Michael B. Robinson - US grants

The amino acids, glutamate and aspartate, are the predominant excitatory neurotransmietters in the central nervous system. The levels of both of these amino acids approach 10 mmol/kg in brain tissue. Under normal conditions, the extracellular concentrations are maintained below .001-.010 M. In many acute neurologic diseases, including ischemia, head trauma, and epilepsy, the extracellular concentrations of these amino acids increase dramatically and the subsequent excessive activation of receptors contributes to the pathophysiology. Many investigators have focused on the postsynaptic mechanisms involved in this "excitotoxicity". The transport process that regulates the extracellular concentration of these amino acids is not well characterized. It is known that the extracellular concentrations of glutamate and aspartate are maintained at low levels by a sodium-dependent high affinity transport process. Data support the presence of at least two subtypes of this transport process with heterogeneous distributions. Studies will be carried out to test the hypotheses that there are subtypes of transport, that these subtypes have different distributions and that these subtypes can be independently regulated. Biochemical, pharmacological, and morphological techniques will be used to characterize these subtypes of transport and their regulation. The long-term goal of these studies is to define the functional relevance of these subtypes of transport systems. The objective of these studies are: 1) To compare the kinetic properties, the ion-dependence, the inhibition by excitatory amino acid analogs, and the sensitivity to sulfhydryl modifying reagents of the transport subtypes. 2) To determine the regional and cellular distribution of these subtypes of transport. 3) To characterize the mechanism(s) that regulate transport activity, including regulation by second messenger systems, including the phosphoinositide and cAMP cascades and regulation by its substrates. Subtypes of transport systems may provide a mechanism for specificity and regulation of synaptic transmission analogous to the specificity of postsynaptic signaling provided by receptor subtypes. Consistent with the diverse functions of these amino acids, metabolic and neurotransmitter pools of the excitatory amino acids are regulated independently in brain tissue. Heterogeneity of the excitatory amino acid transport systems may provide a mechanism for compartmentalization and regulation of excitatory amino acid metabolish. Characterization of the excitatory amino acid transport system(s) will help to define the role of this transport system in synaptic signaling, metabolic compartmentalization, and excitotoxic neuronal degeneration.

Phencyclidine (PCP) is a widely abused drug (e.g. angel dust) which has a variety of actions in the central nervous system. Many of the behavioral effects induced by PCP are thought to result from the blockade of excitatory neurotransmission through the NMDA receptor. The activity of this receptor is critical for many normal brain functions including learning and memory. However, not all compounds that block the NMDA receptor have the same behavioral effects as PCP. Dextromethorphan is another non-competitive NMDA receptor antagonist that is thought to bind to the same site as PCP, but this drug has very different behavioral effects. The two major goals of this project are to understand the molecular interactions between the NMDA receptor and non-competitive antagonists like PCP and to determine the subunit composition and stoichiometry of the NMDA receptor. Several specific questions will be addressed during the course of the project including: l) are there subtypes of NMDA receptors which interact differently with PCP, 2) How are subtypes of NMDA receptors assembled from various subunits, 3) what is the stoichiometry of the NMDA receptor, 4) Which regions of the NMDA receptor bind PCP 5) Which amino acids in the NMDA receptor are most important for drug binding, 6) do amino acids from multiple NMDA receptor subunits interact with PCP, 7) do all non-competitive agonists of the NMDA receptor bind to the same subtypes of NMDA receptor, 8) is it feasible to develop drugs to block PCP binding which will not block the NMDA receptor channel.

