Vittorio Gallo - US grants

A major goal of developmental neurobiology is to define mechanisms that regulate the developmental potential of neural progenitor populations under normal physiological and pathological conditions. The subventricular zone (SVZ) contains a mosaic of neural progenitor cells (NPCs), which maintain their mitotic and differentiation potential throughout their life span. NPCs provide the brain with the potential to replenish damaged glia and neurons through spontaneous gliogenesis and neurogenesis. Glutamic acid decarboxylase 65 (GAD65) and Doublecortin (Dcx)-expressing cells constitute a major progenitor population of the adult SVZ ( ). Under normal conditions, these cells migrate along the rostral migratory stream toward the olfactory bulb to generate inhibitory interneurons. Recent studies demonstrating the ability of NPCs to change their lineage potential after genetic manipulation have led us to hypothesize that pathological stimuli may also induce lineage plasticity in native NPCs. Our preliminary analysis in GAD-GFP and Dcx-GFP transgenic mice indicate that: i) after lysolecithin (LPC)-induced focal demyelination of the corpus callosum (CC), or after perinatal hypoxia (HX), GAD65-GFP+ and Dcx-GFP+ progenitor cells migrate from the SVZ into the CC; ii) GAD-GFP+ and Dcx-GFP+ cells display lineage potential plasticity in CC, as they generate OLs after demyelination or after HX, and iii) chordin is upregulated in the SVZ after demyelination and promotes oligodendrogenesis from GAD-GFP+ and Dcx-GFP+ progenitors in culture and in vivo. Based on these findings, we plan to investigate whether lineage plasticity of both adult and perinatal SVZ neuronal progenitor cells occurs under pathological conditions involving the white matter, and to identify endogenous signals that promote oligodendrogenesis from GAD- and Dcx- expressing progenitors of the SVZ. In order to compare early postnatal and adult neuronal progenitor responses and signals, we will use two distinct animal models of white matter injury, i.e. toxin-induced focal demyelination in adults and perinatal HX. First, we will establish whether SVZ neuronal progenitors generate oligodendrocytes after focal demyelination in the adult brain. Second, we will determine whether SVZ neuronal progenitors generate oligodendrocytes after HX in the early postnatal brain. Finally, we will identify endogenous cellular signals that promote oligodendrogenesis from SVZ progenitors in the early postnatal and adult brain. Together, these studies will not only shed light on crucial cellular and molecular mechanisms of oligodendrogenesis from different NPC populations, but might also lead to the development of new therapeutic approaches aimed at lessening the long-term neurological sequelae of white matter injury.

DESCRIPTION (provided by applicant): Remyelination in the CNS requires oligodendrocyte progenitor cells (OPCs) to divide and migrate towards areas of damage. Some cell migration does occur, but it ultimately ceases and is inadequate. Molecular strategies aimed at promoting post-injury migration of endogenous OPCs could thus improve myelin repair. In the brain, the endothelin (ETs) peptides are synthesized by endothelial cells, neurons and astrocytes. ETs, in particular ET-1, have been implicated in astrocyte and Schwann cell proliferation and maturation; however, their biological effects on oligodendrocyte development are unknown. In both cultured cells and in tissue slices, we will test the hypothesis that ET-1 regulates oligodendrocyte development, namely OPC proliferation, migration and differentiation. Our preliminary results using an in vitro assay indicate that ET-1 promotes OPC migration, but does not affect cell proliferation. We therefore propose to pursue the following avenues of investigation: first, we will establish whether expression of the two main ET-receptors (ET-Rs) found in the brain, ETA-R and ETB-R, is developmentally regulated in cells of the oligodendrocyte lineage in culture and in vivo. With selective ET-R antagonists, we will also define the main signal transduction pathways associated with ETA and ETB-R-activation in OPCs, and their contribution to the activation of specific transcription factor targets. We will investigate the protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and p38-mitogen-activated protein kinase (MAPK) pathways. Second, we will identify the ET-R subtype(s) mediating the functional effects of ET-1 on OPC migration, and define the influence of ET-1 on OPC differentiation. Third, we will extend our studies performed in culture to a more intact system. We will apply multiphoton microscopy to analyze the role of the ET system in OPC proliferation and migration in postnatal brain slices prepared from mice expressing the green fluorescent protein in cells of the oligodendrocyte lineage. Our aim is to define signals that promote OP cell migration and repopulation of demyelinated areas. Our studies will: i) define a role for ET-1 and ET-Rs as migratory signals in oligodendrocyte development, and ii) determine whether novel means of enhancing OP migration could be explored to facilitate myelin repair.

DESCRIPTION (provided by applicant): A cell repair strategy aimed at restoring neuronal function in neurodegenerative disease involves transplantation of neural stem cells either directly into the lesions, or into brain areas from which the grafted cells can migrate toward the desired anatomical destination. The success of this strategy depends on many factors, including the cellular and developmental properties of the neural stem cell/progenitor population used. Cells expressing the proteoglycan NG2 represent the largest population of mitotically active neural progenitors in the postnatal brain. In this proposal, we will isolate and transplant NG2-expressing cells from the (3-actin EYFP transgenic mouse, in which EYFP is uniformly expressed in all NG2+ cells throughout development. We have found that perinatal NG2+/EYFP+ progenitors isolated by fluorescence-activated cell sorting (FACS) are multipotential, i.e. can generate neurons, oligodendrocytes and astrocytes. FACS purified perinatal NG2+/EYFP+ progenitors grafted into the lateral ventricle (LV) of perinatal wild-type mice generate different subtypes of GABAergic intemeurons in distinct regions of the hippocampus, and oligodendrocytes in white matter and cerebral cortex. We propose to use the beta-actin-EYFP mice to establish a transplantation paradigm that will generate hippocampal inhibitory intemeurons from NG2+ progenitors purified from the subventricular zone and hippocampus of perinatal brains, and grafted into the brain of perinatal or adult mice. To study the cell and lineage potential of grafted NG2+ cells, we will employ a multidisciplinary approach, involving cellular, molecular and electrophysiological techniques, to define the phenotypes of hippocampal neuronal populations generated by NG2+ progenitors. By using patch-clamp recording techniques, we will determine whether the EYFP+ neurons derived from grafted NG2+/EYFP+ progenitors are synaptically integrated and display synaptic plasticity. Altogether, these studies will define a novel experimental paradigm aimed at repairing hippocampal GABAergic intemeuron damage in various pathological states, such as epilepsy, stroke, and ischemia

