Michael V. Johnston - US grants

[unreadable] DESCRIPTION (provided by applicant): This is a competitive renewal application that has been revised in response to all the critiques of previous reviewers and includes new preliminary data. Perinatal hypoxic-ischemic brain injuries are an important cause of cerebral palsy. To understand the mechanisms for these brain injuries and develop neuroprotective strategies, we use rodent models in which one carotid artery is ligated at around 7 days of age followed by exposure to a period of hypoxia. Hypoxia-ischemia leads to NMDA receptor-channel opening and activation of neuronal nitric oxide synthase, which synthesizes nitric oxide leading to injury from peroxynitrite. During the last period of funding, we hypothesized that these events lead to brain injury in the neonate by activating poly (ADP-ribose) polymerase-1 (PARP 1). We established a model of unilateral carotid artery ligation plus hypoxia in mice, and used this model to determine if knockout (KO) of the (Parp 1) gene reduced damage. We discovered that Parp 1 KO produced a gene dose-dependent reduction in damage in male, but not female mice. The gender dependent difference we found in neonatal mice has also been confirmed by others in adult rodents. For the next period of support, we propose to focus on the signaling pathways involved in cell death from hypoxia-ischemia and the mechanisms for the gender difference in effect of Parp 1 KO. Recent evidence from neonatal rodent and in vitro models indicates that cell death pathways are sexually dimorphic. We will examine gender differences in the intracellular translocation of apoptosis inducing factor (AIF) and cytochrome C, as well as activation of caspases in male versus female mice that are wild type (wt) vs. KO for Parp 1 and exposed to hypoxia-ischemia. We also plan to examine gender- specific differences in cell death pathways in vitro in cultured cerebellar granule and hippocampal neurons taken from these groups of mice in the neonatal period. This will provide confirmation of the data from mice, and will also allow measurement of mitochondrial membrane potential and levels of ATP, NAD+, and glutathione. Gender dependent differences in vulnerability to NMDA, AMPA and Fas receptor stimulation will also be assessed in vitro. In a third set of experiments, we will determine if female neurons that are not protected by Parp 1 KO can be rescued by treatment with EPO or fibroblast growth factor-1 (FGF-1). We hypothesize that these substances will activate protective pathways in both males and females. These studies are directly relevant to previously reported gender differences in the outcome of perinatal brain injuries in human infants, as well as to the development of clinical therapies to salvage brain tissue in these infants. Detailed information about the molecular pathways that mediate cell death from hypoxia-ischemia is critically important for developing and testing neuroprotective interventions. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This revised competing renewal Institutional National Research Service Award application describes a post-doctoral training program in medical rehabilitation research that focuses on research training in brain injury rehabilitation in children, adolescents and adults. It is proposed to train 4 post-doctoral level trainees each year for a total period of 2-3 years and 3 predoctoral students for 3 months each per year. The training program is based at the Kennedy Krieger Institute (KKI) in Baltimore, a major center focused on the clinical care and research for children with brain injuries and other neurological disorders on the campus of Johns Hopkins University School of Medicine. Trainees will also have the opportunity to work with faculty from the Johns Hopkins University Department of Physical Medicine and Rehabilitation based at the Good Samaritan Hospital and Johns Hopkins Hospital in Baltimore. The goals of the program are to (1) train clinicians and basic scientists who will go on to make important contributions that advance neurological rehabilitation; and (2) to equip these trainees with the skills needed to become independent grant-funded investigators. The focus of the training is a mentored period of hypothesis-driven clinical and/or laboratory-based research in areas related to brain injury. Faculty have expertise in one or more of six major rehabilitation themes (1) cognitive and behavioral recovery; (2) motor recovery; (3) general principles of medical rehabilitation; (4) mechanisms of brain injury and plasticity; (5) neuroimaging; or (6) epidemiology and outcomes of rehabilitation interventions. A weekly series of seminars, rounds, lectures and didactic course work brings together trainees to learn core information about neurorehabilitation in children. [unreadable] [unreadable]

biomedical equipment purchase;