The overall goal of this project is to study the efficacy of in vivo gene therapy with recombinant adenoviruses in two animal models of ornithine transcarbamylase deficiency (OTCD), the sparse fur (Spf) mouse and the abnormal skin and hair (Spf/ash) mouse. The presence of a useful animal model permits us to attempt gene therapy in neonatal and adult animals using various recombinant adenoviruses and detect metabolic and neurochemical changes that will prove useful in the human studies described in Project III. Our studies will revolve around those responses to in vivo gene therapy that will be of greatest significance to the human studies: (1) The metabolic response, particularly with regard to the conversion of ammonia to urea; (2) The safety of adenoviral administration; and (3) The neurochemical/neuropathologic response to gene therapy, since the worst consequence (other than death) of hyperammonemia is brain damage and mental retardation. There are four specific aims: I to develop the metabolic and neurochemical measures that will permit assessment of efficacy of gene therapy; II to study the level, distribution, and time-course of gene expression and to study correction of metabolic abnormalities, including orotate excretion, in vivo urea production with stable isotopes, and steady state levels of amino acids; III to test the efficacy of gene therapy in neonatal Spf and Spf/ash mice to determine duration and the presence of tolerance; and IV to detect if the neurochemical and behavioral alterations and neuropathology induced by hyperammonemia are preventable in neonates and reversible in older animals.

DESCRIPTION: The acidic amino acids are the predominant excitatory neurotransmitters in the mammalian central nervous system (CNS). Most evidence indicates that Na+-dependent high affinity transporters are responsible for the clearance and regulation of extracellular excitatory amino acids (EAA). Pharmacological, biochemical, and molecular biological studies indicate that there are several subtypes of Na+-dependent glutamate (Glu) transporters that are expressed in different brain regions and on different cell types, including the astroglial transporters GLT-1 and GLAST, and the neuronal transporters EAAC1 and EAAT4. This project represents a collaborative effort between two laboratories who have defined the biochemical, pharmacological and pathophysiological properties of Glu transport. The goal of the investigators is to increase understanding of the predominant mechanism that normally protects the brain from excitotoxicity. This information should improve our understanding of the failure of these systems during acute and chronic insults to the CNS, including stroke and head trauma. The investigators will use in vitro and in vivo preparations to test the functional significance of our observations. The investigators have previously established the pharmacology of transporter activity in vitro and in vivo preparations. The investigators hypothesize that the cloned transporters recapitulate the properties observed in these preparations. This hypothesis will be tested by expressing the individual transporters in xenopus oocytes. In addition to this correlative strategy, the investigators propose to use antisense oligonucleotides to "knock-out" subtypes of the transporters to examine the relative contributions of each transporter to activity observed in astrocyte-enriched cultures and in synaptosomes prepared from different brain regions. The investigators hypothesize different transporters contribute to the regulation of extracellular Glu in the pre- and early post-natal period. The investigators propose to study the level of expression and ultrastructural localization of the transporter subtypes during development. Based on the preliminary data, the investigators hypothesize that neurons regulate expression of glial Glu transporters. In vivo and in vitro systems will be used to examine this regulation. The investigators propose that cAMP through activation of protein kinase A is one of the factors that controls expression of the GLT-1 subtype of glial transporter. This will be tested in astrocyte-enriched cultures. The effects of selective antisense "knock-out" of subtypes of transporters on excitotoxicity and the accumulation of extracellular Glu will be examined in vitro. The investigators also propose to study the effects of excitotoxic insults on the expression of subtypes of transporters.

DESCRIPTION: (Applicant's Abstract) Glutamate and aspartate are the predominant rapid excitatory neurotransmitters in the mammalian central nervous system (CNS), and are involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of EAA receptors contributes to brain damage observed in several acute insults to the CNS, including stroke and head trauma. The extracellular concentrations of these excitatory amino acids (EAAs) are controlled by a family of NA+-dependent high-affinity transporters. We have recently developed evidence that platelet-derived growth factor (PDGF) increases cell surface expression of EAAC1, one of the neuronal glutamate transporters. We have also found that wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreases cell surface expression of this same transporter. The overall goal of this project is to study the mechanisms that control this regulation, and to define the functional consequences of this altered cell surface expression. Based on our preliminary data, these alterations in cell surface expression are due to trafficking between intracellular vesicles and the plasma membrane. We hypothesize that PDGF increases cell surface expression through PI3-K and a serine-threonine kinase called Akt (also known as protein kinase B). We hypothesize that SNAREs are involved in the trafficking to the cell surface, and that dynamin, a GTPase, is important for endocytosis of the transporter. We hypothesize that this regulation is specific for the EAAC1 subtype of transporter, and that chimeras will help to delineate both the mechanisms involved in this regulation and the portions of EAAC1 that govern this regulated trafficking. Finally, we hypothesize that this PDGF-mediated increase in cell surface expression may contribute to its previously documented neuroprotective activity. These hypotheses will be tested using C6 glioma, a model system that only expresses the EAAC1 subtype of transporter and primary neuron-enriched cultures. We propose using a variety of complimentary biochemical, molecular biological, pharmacological, and cell biologic approaches to address these hypotheses. Since EAAC1 is enriched in cortex and hippocampus, two areas that are particularly vulnerable to excitotoxic insults, the proposed studies may provide opportunities to develop new strategies to limit excitotoxic brain damage.