DESCRIPTION (provided by applicant): The purpose of this application is to establish a post-doctoral training program in the Mental Retardation and Developmental Disabilities (MRDD) Research Center at the Children's Research Institute, of Children's National Medical Center (CNMC) in Washington, D.C. The focus of this program will be on five areas of inquiry associated with MRDD: autism, learning disabilities (developmental dyslexia), traumatic brain injury, epilepsy, and urea cycle disorders. This will be a multidisciplinary effort that draws on 14 faculty preceptors in the areas of neuroscience, neurobehavioral science and genetics from seven Departments at CNMC and Georgetown University School of Medicine. CNMC is particularly well positioned to lead this program, based on: i) its strengths in basic, translational and clinical research, and in mentorship in all the proposed areas of inquiry;ii) its established strong collaborations with Georgetown University, and iii) its leading role in a number of NIH Center Grants focusing on conditions causing MRDD. We propose to enter 2 postdoctoral trainees (MD and PhD) per year in the 3 year training program. A unique aspect of the training will be that each area of inquiry will have genetic, neuroscience and neurobehavioral components, and each fellow will chose a mentor's laboratory that focuses on one of these areas but will rotate through each component to acquire Interdisciplinary training in the specific disorder being studied. The objectives of the program are to encourage MD's to develop as researchers in the field of MRDD and to stimulate greater participation of promising PhD researchers in this area. Each trainee will be carefully mentored through the program to ensure that he/she fully exploits the range of opportunities of the program.

DESCRIPTION (provided by applicant): Permanent demyelination, the major pathology of multiple sclerosis, involves the disruption of axonal myelin and neuronal damage. Effective repair requires the repopulation of lesions with oligodendrocytes capable of differentiating and remyelinating damaged neurons. Mature oligodendrocytes originate from progenitor cells (OPCs), whose developmental pathways are poorly understood. Using DMA microarray analysis, we have established a developmental gene expression profile of EGFP+ oligodendrocyte lineage cells FACS-purified from postnatal day 2 (P2)-P30 CNP-EGFP mice. A gene sub-cluster identified by a peak of expression at P15 was found to contain the SoxF family members (Sox7/Sox17/Sox18), whose expression and function in oligodendrocytes are unexplored. Developmental expression of Sox17 displayed temporally-related coordinate clustering with myelin genes. In FACS-purified EGFP+ cells, Sox17 expression increased between P2 and P15, coincident with the onset of myelination and downregulation of cell cycle genes, and decreased thereafter. In white matter, Sox17 was expressed at higher levels in O4+ cells. In cultured OPCs, a transient increase of Sox17 expression correlated with i) increases in p21Cip1/Waf1, p27Kip1 and MBP expression, and ii) a subsequent decrease in proliferating cell nuclear antigen. siRNA-induced Sox17 downregulation increased OPC proliferation and decreased differentiation. Reporter assays showed that Sox17 can activate the MBP promoter. We plan first to characterize the expression and regulation of Sox17 in OPCs cultured under conditions that promote cell differentiation, to associate changes in Sox17 expression with specific stages of OPC development and the expression of cell cycle regulators. We will define a functional role for Sox17 in cell cycle regulation and OPC maturation by studying the effects of: i) Sox17 siRNA on OPC gene expression in culture by microarray analysis and ii) Sox17 overexpression in vivo in a CNP-Sox17-IRES-EGFP transgenic mouse strain. We will identify Sox17-responsive regions in the p21 and MBP gene promoters by reporter assays, and analyze DMA binding activity at these sites. Finally, we will characterize Sox17 expression in the oligodendrocyte lineage in vivo during normal development and during remyelination following chemical demyelination. Our proposed studies hope to shed light on crucial regulators of oligodendrocyte maturation that are potentially amenable to therapeutic intervention.

DESCRIPTION (Provided by applicant): The Children's National Medical Center (CNMC), in collaboration with Georgetown University Medical Center (GU) and the George Washington University School of Medicine and Health Sciences (GWU), is submitting a competitive renewal for its Mental Retardation and Developmental Disabilities Research Center (MRDDRC). This center focuses on developmental and cellular aspects of brain development and dysfunction and on the molecular basis of genetic diseases causing intellectual and other developmental disabilities. The 84 NIH/NSF funded grants that form the investigator group for the MRDDRC have annual direct costs of more than $21 million, over $7 million from NICHD. The projects cover 23 of the 31 areas of focus in the MRDDRC RFA: HD-05-030. A total of 69 faculty members are affiliated with the MRDDRC as Center Investigators. These faculty represent 23 departments/divisions from the collaborating institutions. We propose to support an Administrative and 5 scientific cores: Molecular Genetics, Proteomics and Biochemical Analysis Core (MGPBAC);Cellular Imaging Core (Cl);Neuroimaging Core: Neurobehavioral Evaluation Core (NEC);and Biostatistics and Informatics Core (BIC). The Director of the MRDDRC, Mark L. Batshaw, M.D., was previously the founding Director of the Children's Hospital of Philadelphia MRDDRC (1990-1998). He will also direct the Administrative Core that functions as the hub of the Center, providing strategic planning, financial oversight and administrative services to cores and users. MGPBAC provides access to and training in advanced molecular biologic technologies including expression microarrays, quantitative PCR and automated DNA sequencing, proteomic technology and biochemical analysis by tandem mass spectrometry. The Cellular Imaging Core focuses on classic neuropathology (light and EM), immunohistochemistry, and innovative techniques such as confocal microscopy. The Neuroimaging Core provides access to structural and functional brain imaging and image processing. The Biostatistics and Informatics Core assists investigators in study development, use of statistical methodology, and data storage management and analysis. Finally, the Neurobehavioral Evaluation Core provides support for neuropsychological testing and development of behavioral tasks for functional imaging. With the continued support of the MRDDRC, we expect to further enhance research productivity in the area of intellectual and other developmental disabilities.