Rett Syndrome (RS) may involve a genetic defect in development of specific groups of neurons in the immature brain. This project will extend studies of the abnormal neurotransmitter circuitry in RS by examining postmortem brain tissue using neurotransmitter receptor autoradiography and by assessing the effects of neuronal degeneration in a rodent model. Following up on neurochemical observations made by Dr. Wenk in the previous grant period (Project 3a), postmortem RS brain, along with age- and sex-matched control brain will be prepared to visualize binding sites for excitatory amino acid receptor subtypes and messenger RNA for these receptors, D1 and D2 dopamine receptors, dopamine reuptake sites, muscarinic receptors and nicotinic receptors. These autoradiographic studies will allow us to assess abnormalities in both the quantity and distribution of receptors for specific groups of neurons that may participate in the neurodegenerative stage of RS. Experiments utilizing a model of neuronal degeneration in dopaminergic and cholinergic projections from the brainstem and diencephalon to the basal ganglia and cerebral cortex in neonatal mice will allow us to interpret the results of analyzing the human tissue. The animal model will also provide information about the effects of injury to these projections on neurons in their terminal fields. Potential therapeutic interventions including supplementation with the growth factors, nerve growth factor (NGF), and brain derived neurotrophic factor (BDNF) will also be tested in the rodent model. The model will also be useful for understanding molecular mechanisms involved in neuronal degeneration and regeneration that may be disrupted in RS.

Project 2, entitled "the Neurobiology of Rett Syndrome" focuses on the pathogenesis of the neuronal abnormalities in the brains of girls with Rett Syndrome (RS) utilizing postmortem tissue and a neonatal mouse preparation with cerebral cortical changes that resemble those in RS. Study of both the postmortem tissue and the mouse model in the same project is synergistic because the mouse model can be controlled and manipulated in ways that are impossible with postmortem tissue, and our work over the last period of support has uncovered striking parallels between the two. The project is divided into two subprojects, Project 2a is primarily concerned with changes in selected neurotransmitter synaptic markers and Project 2b focuses on cytoskeletal changes, especially in the dendritic marker microtubule associated protein. The specific aims of proposed autoradiographic, Western Blot and immunocytochemical experiments in Project 2a will test the hypothesis that: I. Disorders of cholinergic, glutamatergic and other neurotransmitter synapses play a fundamental role in the pathogenesis of RS. In human postmortem tissues, the abnormalities will be most dynamic in early infancy and childhood cases. II. Early molecular events after cholinergic denervation in cerebral cortex contribute to the elevations in GluRs observed in the youngest cases of RS, and possibly for other abnormalities in synapse-related proteins including glutamate transporters. Cortical cholinergic denervation will be modeled by neonatal nucleus basalis lesions in mice. III. Neonatal nucleus basalis lesions plus elevated extracellular glutamate induced by administering ammonium acetate will produce encephalopathy, seizures, and histologic changes that strongly resemble RS. IV. Enhancing cholinergic neurotransmission, either by increasing acetylcholine levels or enhancing the regrowth of nucleus basalis neurons, in the neonatal period may reverse cortical pathology in the models. We focus on the neurobiology of RS in brain tissue both to understand basic mechanisms and to design rational therapy.

The Neurochemistry Core provides a service function for standard analytical techniques. Microdialysis is used extensively to monitor changes in extracellular fluid neurotransmitter and metabolite concentrations in multiple time points in each experiment. The concentration of amino acids, including glutamate, aspartate glycine and GABA, will be measured by HPLC. Another assay used extensively is the conversion of radiolabeled arginine in radiolabeled citrulline a marker of nitric oxide production. This production is also applied to microdialysis samples in which labeled arginine is measured in the dialysate. The Core will serve by performing radioimmunoassays for estradiol, progesterone and cyclic GMP. By pooling all of these analytical techniques into a common core, personnel trained in benchwork biochemistry can generated high quality results in a cost-efficient manner. Moreover, the analytical workload within individual projects can fluctuate from year-to-year. Pooling the workload from all projects will result in a more efficient utilization of personnel time. Lastly, the Core facility provides a resource for introducing new assays into the laboratory when a study may require unforseen measurements to strengthen the conclusions of the study.

The Neuroscience Core was a part of our MRDDRC during the initial period of support from 1987-1992, and consisted of a synaptic neurochemistry unit directed by Dr. Joseph Coyle at Johns Hopkins Hospital and a lipid biochemistry unit directed by Dr. Yasuo Kishimoto at the Kennedy Institute. The synaptic neurochemistry core provided biochemical assays for neurotransmitter enzymes, high performance liquid chromatography (HPLC) assays for a variety of neurotransmitters including amino acids and receptor binding assays for neurotransmitter receptors in the brain. The lipid biochemistry unit provided HPLC, gas liquid chromatography (GLC) and GLC-mass spectrometer assays used to characterize inborn errors of metabolism including sphingolipidoses, peroxisomal disorders such as adrenoleukodystrophy and Refsum's disease and lysosomal disorders. Dr. Michael Johnston replaced Dr. Coyle as director of the Neuroscience Core and head of the synaptic neurochemistry unit during the next period of support from 1993-1998 after Dr. Coyle left Hopkins to become the chairman of psychiatry at Harvard Medical School. Dr. Johnston recruited Dr. Mary Blue as a coinvestigator of the sysnaptic neurochemistry core to develop a new neuroimaging unit for brain histology. During this period the synaptic neurochemistry unit continued to provide HPLC assays for neurotransmitters and expanded sophisticated imaging techniques for immunocytochemistry and analysis of neurotransmitter receptors and messenger RNA's in tissue sections using quantitative morphometric techniques. At the same time Dr. Paul Watkins replaced Dr. Kishimoto as director of the lipid neurochemistry core. This unit continued to develop advanced techniques for studying peroxisomal and related lipid disorders and added gas chromatography/mass spectrometry services for studying fatty and organic acid disorders under the direction of Dr. Richard Kelley. Leadership of the Neuroscience Core has remained stable for two additional periods of support from 1998-2008. The Core has continues to provide the most modern technology for neuroscience research including HPLC equipment, several systems for computerized morphometry and stereology, and most recently has added a confocal microscope system. Equipment in the lipid biochemistry core has also been updated to provide expanded research on peroxisomal and organic acid disorders.