DESCRIPTION: (Applicant's Abstract) Glutamate and aspartate are the predominant rapid excitatory neurotransmitters in the mammalian central nervous system (CNS), and are involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of EAA receptors contributes to brain damage observed in several acute insults to the CNS, including stroke and head trauma. The extracellular concentrations of these excitatory amino acids (EAAs) are controlled by a family of NA+-dependent high-affinity transporters. We have recently developed evidence that platelet-derived growth factor (PDGF) increases cell surface expression of EAAC1, one of the neuronal glutamate transporters. We have also found that wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreases cell surface expression of this same transporter. The overall goal of this project is to study the mechanisms that control this regulation, and to define the functional consequences of this altered cell surface expression. Based on our preliminary data, these alterations in cell surface expression are due to trafficking between intracellular vesicles and the plasma membrane. We hypothesize that PDGF increases cell surface expression through PI3-K and a serine-threonine kinase called Akt (also known as protein kinase B). We hypothesize that SNAREs are involved in the trafficking to the cell surface, and that dynamin, a GTPase, is important for endocytosis of the transporter. We hypothesize that this regulation is specific for the EAAC1 subtype of transporter, and that chimeras will help to delineate both the mechanisms involved in this regulation and the portions of EAAC1 that govern this regulated trafficking. Finally, we hypothesize that this PDGF-mediated increase in cell surface expression may contribute to its previously documented neuroprotective activity. These hypotheses will be tested using C6 glioma, a model system that only expresses the EAAC1 subtype of transporter and primary neuron-enriched cultures. We propose using a variety of complimentary biochemical, molecular biological, pharmacological, and cell biologic approaches to address these hypotheses. Since EAAC1 is enriched in cortex and hippocampus, two areas that are particularly vulnerable to excitotoxic insults, the proposed studies may provide opportunities to develop new strategies to limit excitotoxic brain damage.

positron emission tomography; neurochemistry; spectrometry; mental retardation; analytical chemistry; biomedical facility; high performance liquid chromatography; nuclear magnetic resonance spectroscopy; gas chromatography mass spectrometry;