A. OBJECTIVES This core was initially called the Cellular Neuroscience Core and had sites at CNMC, Georgetown and GW. It was directed by Dr. Alan Faden, a neurologist and neuroscientist. There was a broad range of services offered including neuropathology, immunohistochemistry, access to neural cell culture and cell lines, preparation of samples, and confocal and electron microscopy. In the review of our proposal in 2001 the study section recommended that the scope be narrowed and that cutting edge technology be purchased. We have responded to both suggestions. We found that the most valued services were electron and confocal microscopy, and this is now the focus of the core which has been renamed Cellular Imaging. At its founding there was no site at CNMC despite the principal users being there. We therefore recruited Tarik Haydar, Ph.D. from Yale to develop a section of the core focusing on confocal and time-lapse multiphoton microscopy at CNMC. We also purchased, through institutional funds, cutting edge cellular imaging equipment. With the planned sabbatical of Dr. Faden, Dr. Haydar was recently elevated to core co-director with Dr. Gallo. In this competitive renewal application we propose that the Core continue to focus on providing MRDDRC investigators with access to sophisticated cellular imaging instrumentation and with the requisite training for cellular/subcellular visualization and analysis. The core houses and maintains high-performance equipment for manipulating, visualizing, and analyzing nervous system cells. We have transmission electron microscopy and instrumentation for optical imaging, as well as associated image analysis software, permitting evaluation of neuronal activity within functional ensembles of neurons in culture, brain slices, or intact brain. Facilities also include equipment for obtaining data based on histochemistry, immunocytochemistry, in utero electroporation, and transplantation of neural progenitor cells.

DESCRIPTION (provided by applicant): Children's National Medical Center (CN) is submitting a competitive renewal for its Intellectual and Developmental Disabilities Research Center (IDDRC). The focus of this Center is on cellular, physiological and neurobehavioral aspects of brain development and dysfunction, and on the mechanisms of genetic diseases causing intellectual and other developmental disabilities. The Center comprises 151 NIH and other federally funded projects, with a total annual direct costs of more than $50 million, of which over $12 million is from NICHD. The projects that are part of the IDDRC cover 27 of the 31 areas of focus in the IDDRC RFA:HD-10-022. A total of 96 faculty members from 25 departments/divisions at the participating institutions (CN, GU, GWU, HU) are affiliated with the IDDRC as Center Investigators. We propose to support an Administrative Core (Administrative Core) and 5 scientific cores: Biostatistics and Informatics (Biostatistics and Informatics Core), Cellular Imaging (CIC), Proteomics and Genomics (PGC), Neuroimaging (NIC) and Neurobehavioral Evaluation (Neurobehavioral Evaluation Core). The Director of the IDDRC, Vittorio Gallo, Ph.D. is also Director of the Centerfor Neuroscience Research at CN, where he developed a research program on neurodevelopmental disorders. He will direct the Administrative Core that functions as the heart/coordinating core of the entire IDDRC. The Administrative Core will promote growth of the IDDRC by providing advocacy, management and administration, communication and training, compliance and quality assurance, strategic planning and recruitment. Biostatistics and Informatics Core provides a broad range of study design, information management, and statistical support services extending from the design of research studies to publication of results. CIC assists investigators with a broad array of imaging tools for cellular analysis, and provides advisory and training services. PGC provides advanced genomics and proteomics technologies, genetic counseling as well as specialized statistical analyses for DNA, mRNA and proteomics studies. NIC provides support for image acquisition, image sequence selection and implementation, paradigm design for functional studies, software development, image processing/analysis. Neurobehavioral Evaluation Core provides sophisticated neurobehavioral and neuropsychological research support to define developmental and behavioral phenotypes and to develop or validate new methods for this purpose. As demonstrated in the past funding cycle, the continued support to our IDDRC will further enhance recruitment of new IDD investigators, and will promote interdisciplinary IDDR to generate innovation, resulting in major advancements in IDDR.

The Genomics/Proteomics Core (GPC) of the IDDRC at Children's National Medical Center (Children's National, CN) has been highly active, with over 7,000 samples processed during the last award period and support for more than 40 investigators. Two key aspects of the success of the Core include innovation (new equipment and assays offered each year), and full service (including assistance with experimental design, sample processing, technical support and training, bioinformatics assistance, and help with grants and manuscripts). New technologies and services that will be implemented during the proposed new award period include emulsion PCR (RainDance custom panels), epigenomics scans (both Illumina and Pacific Biosciences single molecule whole genome), nextgen sequencing (Illumina and Pacific Biosciences), glycoproteomics, and phosphoproteomic scans. Objectives of the Core are: 1. Supply full service and innovative genomic and proteomic research tools to IDDRC investigators from project planning to data interpretation and manuscript writing with no service fees (users pay only for the cost of reagents). 2. Rapid acquisition and implementation of cutting-edge high-content technology. 3. Support of multi-scale projects inclusive of DNA, mRNA, microRNA, epigenomics, proteomics, and integrative and systems biology bioinformatics approaches. 4. Offer group and individualized training to increase Core capacity and provide flexibility to investigators. 5. Leverage Core support to users through synergism and integration of commensurate NIH- and philanthropy-supported genomics/proteomics/bioinformatics Cores: NICHD's National Center for Medical Rehabilitation Research (NCMRR-DC) Core for Molecular & Functional Outcome Measures in Rehabilitation Medicine, the CTSI-CN Genomics/Proteomics Core for Children's Translational Medicine, and the Sheikh Zayad Institute for Surgical Innovation. These synergisms enable introduction of advanced new and costly equipment into the IDDRC Genomics/Proteomics Core, and offer unique and substantial opportunities for resource sharing and collaboration for IDDRC investigators. In the previous Summary Statements of the 2006 review, the Genomics and Proteomics Core (previously called MGPAC) received an outstanding evaluation. The previous reviewers felt that bioinformatics support could be bolstered further, and better integrated with the Statistical Core. Towards this end, we have added two faculties to the Genomics/Proteomics Core personnel: statistical geneticist Dr. Gordish-Dressman and biomedical engineer and bioinformatics specialist Dr. Wang, and have ongoing meetings with the Statistical Core to integrate efforts. We have also submitted grants together with bioinformatics researchers at Georgetown University and Virginia Tech to continue our methods development work in bioinformatics support. Our Core experienced considerable expansion in the number of samples and complexity of user projects over the last funding cycle. A formal survey of anticipated user needs showed emerging strong demand for epigenomics, miRNA expression profiling, ELISA bead assays, next-generation sequencing, quantitative proteomics using stable isotope labeling and label free strategies, phosphoproteomics and glycoproteomics. To meet these needs, we significantly increased service offerings, implemented new equipment and new expertise. Our emphasis remains on offering highly specialized, innovative, full service support for a broad spectrum of genomic, proteomic and bioinformatic technologies.