DESCRIPTION (provided by applicant): This is the first resubmission of an application for renewal of an Institutional National Research Service Award for a successful postdoctoral training program in medical rehabilitation research that focuses on brain injury and neurological disability. The PIs propose to train 4 postdoctoral level trainees per year, each for a total duration of 2-3 years. The training program is based in the Johns Hopkins Department of Physical Medicine and Rehabilitation (PM&R) and in the Kennedy Krieger Institute in Baltimore. The Kennedy Krieger Institute is a major center for clinical care and research on neurological disabilities in children and young adults on the Johns Hopkins University medical campus. These programs complement the clinical and research programs of the Department of PM&R that serve adults at the Johns Hopkins Hospital, the Good Samaritan Hospital in Baltimore and the Johns Hopkins Bayview Medical Center. Trainees have the opportunity to work with faculty advisors from the Kennedy Krieger Institute, the Department of PM&R, the Johns Hopkins Bloomberg School of Public Health, and the Departments of Neurology, Neuroscience, Biomedical Engineering and Mechanical Engineering. The goals of the program are to: 1) train clinicians and basic scientists who will go on to make important contributions that advance the rehabilitation of patients with brain and spinal cord injuries and other neurological disabilities, and 2) equip these trainees with the skills needed to become independent grant-funded investigators. The focus of the training program is on a mentored period of hypothesis-driven clinical and/or laboratory based research. Faculty have expertise in one or more of six major rehabilitation themes: 1) brain-behavior relationships and rehabilitation for pediatric traumatic brain injury;2) the continuum of care from intensive care to rehabilitation;3) physiology of swallowing and recovery from dysphagia;4) brain plasticity and recovery of function;5) spinal cord rehabilitation and regeneration;and 6) epidemiology and rehabilitation outcomes. A training program management committee provides specific goals for progress of trainees to achieve core research competencies, and it monitors progress closely. The program provides a strong curriculum of weekly conferences, journal clubs and didactic lectures that reflect the research and scholarly environment at Johns Hopkins. The program has produced more than 30 researchers and university faculty members who are active in rehabilitation research, and several who have high profile careers and hold two R01 grants. Six graduates who completed the program in the last three years are currently supported by NIH career development awards. This training program is directly related to improving treatment and reducing disability for millions of children and adults with disorders including traumatic brain injury, spinal cord injury, stroke, multiple sclerosis, autism, Parkinson's disease and numerous related disorders.

DESCRIPTION (provided by applicant): The goal of this Neuroscience Academic Development Award (NSADA, K12) renewal application is to continue to train pediatric neurologists who have completed their clinical training to be translational neuroscientists who will become leaders in academic Pediatric Neurology and make groundbreaking discoveries that can be used in clinical practice. The combined Hopkins child neurology training programs located at The Johns Hopkins Hospital (JHH) and The Kennedy Krieger Institute (KKI) are well suited to provide integrated clinical and research training. Strengths of this application include excellent clinical training programs in pediatric neurology and neurodevelopment disabilities at JHH and KKI, strong academic resources for basic and clinical research, an exceptional concentration of research infrastructure focused on neurological disorders of children, and a senior faculty with an established record of successfully training young investigators. In this proposal, formal training in research will begin after three years of clinical child neurology training. The faculty for this NSADA program are clinical/basic science researchers who are established, well-funded, and have been chosen because of their enthusiasm and experience as educators. In addition to clinical and/or laboratory based research, trainees will take courses in biostatistics, experimental design, clinical trials management, epidemiology and responsible conduct of research at the Johns Hopkins Bloomberg School of Hygiene and Public Health or the Johns Hopkins School of Medicine. With the guidance of the program Director and Co- Director Trainees can choose from several fields in neuroscience relevant to pediatric neurology including neurogenetics, neurodevelopment disabilities, epilepsy, neuromuscular disorders, demyelinating disorders, magnetic resonance neuroimaging, spinal cord injury, regenerative medicine and basic neuroscience. Candidates will be chosen from pediatric neurologists completing their training in either pediatric neurology or neurodevelopment disabilities at JHH or KKI, or from outstanding candidates from other institutions across the United States. It is anticipated that during the 5 year award period, a total of 4 appointments will be made. Our record for recruiting minorities to our clinical training program is excellent. This program will provide the field of pediatric neurology with academic physicians well trained in basic and clinical research. PUBLIC HEALTH RELEVANCE: This project will train clinical pediatric neurologists early in their careers to learn how to conduct research that will discover new therapies for pediatric disorders of the nervous system. Emerging basic science research is uncovering mechanisms for these disorders, but clinicians with research expertise are required to translate these discoveries into effective cures.

