellectual and developmental disabilities at Boston Children's Hospital and Harvard Medical School. Our specific aims are to: 1) Maintain and introduce new ?state of the art? core facilities that can be shared by Intellectual and Developmental Disabilities Research Center (IDDRC) investigators, providing core research services not possible in a single laboratory; 2) Use the framework of the core laboratories to enhance and encourage collaboration between IDDRC investigators; 3) Provide core services to aid in the training of young investigators and trainees and 4) Use the core services provided by the IDDRC program to leverage collaboration outside the institution, particularly with other IDDRCs, to speed the development of effective clinical interventions in intellectual and developmental disabilities. The Center supports 105 research projects and 64 investigators who receive approximately $71M in external funding, $46M of which is from the NIH. Research in the Center occurs in over 125,000 sq.ft. of space in research buildings at Boston Children's Hospital and Harvard Medical School affiliated institutions in the Harvard Longwood Medical Area. Our research focuses on three programmatic areas- Basic Neuroscience, Clinical / Translational Neuroscience and Genetics- and our primary goal is to identify the causes of and develop therapies for children with intellectual and developmental disabilities. The research of this IDDRC encompasses laboratory research on fundamental processes of normal and abnormal neurodevelopment and plasticity, as well as clinical and behavioral studies directed at disorders including, but not limited to, autistic spectrum disorders, brain injury, neuro-oncology, learning and cognitive disabilities, the effects of surgery and environmental toxins on neural development, and multiple neurogenetic disorders, including those that affect neural formation, migration, specification and synaptic connectivity, as well as muscular dystrophy.

Abstract: Intellectual disability (ID) affects 1-­3% of the population, resulting in cognitive and adaptive behavioral  deficits. Many genes are associated with ID, including multiple mutations in KDM5C, an X-­linked gene which we  discovered to be a histone demethylase a decade ago. In the past funding cycle, we found that KDM5C patient  mutations  reduce  both  protein  stability  and  catalytic  activity.  We  showed  that  a  mouse  Kdm5c  knock-­out  (KO)  model  recapitulated  the  cognitive  and  behavioral  deficits  seen  in  human  patients.  Kdm5c  bound  primarily  at  promoters  in  terminally  differentiated  mouse  neurons  to  modulate  methylation  at  lysine  4  of  histone  3  (H3K4),  and Kdm5c loss affected the expression of neuronal genes in the amygdala. Further studies of conditional mouse  Kdm5c KOs have suggested that Kdm5c potentially plays a neurodevelopmental role. To study the function of  KDM5C during neurodevelopment in a human model, we have generated patient-­derived iPS cell lines bearing  KDM5C  mutations,  and  isogenic  lines  with  the  mutations  corrected,  both  of  which  can  undergo  neuronal  differentiation in culture. These cell lines provide an unprecedented opportunity to explore the effects of KDM5C  in a well-­defined and experimentally accessible human developmental system.    The  goals  of  this  work  are  to  obtain  a  comprehensive  molecular  and  cellular  understanding  of  how  KDM5C  regulates human neurodevelopment. We will conduct high-­resolution time-­course analyses to determine exactly  which  stages  of  neuronal  differentiation,  and  which  cell  types,  are  compromised  by  KDM5C  mutation.  We  will  investigate the functionality of KDM5C-­mutant neurons by interrogating the expression of synaptic markers and  electrophysiology. Because some aspects of brain development (e.g. formation of multiple cell types and their  organization) are not recapitulated in a 2D culture system, we will use 3D human brain organoids generated from  mutant  and  corrected  iPS  cells  to  investigate  the  roles  of  KDM5C  in  promoting  brain  growth,  generating  the  appropriate  diversity  of  neural  cell  types,  and  facilitating  neuronal  network  connectivity.  Our  findings  will  be  validated in vivo during embryogenesis of WT and Kdm5c KO mice, and the critical timing and location of Kdm5c  activity determined by expressing or deleting Kdm5c in specific stages/ cell types. To investigate the molecular  mechanisms  of  the  neuronal  differentiation  defects  in  KDM5C  mutant  cells,  we  will  determine  transcriptional  profiles  of  mutant  and  corrected  iPS  cells  during  differentiation  in  2D  and  3D  cultures.  Because  the  critical  genomic  targets  of  KDM5C  (e.g.  promoters,  enhancers)  during  neurodevelopment  are  unknown,  we  will  map  KDM5C  binding  sites  genome-­wide  during  2D  neuronal  differentiation,  and  determine  how  KDM5C  mutations  alter  the  chromatin  landscape.  Finally,  we  will  use  biochemical  approaches  to  identify KDM5C  regulators,  and  investigate their effects on KDM5C function and neurodevelopment. These studies will shed critical new light on  KDM5C function during neurodevelopment, and provide a basis for designing therapeutic strategies to treat ID.  

ABSTRACT Our overarching vision of the Boston Children?s Hospital and Harvard Medical School Intellectual and Developmental Disorders Research Center (IDDRC) is to improve the lives of individuals with IDD with timely and efficient translation of scientific research through collaboration among our institutions? exceptional investigators and clinicians in partnership with the external IDD community. To achieve our broad vision, we organize the Center?s research around four clearly defined themes: 1) Discovery of genetic and non-genetic causes of IDD; 2) Determination of the cellular bases of IDDs using advanced imaging and analysis tools; 3) Identification of translational phenotypes in animal models of IDD to validate therapeutics; 4) Accelerated translation of research discoveries into new prevention and treatment strategies for IDDs. The Center currently supports 106 research projects and 68 investigators through the Administrative Core and the four scientific Cores. The Administrative Core is the hub of the Center as it provides both scientific and administrative leadership which promotes synergistic, interdisciplinary interactions that address IDD-related issues at multiple levels, trains the next generation of young investigators and facilitates outreach and dissemination of IDD research to diverse audiences. The Genetic Analysis and Editing Core (GAEC) provides access to the latest technological advances both in genetic analysis and in gene editing. The Cellular Imaging Core (CIC) facilitates the study of cellular and circuit biology through state-of-the-art imaging and image analysis services which enable visualization of fixed tissue, in vitro organ explants and in vivo awake behaving model organisms. The Animal Behavior and Physiology (AB&P) Core provides investigators with access to a wide range of validated technologies and scientific expertise for in vivo rodent behavioral, biochemical and physiological measures in a well-controlled and rigorous preclinical setting. Finally, the Clinical Translational Core (CTC) provides full access for IDDRC PIs to all services required for translation of research discoveries into clinical innovation; this ranges from biosample collection and storage, generation of patient-derived stem cell models of diseases, drug screening platforms on the preclinical side to neurobehavioral and electrophysiological assessment of IDD patients of different age groups as well as statistical and regulatory support of clinical trials. The Cores all interact through shared projects, providing complementary expertise and tools to address unique aspects of central scientific questions, and the Core directors meet to exchange ideas and optimize resource utilization in the monthly Executive Committee meetings. This integrated approach aims to enhance the translational potential of basic research in IDD by putting patients at the center of the drug discovery cycle starting with genetic and molecular screens through clinical trials. The Center engages frequently with patient- advocacy groups and the IDD community in a bi-directional manner to ensure that their needs and concerns steer the Center?s efforts.