A. Objective The IDDRC Neuroimaging Core provides support for all aspects of human neuroimaging research necessary for the IDDRC mission. The core supports a growing group of investigators that have unique experience in imaging normal brain development and children with a wide range of intellectual and developmental disabilities. We also place an emphasis on young investigator development. Advanced neuroimaging technology provides a critical means for evaluating the structural anatomy, biochemistry, and functional anatomical abnormalities that underlie developmental disabilities in addition to identifying disease/disorder biomarkers. Furthermore, imaging provides objective measures for examining the consequences of these disorders on brain structure and function, and providing a non-invasive means for exploring human neuroplasticity, adaptation in response to disease, and response to neuro-intervention. The Neuroimage Core: 1. Provides consultation in identifying efficient and effective imaging protocols to investigate experimental questions in specific clinical populations and during normal development 2. Provides consultation in processing and data analysis of structural and functional MRI scans in studies designed to detect alterations in brain structure, connectivity, or regional cortical activation 3. Provides access to and specific training in the use of the 3.0T MRI systems for structural, functional, and metabolic imaging 4. Supports investigators in developing and utilizing functional imaging tasks (paradigms) focusing on visual, auditory, and sensorimotor processing, memory, language, executive function, and visuospatial skills, among others 5. Implements and supports techniques to desensitize anxious research subjects to the staff, settings, equipment and procedures associated with neuroimaging 6. Assists in selection, and implementation, and interpretation of statistical plan (with statistical core) 7. Performs imaging assessments of children, adolescents and adults 8. Assists in manuscript preparation for studies involving imaging 9. Assists in the monitoring of outcomes (efficacy, safety) in clinical trials utilizing imaging measures 10. Provides support and training for new investigators (and research assistants/personnel) entering the neuroimaging field.

A. Objective The primary objectives of the Neurobehavioral Evaluation Core (NEC) are (a) to provide sophisticated neurobehavioral/ neuropsychological research support to IDDRC projects, and (b) to develop/validate new measures and methods for this purpose. Scientific advancement of our understanding of complex neurobehavioral disorders of development is greatly facilitated by our ability to define reliably and precisely developmental and neurobehavioral phenotypes. Measurement of neurobehavioral and cognitive variables is crucial in studies of both disease and normal development and over the past 5 years, the NEC has supported 38 IDDRC projects resulting in 54 peer-reviewed publications. The aims of these studies have focused on developmental, cognitive, and behavioral outcomes in children who have a variety of neurogenetic, neurodevelopmental, or acquired neurological disorders that affect cognition and behavior. In addition to a rigorous study of the underlying medical condition (e.g. urea cycle disorders or epilepsy. Table 1: Project#9,13), these investigations aim to describe the impact on the child's neurocognitive and neurobehavioral development. Newer neuropsychological tests and methods allow a more in depth focus into cognitive developmental factors that include attention, executive function, speed of processing, and memory. In addition, the explosion of the developmental neuropsychological knowledge base linking underlying neurological developmental disorders with behavioral and cognitive development has further fueled the need for pediatric research to include sophisticated models of neurobehavioral and/or neuropsychological function. In turn, this requires the continued development of new methods to measure neuropsychological functioning. The primary goal of the NEC is to improve access to state of the art measures and to enhance the overall quality of neurobehavioral research being conducted throughout the IDDRC. The NEC supports and enhances studies that can benefit from the examination of neuropsychological and neurobehavioral variables, enhancing our understanding of the functional outcomes of medical and neurological diseases and disorders. The Core is able to bring state of the art methods and measures to these studies, improving efficiency by providing access to expert personnel and/or testing materials that maximize cost effectiveness. The NEC provides (1) access to an extensive battery of neuropsychological testing services, (2) consultation to IDDRC investigators on the proper design, use and interpretation of neurobehavioral/neuropsychological measures in research studies, (3) training of investigators and their staff in the use of these measures, and (4) assistance in the administration of neurobehavioral tasks. This increased sophistication in neurobehavioral assessment allows investigators to assess specific, yet complex, neuropsychological processes. These methods can be joined with studies exploring etiologic factors at the genetic level (utilizing the Molecular Genetics Core) and neuropathophysiologic level (using the Neuroimaging Core). As our Core is also involved in developing novel test materials, especially those focusing on executive function, memory and processing speed, the Core benefits the entire IDDRC network. Several issues were raised regarding the NEC in the 2006 renewal that have been explicitly addressed. These were: i) time allocation and prioritizing services; ii) specifics regarding collaboration with other Cores, and iii) testing provided in other languages than English. Time Allocation As a new Core established in 2006, the NEC has actively worked to establish its role and presence with a variety of research protocols. In order to deliver the array of services, especially direct testing support, we prioritize studies in terms of the particular study's needs and Core resources. Each study is evaluated with respect to its needs from the Core. The first service option is study consultation. The next level of service involves training staff on specific neuropsychological tests or methods. Some studies may need greater direct service, requiring the third service option, which is assisting with data collection. Finally, some studies, such as a K award with fewer resources, will receive the fourth service option, a relatively greater amount of direct service. A study with greater resources (R01 or center grant) would receive less direct service and more consultative/training service. Studies with lower service needs (e.g., multi-center study with a low number and/or sporadic recruitment of study participants, such as the Cool Kids protocol. Table 1: Project#37) would also receive direct service, assuming no direct study staff to perform the testing is available. Finally, projects that require a significant and ongoing direct involvement of the NEC staff provide support within the grant for this involvement, allowing the NEC to expand its staff to provide this service (e.g. Table 1, Proj. 9). Collaboration with Other Cores There is a strong collaboration between the NEC and a number of the other IDDRC Cores. NEC develops activation task protocols with the NI Core and works closely with the BI Core in validating new studies. Many of the genetically based projects involve close collaboration between the GP Core and NEC to establish genotype/phenotype relationships. In many of these studies, key clinical outcomes involve neurobehavioral or neuropsychological performance, necessitating reliable and valid measures. These collaborations have markedly advanced since the establishment of the NEC 4 years ago. Testing in Other Languages This is clearly a challenging issue in all of research as many of the standard neuropsychological tests have limited validated foreign language translations. In our DC population, the principal second language is Spanish. The NEC has purchased and maintained Spanish translations for a variety of tests and protocols. In addition, there is the availability of a Spanish-English bilingual neuropsychologist within the Division of Pediatric Neuropsychology for consultation on these issues when necessary. For verbal interviews in a foreign language, a translation service can be accessed. Nevertheless, valid neuropsychological/cognitive assessment of non-English speakers remains a challenge for many IDDRC studies, and the NEC plays an important role in educating investigators about the challenges and advising them on their options. For some studies, the only realistic option is to require English fluency.