RFA-HD-13-002 requires the inclusion of a Clinical Translational Core within each IDDRC, addressing a combination of activities that includes: (a) recruitment of research participants, (b) subject assessment, phenotyping, or treatment, (c) development of biomarkers, (d) manufacturing services, (e) highthroughput screening, and/or (f) collection of samples from individuals with IDD for biobanking. As described in our Introductory Overview and the following Core descriptions, many of these activities are supported by other Cores and existing resources within the large and multidisciplinary research environment developed within our Institutions over the years. Therefore, the overall objective of the Clinical Translational Core is to complement those resources and coordinate access for IDDRC-supported investigators. This will contribute to our primary goal of enhancing the conversion of basic science discoveries into effective, commercially available disease-altering therapies for children and adults with genetic and acquired intellectual and developmental disabilities. The Core will be structured to complement our Clinical Trials Unit (CTU), a program established three years ago in response to the increasing emphasis on translational research within our IDDRC and the broader research program it supports. The CTU is focused on the development of treatments targeting developmental disorders, including conducting investigational dmg trials at all phases. The Clinical Translational Core will expand upon CTU capabilities to provide expert resources including consultation on biostatistics and protocol design, pre-review prior to submission of protocols to the institutional review board (IRB) and hands-on advice and help with carrying out investigational protocols by experienced clinical trials faculty, research coordinators and administrative staff.

Project Summary / Abstract This is a proposal to provide intensive, mentored research training for pediatric neurologists who are within 5 years after finishing their clinical training in pediatrics and pediatric neurology, and who want to continue in an academic/research position. The name of the program will be the Child Neurologist Career Development Program (CNCDP) that will be sponsored by the NIH/NINDS and it is intended that CNCDP will replace a similar program of mentored research for pediatric neurologists called the Neuroscience Academic Development Award (NSADA). The NSADA program has been operating since 1992, and it has fostered the education of numerous child neurologists in academic positions, but less than half of the graduates of the NSADA program have gone on to become PI's of independent career development grants such as the K08, K23 or R01 awards or continued in the role of independent clinician-scientists. The CNCDP is intended to correct the shortcomings of the NSADA, and the most important change will be that the CNCDP will be a unified national program directed by a single PI with a Steering Committee and a National Advisory Committee (NAC), referred to as he CNCDP leadership. This plan is different from the NSADA program in which nine individual PI's of separate NIH awards reported directly to NIH-NINDS. The new CNCDP structure will include two layers of mentorship: a mentorship team at the scholar's home institution and mentorship from the national CNCDP leadership. Under the CNCDP plan, individual pediatric neurologists and mentorship teams will be able to apply for CNCDP support from any academic institution in the US with a pediatric neurology training program. The CNCDP Steering Committee will work with prospective candidates to find appropriate mentors and to apply for CNCDP support using the same form as for the NIH K08 Award. A separate NAC that includes national and international leaders in pediatric neuroscience will review the applications and choose up to 6 of the most meritorious for funding per year. Over 5 years, a total of 30 trainees are planned for the program. There is a transition plan for current NSADA scholars to complete their mentored research. Successful applicants to the program will present a training program that links scientific research training with a clinical career in pediatric neurology so that they are not separate but integrated. The scientific merit of the program and the quality of the mentors will be outstanding. The program will include training in experimental design, biostatistics and the responsible conduct of research, but generally will not include prolonged didactic work that can detract from the scholar's research and mentorship. Then the CNCDP leadership will continue to participate in the scholar's mentorship through an initial site visit to the scholar's institution, frequent webinar meetings with the scholar and mentoring committee, and a yearly two day retreat meeting in which scholars present their work to the CNCDP leadership and outside experts. CNCDP leadership will strictly enforce the rule that scholars must be protected to do research for 75% of their time.