[unreadable] DESCRIPTION (provided by applicant): It has been estimated that developmental disabilities affect 10% of all families in the USA. Researchers are urgently needed who are willing and able to apply modern research methods to elucidating the pathogenesis and pathophysiology of these disorders, so that new and more effective therapeutic interventions can be identified. The aim of our interdisciplinary Institutional Post-doctoral Training Program in Neurodevelopmental Disabilities, based at The Children's Hospital of Philadelphia (CHOP) and The University of Pennsylvania (U of P), is to train MD and PhD post-doctoral fellows in research focused on genetic and acquired disorders that cause mental retardation and developmental disability. Nineteen Mentors will participate in this first competitive renewal of this program; 13 are based primarily at CHOP, and 6 primarily at the U of P. All are heavily involved in biomedical graduate education and are closely interlinked by mutual research projects and grants. In addition to mentored research training, our curriculum emphasizes a clinical practicum requirement for both MDs and PhDs, formal course work through the U of P Graduate Studies Program, and training in bioethics, biostatistics, and skills in scientific writing and presentation. During the first 4 years of our program, we accepted 8 Trainees, of whom 2 were MDs, 5 PhDs, and 1 and MD/PhD. Of the 5 who have thus far completed our program, 2 have obtained junior faculty positions, 2 have entered clinical training programs (1 in neurology, 1 in neurosurgery) prior to returning to an academic career, and 1 accepted a position in a biotechnology firm. Two additional Trainees will complete our program during the coming year; each has been offered and accepted a medical school assistant professorship (1 in Neurology and Pediatrics, U of P, the other in Psychiatry, UCLA). We were approved for 4 post-doctoral fellows/year during our first 5 years, and request the same level of funding for the next 5 years. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Project Summary: The aim of our interdisciplinary Institutional Training Program in Neurodevelopmental Disabilities, based at The Children's Hospital of Philadelphia (CHOP) and The University of Pennsylvania (U of P), is to train MD and PhD pre-/post-doctoral fellows in research focused on genetic and acquired disorders that cause mental retardation and developmental disability. Twenty-seven mentors will participate in this second competitive renewal of this program; 20 are based primarily at CHOP, and 7 primarily at the U of P. All are heavily involved in biomedical graduate education and are closely interlinked by mutual research projects and grants. In addition to mentored research training, our curriculum emphasizes: 1) a clinical practicum requirement for both MDs and PhDs, 2) formal course work through the U of P Graduate Studies Program, 3) training in responsible conduct of research, 4) training in biostatistics and 5) workshops that cover a variety of important survival skills, including scientific writing, public presentations, grant writing workshops, laboratory management, and career advancement skills. During the first 9 years of our program, we accepted 19 Trainees; 4 were MDs, 14 were PhDs, and 1 was a MD/PhD. 11 of these trainees were female and 4 of these trainees were under-represented minorities. Of the 14 who have completed training, 8 are in faculty positions, 2 are continuing their training at other academic institutions, 1 is a senior research scientist at a pharmaceutical company, 1 is a scientific administrator, and 1 is in private practice. We request continued support for 4 postdoctoral fellows/year and would like to expand our program to include support for 2 pre-doctoral fellows/year. Our goal remains focused on providing a training program unlike any other here at the CHOP or the U of P that is focused on acquired and genetic causes of developmental disability. This program benefits from being in an outstanding environment that commits substantial resources to training, to basic biomedical research, and to a true 'bench to bedside' approach to translational research. Project Relevance: It has been estimated that developmental disabilities affect 10% of all families in the USA. Researchers are urgently needed who are willing and able to apply modern research methods to elucidating the pathogenesis and pathophysiology of these disorders, so that new and more effective therapeutic interventions can be identified. This program strives to fill this need.

The goals of this Core are to: (1) Measure "small molecules" (primarily amino acids and biogenic amines) of central importance to brain chemistry. (2) Assay stable isotope enrichment with mass spectrometry in order to perform kinetic studies of metabolic flux, both in vitro and in people with developmental disabilifies. (3) Idenfify and characterize pepfides and proteins that are important to neurologic function. (4) Perform cell-based assays of physiologic processes that are important to brain funcfion, including assays of Ca2+, calpain, caspase and ROS;(5) Assist users with regard to experimental design and data interpretation. The rationale for the core is that neurochemical derangements occur in many developmental disabilifies, including metabolic diseases, hypoxic injury, epilepsy, neurotoxicity, etc.