Long term consequences of white matter damage by perinatal hyperoxia PI: Vittorio Gallo, PhD PROJECT SUMMARY Survivors of premature birth frequently develop cerebral palsy and cognitive impairment. The primary pathological hallmark is white matter (WM) atrophy resulting from loss of myelin and oligodendrocytes. The cellular pathophysiology underlying the delayed development of WM is not fully understood, but possible causes include hypoxia-ischemia and perinatal infection which impact vulnerable neural cells. High tissue oxygen tension or hyperoxia (HO) has also recently been reported to lead to poor neurological outcome. Premature infants express lower levels of antioxidant enzymes than term infants and experience a greater than two-fold change in oxygen tension at delivery. This exposes WM oligodendrocyte progenitors (OP) to hyperoxic injury, and therefore elucidation of mechanisms of WM damage is critical to strategies for improving neurological outcome. The proposed aims will address the overall hypothesis that delayed WM development after neonatal HO leads to persistent abnormalities in myelin sheath formation, axonal conduction and functional behavior. We have established a mouse model of neonatal HO injury, in which exposure of newborn pups to 80% oxygen causes i) initial myelin deficiency in the WM, ii) loss of progenitors and mature oligodendrocyte cells, and iii) subsequent recovery of myelin protein and oligodendrocytes. However, when WM is analyzed by diffusion tensor imaging later in adulthood, significant abnormalities in corpus callosum are detected which indicate long term changes in myelination. In addition, we have found dyscoordinated regulation of myelin protein expression and reduced Contactin-associated protein in the axon after apparent recovery when oligodendrocyte numbers had returned to control levels. Based on these findings, we plan to analyze progression of the HO injury to characterize the time course of myelin loss and recovery, and determine whether axon damage is observed. We will perform immunochemical analysis for multiple myelin and axonal proteins, and electron microscopy to investigate possible ultrastructural changes in myelin integrity and axon-glial interaction after delayed WM development. We will also determine the effect of HO damage on i) physiological function of axons of the corpus callosum by measuring axonal conduction properties such as velocity and compound action potentials, and ii) changes in motor behavior using the motor skills sequence task test. These studies will not only shed light on long-lasting HO-induced structural and functional alterations in WM resulting from delayed myelination, but also identify changes in oligodendrocyte proteins which may underlie the long-term neurological sequelae of premature birth.

The Administrative Core (AC) of the District of Columbia Intellectual and Developmental Disabilities Research Center (DC-IDDRC) provides leadership and coordinates all DC-IDDRC activities at the four partnering institutions: Children's National Hospital (CNH), George Washington University (GWU), Georgetown University Medical Center (GU) and Howard University (HU). This core plays a crucial role to ensure that our DC-IDDRC is an efficient, effective, cohesive and integrated interdisciplinary program for the advancement of IDDR. The primary objectives of the AC are to provide: (a) Overall vision, strategic planning and direction of the DC- IDDRC and supervision, integration and management of the research cores; (b) Educational and training programs; (c) A home for the IDDR community (?centeredness?) at each of the member institutions by bringing together a diverse group of investigators; (d) Dissemination of new knowledge; (e) Advocacy and engagement with philanthropic agencies/foundations, governmental agencies and patient advocacy groups to enhance support for IDDR; (f) Collaborations with other IDDRCs within the Network. Our specific aims to reach these goals include: (a) assure that all DC-IDDRC investigators have access to state-of-the art, high quality, cost- effective research core support; (b) promote integration of basic/applied, translational and clinical investigation; (c) closely monitor effectiveness, cost/charge backs and needs for support of research cores; (d) ensure quality control, coordination and integration of services between cores; (e) evaluate and develop new technology required by DC-IDDRC investigators; (f) enhance visibility of core laboratory services to our research community and potential users, and promote recruitment of new investigators at DC-IDDRC institutions; (g) assist DC-IDDRC investigators in familiarizing themselves with core functions; and (h) provide leadership and be an active participant in the national IDDRC Network. The DC-IDDRC educational goals include: (a) supporting DC-IDDRC seminars and related lecture series; (b) attract and recruit outstanding junior investigators to IDD-related research programs through training grants and fellowships that are specifically targeted to this cohort; and attract/recruit senior investigators into IDDR; (c) disseminate and communicate DC- IDDRC research findings; and (d) support/promote the overall goals of the DC-IDDRC and of the IDDRC Network through networking and advocacy. In summary, the AC will play a pivotal role in future development and expansion of the DC-IDDRC through Administration, Research, Education and Training, Advocacy, and Dissemination.

DESCRIPTION (provided by applicant): This application is to request support for the 2014 Gordon Research Seminar (GRS) and Conference (GRC) on Myelin, to be held in Ventura, California from March 22 to March 28, 2014. The 2014 Myelin GRC will reflect the dynamic expansion of knowledge in myelin biology, physiology and pathology by bringing together researchers who are at the cutting edge of myelin biology to discuss the latest and most significant progress in the field. Since the last Myelin GRC in Barga, Italy in April 2012, major new insights have emerged concerning: i) reprogramming progenitor and pluripotential stem cells into myelinating glia; ii) the importance of myelinating glia in axonal support, and iii) the role of myelination in behavior during development and in the adult. There is also increasing appreciation of axonal signals in regulating developmental myelination and remyelination. The scientific program of the 12th GRC Myelin: Biology, physiology and pathology of myelinating glia is designed to create the best forum to bring together investigators in the field to emphasize these and other cutting-edge research, to foster a dynamic and vigorous exchange of ideas and to expand research through synergistic interactions among attendees. Emphasis will be on the relationships between myelin development, neonatal and adult disease and regeneration. We will highlight basic cellular and molecular mechanisms of significant clinical relevance. We have focused on including newly independent scientists to promote and support the next generations of myelin investigators. A number of young investigators will give regular and short talks, together with more senior investigators who will additionally serve as session chairs. We have made an effort to maintain appropriate gender balance among the roaster of speakers. The newest and most cutting-edge posters will be selected for short talks. The 2014 GRC will also begin with a Gordon Research Seminar (GRS) on myelin, which builds on the exceptional success of its first meeting that preceded the 2012 GRC. We expect more than 50 young investigators, including graduate students and postdoctoral fellows to attend the GRS and the GRC. The focus of this year's GRS is on myelin development and disease, and will provide a unique venue for young investigators to interact, present their most current research and develop collaborations. As funds will permit, we will offer support for travel and registration to trainees in myelin biology. All participants to the GRC will contribute an oral or poster presentation. We will emphasize poster sessions as key elements in promoting and nurturing productive interactions among scientists with different skills and backgrounds. This and all other activities included in the GRC program will contribute to reach the main goals of the conference, i.e. to provide stimulate and accelerate progress in the field of myelin biology, physiology and pathology.

DESCRIPTION (provided by applicant): Damage to the myelin sheath (demyelination) in multiple sclerosis (MS) retards the propagation of nerve impulses, with devastating neurological consequences. Remyelination restores myelin through the proliferation, migration and maturation of oligodendrocyte (OL) progenitor cells (OPCs). The failure of remyelination by OPCs in MS lesions is not understood, and the identification of inhibitory factors in lesions is critical to improve repair. Demyelination results in reactive gliosis, an astrocytic response that impedes OL development and regeneration. Strategies that target inhibitory factors of reactive gliosis could improve remyelination by OPCs. We have shown that: i) Endothelin-1 (ET-1) is expressed at high levels by reactive astrocytes in MS lesions; ii) in an animal model of demyelination, astrocyte-derived ET-1 delays OPC differentiation and remyelination, and iii) inhibition of ET-1 signaling accelerates myelin repair. ETA- and ETB-receptors (ET-Rs) are expressed by astrocytes and OPCs, but the specific contribution of each receptor and each cell type to the remyelination phenotype is unknown. Our preliminary data show that pharmacological inhibition of ETB-Rs or selective ETB-R deletion in astrocytes accelerates remyelination, suggesting a major functional role for this ET-R subtype. We will use two novel conditional knock-out mouse strains, hGFAP-Cre ;ETBflox/flox and PDGF a- R ERT2 Cre ;ETBflox/flox, in which the ETB-R is inducibly ablated in astrocytes or OPCs. We will test the ERT2 hypothesis that ET-1 acts predominantly through the ETB-R on astrocytes to inhibit remyelination. We will determine: i) the functional role of ET-Rs in remyelination and determine whether the ETB-R is the predominant functional receptor involved in this process; ii) the specific role of ETB-R signaling on astrocytes during remyelination; iii) the specific role of ETB-R signaling on OPCs during remyelination, and iv) whether the inhibitory effects of ET-1 on OPCs and remyelination are direct or mediated by astrocytes. Our studies will provide crucial insight into the mechanisms by which ET-1 limits remyelination, and will identify new potential therapeutic targets to promote OL regeneration and myelin repair in demyelinating diseases.

? DESCRIPTION (provided by applicant): Developmental brain injury is a major risk factor for neurological sequelae, including cognitive impairment, learning disability, Attention Deficit/Hyperactivity Disorder and cerebral palsy. Susceptibility to injury is especially high in prematurely born neonates. The cellular and physiological mechanisms underlying long-term consequences of premature birth on brain development are poorly understood, in particular damage to specific neural circuits. Diverse insults to the preterm brain contribute to injury, but little is known about the neurological effects of high tissue oxygen tension or hyperoxia (HO), which is associated with poor neurological outcome. Premature infants express lower levels of antioxidant enzymes than term infants, and lack adequate defenses against oxidative stress arising from the transition to increased oxygen tension at delivery. Our mouse model of perinatal HO-induced brain injury, using short-term exposure to high oxygen tension (80%) at P6-P8, shows delayed white matter development, disrupted integrity of axonal myelin, motor hyperactivity and impaired motor coordination. Learning disability and hyperactivity in survivors of preterm birth suggest damage to brain structures critical for memory formation. The hippocampus is a brain structure central to cognitive processing. As this brain region remains active in postnatal and adult neurogenesis, and in remodeling/synaptic plasticity, it is particulary vulnerable to insults. Our preliminary findings in the hippocampus indicate that perinatal HO generates reactive oxygen species, reduces parvalbumin- and GAD65-expressing interneuron populations, reduces GABA-ergic and disinhibits glutamatergic excitatory neurotransmission. These changes in neurotransmission, together with reduced adult dentate gyrus neurogenesis, are accompanied by adult memory and learning deficits. We therefore hypothesize that HO impairs the long-term capacity of the hippocampus for neurogenesis and remodeling, as well as development of specific hippocampal GABAergic circuitry. These changes disrupt the balance between excitatory and inhibitory (E/I) neurotransmission, which reduces synaptic plasticity and cognitive performance. Our proposed studies will test these hypotheses in two Specific Aims. In Aim 1, we will determine how HO attenuates the long-term neurogenic capacity of the hippocampus through cellular and gene expression changes. We will also perform electrophysiological studies to determine the effects of HO on disrupting E/I balance and the capacity for long-term potentiation. In Aim 2, we will define behavioral correlates of altered hippocampal remodeling, using tests of learning, memory and cognitive flexibility. Finally, we will determine whether pharmacological restoration of GABA neurotransmission improves E/I balance and cognitive performance following HO injury. Our study will establish functional relationships between HO-induced cellular changes, GABAergic interneuron dysfunction, long-term neurogenesis and cognitive deficits in a developmental model of neuronal injury. These will provide insights into injury mechanisms and functional readouts for future therapeutic intervention.

Funded since 2001, the mission of the DC-IDDRC is to expand our understanding of the causes underlying intellectual and developmental disabilities (IDD), develop innovative therapies, and prevent or attenuate the full effects of these disorders, so that each child can achieve her/his full physical and intellectual potential. To realize this mission, we provide a rich environment for performing fully translational IDDR in the four collaborating DC academic medical centers (Children?s National Health System (lead), George Washington University, Howard University, and Georgetown University). In this proposal, our specific aims are: 1) To identify the causes and develop new clinical approaches for the prevention or amelioration of IDD; 2) To provide accessible, state-of-the-art and cost-effective core facilities for cohesive, multidisciplinary research and education/training in IDDR; 3) To create an intellectual home for investigators engaged in IDDR; and 4) To implement an innovative Research Project that addresses one of the five IDDR themes (Developing Biomarkers of Premature Brain Injury). The Director of the DC-IDDRC, Vittorio Gallo, PhD, is an internationally renowned neuroscientist who will continue to direct the Administrative Core that functions as the organizing nexus of the DC-IDDRC, providing management, administration, communication and training, assuring compliance and quality assurance, directing strategic planning and recruitment, and promoting the growth of the DC-IDDRC. The scientific cores provide an integrated platform and synergy for investigation as required by truly translational IDDR. The Clinical Translational Core (CTC) is designed to serve as a ?one-stop-shop? for IDDR investigators, providing assistance at each stage of the clinical and translational research spectrum and optimizing the efficient, high quality implementation of fundamental research. The Genomics and Proteomics Core (GPC) provides advanced genomics and proteomics technologies, as well as specialized statistical analyses for DNA, mRNA and proteomics studies. The Cell and Tissue Microscopy Core (CTMC) supports investigators with a broad array of advanced cellular and molecular imaging tools for state-of-the-art neuroscience studies. The Human and Animal Imaging Core (HAIC) provides scientific and technical support for in vivo and ex vivo whole brain imaging, image processing, and image analysis for both human and animal studies. The Neurobehavioral Evaluation Core (NEC) provides sophisticated neurobehavioral and neuropsychological research support to define developmental and behavioral phenotypes in humans and animals. Our hypothesis-driven project is entitled ?The vulnerable preterm cerebellum: Elucidating mechanisms and consequences of injury?. It utilizes the HAIC, NEC and CTC functions. Through these components, the DC-IDDRC will enhance the recruitment of investigators, generate innovation, and promote transdisciplinary research that together will facilitate the development, implementation, and dissemination of new diagnostic and therapeutic advances for the care of individuals with IDD.!

CORE A ? ADMINISTRATIVE CORE Vittorio Gallo, PhD Core Director Director, DC-IDDRC Children?s National Health System William Gaillard, MD, PhD Associate Director, DC-IDDRC Children?s National Health System Kerstin Hildebrandt, MSHS Director of Operations, Children?s Research Institute Children?s National Health System Nikkie Adesida, BA Project Coordinator Children?s National Health System Abstract The Administrative Core (AC) of the District of Columbia Intellectual and Developmental Disabilities Research Center (DC-IDDRC) provides leadership and coordinates all DC-IDDRC activities at the four partnering institutions: Children?s National Health System (CNHS), George Washington University (GWU), Georgetown University Medical Center (GU) and Howard University (HU). This core plays a crucial role to ensure that our DC-IDDRC continues to be an efficient, effective, cohesive and integrated interdisciplinary program for the advancement of IDDR. The primary objectives of the AC are to provide: (a) Overall vision, strategic planning and direction of the DC-IDDRC and supervision, integration and management of the research cores; (b) Educational and training programs; (c) A home for the IDDR community (?centeredness?) at each of the member institutions by bringing together a diverse group of investigators; (d) Dissemination of new knowledge; (e) Advocacy and engagement with philanthropic agencies/foundations, governmental agencies and patient advocacy groups to enhance support for IDDR; (f) Collaborations with other IDDRCs within the Network. Our specific aims to reach these goals include: (a) assure that all DC-IDDRC investigators have access to state-of- the art, high quality, cost-effective research core support; (b) promote integration of basic/applied, translational and clinical investigation; (c) closely monitor effectiveness, cost/charge backs and needs for support of research cores; (d) ensure quality control, coordination and integration of services between cores; (e) evaluate and develop new technology required by DC-IDDRC investigators; (f) enhance visibility of core laboratory services to our research community and potential users, and promote recruitment of new investigators at DC- IDDRC institutions; (g) assist DC-IDDRC investigators in familiarizing themselves with core functions; and (h) provide leadership and be an active participant in the Mid-Atlantic IDDRC Consortium with Children?s Hospital Philadelphia and Kennedy Krieger Institute, and more broadly for the national IDDRC Network. The DC-IDDRC educational goals include: (a) supporting DC-IDDRC seminars and related lecture series; (b) attract and recruit outstanding junior investigators to IDD-related research programs through training grants and fellowships that are specifically targeted to this cohort; and attract/recruit senior investigators into IDDR; (c) disseminate and communicate DC-IDDRC research findings; and (d) support/promote the overall goals of the DC-IDDRC and of the IDDRC Network through networking and advocacy. In summary, the AC will play a pivotal role in future development and expansion of the DC-IDDRC through Administration, Research, Education and Training, Advocacy, and Dissemination.

PROJECT SUMMARY Our recent transcriptome analysis of the brains of people with Down syndrome (DS), conducted from fetal stages to 40 years old, identified approximately 800 dysregulated genes across all chromosomes, each with specific temporal and regional profiles. These altered genes form co-expression networks, the most prominent of which indicates defective oligodendrocyte (OL) development and myelination. This finding is consistent with imaging studies demonstrating reduced white matter integrity in individuals with DS. In this collaborative study between the Haydar and Gallo labs, we will answer key questions regarding the timing and source of OL dysmaturation, and particularly whether cell-autonomous or non-cell autonomous mechanisms lead to altered cellular differentiation and myelination. The Aims of the project progress from defining the developmental time course of OL dysmaturation to comprehensive and integrated transplantation, behavioral and functional tests to evaluate the mechanism(s) of the defect. Whether these changes can be rescued by gene dosage normalization or by using newly identified pharmacological tools to prompt OL maturation will be studied in the last Aim.

A major cause of chronic disability in survivors of premature birth is diffuse white matter injury (DWMI) and hypomyelination. Altered development of the WM is directly associated with adverse outcomes, including cerebral palsy, cognitive delay and neurobehavioral problems. The cellular pathophysiology underlying DWMI and abnormal myelination is complex and not fully understood. WM glia, and particularly oligodendrocytes (OLs) and their progenitors (OPCs), are susceptible to injury that often occurs in premature birth. We have previously used an animal model of hypoxia (HX)-induced global WMI to demonstrate that OPCs display delayed maturation, which results in abnormal myelination and altered WM function. We have uncovered major aspects of the cellular dysmaturation pathology underlying HX-induced delayed myelination in corpus callosum, including enhanced OPC proliferation associated with decreased OL differentiation, and disrupted myelin ultrastructure. Our recent analysis of OL development in corpus callosum (CC) demonstrates that: i) the prolonged proliferative state of OPCs and delayed OL differentiation in HX is a result of changes in HIF1?- dependent expression and activity of the histone deacetylases Sirt1 and Sirt2, respectively; ii) HX reduces synaptic glutamate (Glu) release from cortical pyramidal neurons on OPCs, which normally downregulates OPC proliferation, and iii) HX compromises axonal integrity and function in SCWM, resulting in altered axon/myelin interactions. Based on these results, we now propose to test the hypothesis that HX-induced protracted WM immaturity arises from intrinsic (Sirt1 and Sirt2) and extrinsic (synaptic) dysregulation of OPC proliferation and OL maturation, thus affecting axonal integrity and function. Firstly, we will establish the role of Sirt2 as a crucial mediator of HX-induced delayed OL maturation in CC. Secondly, we will define the role of non-cell autonomous, glutamate-mediated synaptic changes in regulating OPC proliferation and delayed OL maturation in CC after HX. Finally, we will define the effects of HX and the role of delayed OL maturation on axonal integrity/function in CC. Together, these studies will not only shed light on crucial cellular mechanisms of HX-induced delay in WM maturation, but might also lead to the development of new therapeutic approaches aimed at lessening the long-term neurological sequelae of premature birth.

A major cause of chronic disability in neonates is diffuse white matter injury (DWMI) and hypomyelination. Altered development of the WM is directly associated with adverse outcomes, including cerebral palsy, cognitive delay and neurobehavioral abnormalities. The cellular pathophysiology underlying DWMI and defective myelination is complex and not fully understood. Our lab has extensively published on the effects of neonatal brain injury on white matter development, and demonstrated that OL progenitor cells (OPCs) display delayed maturation into OLs, which results in aberrant myelination, altered WM function and behavioral abnormalities. In the postnatal and adult brain, OPCs arise from radial glial cells (RGCs) of the subventricular zone (SVZ), a major gliogenic and neurogenic region of the brain. OPC proliferate in the SVZ and migrate throughout the brain to gray and WM, where they mature into myelinating OLs. While some important signaling pathways have been characterized, much remains unknown about homeostatic regulation of OPC proliferation and maturation in the SVZ, both during normal development and after injury. Furthermore, although it is established that the proliferative response of endogenous OPCs to injury is crucial for expanding this progenitor pool and for regenerating a normal number of OLs, the endogenous molecular signals involved in the regulation of OPC proliferation in the SVZ are still largely undefined. We utilized our previously generated Endothelin-1 (ET-1) and ET-1 receptor (Ednr) mouse mutant lines, and discovered that, in the postnatal brain, RGC-derived ET-1 plays a novel and different role, i.e. regulates OPC proliferation. In this proposal, we will test the hypothesis that ET-1 signaling between RGCs and OPCs plays a crucial role in SVZ developmental homeostasis and regeneration. We will use an integrated approach in a mouse model and in a larger mammal (piglet), in which the SVZ displays a structure and a cellular composition identical to the human brain. Firstly, we will define the role of RGC-derived ET-1 and specific Ednr(s) in SVZ OPC proliferation in mouse and piglet during normal development. Secondly, we will determine the role of ET-1 in OPC proliferation and differentiation after HX. Finally, we will define the molecular pathways involved in HX- induced alterations in SVZ OPCs, in particular genes that are downstream of Ednr activation and are involved in OPC proliferation, cell-cycle exit and cell differentiation. Together, these studies will not only shed light on crucial cellular mechanisms of HX-induced delay in WM maturation, but might also lead to the development of new therapeutic approaches aimed at lessening the long-term neurological sequelae of HX-induced neonatal brain injury. !

The mission of the DC-IDDRC is to expand our understanding of the causes underlying intellectual and developmental disabilities (IDDs), develop innovative therapies, and prevent or attenuate the full effects of these disorders, so that each child can achieve her/his full physical and intellectual potential. To realize this mission, we provide a rich environment for performing fully translational IDDR in the four collaborating DC academic medical centers (Children?s National Hospital (lead), George Washington University, Howard University, and Georgetown University). Our specific aims are: 1) To identify the causes and develop new clinical approaches for the prevention or amelioration of IDDs; 2) To provide accessible, state-of-the-art,cost- effective core facilities for cohesive, multidisciplinary research and education/training in IDDR; 3) To create an intellectual home for investigators engaged in IDDR; and 4) To implement an innovative Research Project that addresses two IDDR themes (Interventions and Management of Co-morbid Mental Health Conditions; Outcome Measures or Biomarkers for Interventions or Treatments). The DC-IDDRC Director, Vittorio Gallo, PhD, is an internationally renowned neuroscientist who will direct the Administrative Core that functions as the organizing nexus of the DC-IDDRC, providing management, administration, communication and training, assuring compliance and quality assurance, directing strategic planning and recruitment, and promoting the growth of the DC-IDDRC. The scientific cores provide an integrated platform and synergy for investigation, as required by truly translational IDDR. The Clinical Translational Core (CTC) is designed to serve as a ?one- stop-shop? for IDDR investigators, assisting at each stage of the clinical and translational research spectrum and optimizing the efficient, high quality implementation of fundamental research. The Genomics and Bioinformatics Core (GBC) provides advanced genomics technologies and specialized statistical analyses for DNA and mRNA studies. The Cell and Tissue Microscopy Core (CTMC) supports investigators with a broad array of advanced cellular/molecular imaging tools for state-of-the-art neuroscience studies. The Human and Animal Imaging Core (HAIC) provides scientific and technical support for in vivo/ex vivo whole brain imaging, image processing, and image analysis for human and animal studies. The Neurobehavioral Evaluation Core (NEC) provides sophisticated neurobehavioral research support to define developmental and behavioral phenotypes in humans and animals. Our project ?Intervention-induced plasticity of flexibility and learning mechanisms in ASD? utilizes the CTC, HAIC and NEC. Through these components, the DC- IDDRC will enhance the recruitment and training of investigators, generate innovation, and promote transdisciplinary research to facilitate the development, implementation, and dissemination of new diagnostic and therapeutic advances for the care of individuals with IDDs.