DESCRIPTION (provided by applicant): Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. An extracellular accumulation of glutamate causes excessive activation of glutamate receptors and cell death through excitotoxic mechanisms. Unlike other classical neurotransmitters, that are recycled directly into the presynaptic nerve terminal, most glutamate is cleared by two astroglial glutamate transporters, called GLAST and GLT-1 (or EAAT1 and EAAT2). These transporters maintain very low synaptic concentrations of glutamate, estimated at ~25 nM, in an environment that contains millimolar concentrations of glutamate. These transporters are enriched on the fine processes of astrocytes that sheath synapses. We recently developed physical evidence (co-immunoprecipitation, mass spectrometry, reverse immunoprecipitations) and anatomic evidence (co-localization in individual astrocytes in organotypic slice cultures) that these transporters exst in a complex with the Na+/K+ ATPase, most of the enzymes in glycolysis, and mitochondria. This complex is observed in fine processes of astroglia. In the first aim, we will identify specifi domains of GLT-1 and GLAST that support the interactions/co- compartmentalization. We wish to identify potential scaffolding proteins that may form a linkage between the transporters and mitochondria. As has been observed with mitochondria at synapses or at nodes of Ranvier, we propose that neural activity recruits mitochondria to regions where transporters are enriched. In the second aim, we will study the effects of neuronal activity on this co-compartmentalization and define the mechanisms involved. Finally, we will test the hypothesis that formation of these complexes is required for glutamate-dependent changes in glycolysis and a shift in glutamate metabolism (from conversion to glutamine to glutamate oxidation). Compartmentalization of the astroglial glutamate transporters with these proteins and mitochondria provides an opportunity to spatially match energy production and buffering capacity. It also has implications for disposition of the glutamate. Therefore, our proposed research will impact our understanding of fundamental aspects of glutamate handling and metabolism. They will also define a novel molecular mechanism that matches astroglial energetic demands to changes in neuronal activity. PUBLIC HEALTH RELEVANCE: For approximately two decades, it has been clear that failure to clear glutamate contributes to the brain damage observed in stroke, neurodevelopmental disorders, and neurodegenerative diseases. This failure to clear glutamate occurs, at least in part, because of impaired energy mobilization. Our long-term goal is to understand how extracellular glutamate is controlled so that it will be possible to intervene and prevent the debilitating effects of these disorders.

Project Summary/Abstract The aim of our interdisciplinary Institutional Training Program, based at The Children's Hospital of Philadelphia (CHOP) and The University of Pennsylvania (Penn), is to train MD and PhD post-doctoral fellows in research focused on Neurodevelopmental Disabilities (NDD). The rationale for this NDD T32 program is three-fold. First, ~10% of households in the United States live with an individual with a neurodevelopmental disability; thus, these disorders are a significant financial and emotional burden in our society. Second, the causes of neurodevelopmental disability range from genetic to acquired insults; this necessitates an interdisciplinary approach. Finally, there is substantial overlap of symptoms amongst the various neurodevelopmental disorders, suggesting overlapping mechanisms. Trainees and their mentors use state-of-the-art techniques, including genetic, cellular/molecular, behavioral, and structural/dynamic imaging to pursue basic and translational research related to these disorders. Twenty-seven mentors help support the careers of the trainees. There is a high degree of collaboration among the mentors and trainees with shared publications and grants. In addition to mentored research training, our curriculum emphasizes: 1) a clinical practicum requirement for both MDs and PhDs, 2) formal course work through Penn Graduate Studies Program, 3) training in responsible conduct of research, 4) training in biostatistics and rigor and reproducibility, 5) grants club, and 6) forums that teach a variety of important survival skills, including scientific writing, public presentations, grant writing, laboratory management, mentoring skills, and becoming knowledgeable about career options. During the first 19 years of the NDD T32 program, we have enrolled 46 trainees; 5 are MDs, 4 are MD/PhDs, and 37 are PhDs. Thirty-two (69%) of these trainees are female and 10 (22%) of these trainees are from under-represented minority groups. Thirty-three different NDD T32 mentors have supervised trainees. Of the 36 who have completed training, 16 are in faculty positions, 2 are in instructor positions, 4 are senior research scientists at pharmaceutical companies, 3 are scientific administrators, 2 work in Clinical Genomics, 4 are in clinical practice, 1 is a lecturer, and 3 are senior scientists in academia. We request continued support for 6 postdoctoral fellows/year who participate in a program that is designed to be 3 years in length. This number of trainees allows us to maintain a critical mass to support a diverse trainee pool that can learn from one another and is easily justified by the large number of outstanding trainees who seek admission. This NDD T32 program continuously evolves. A Grants Club and closer monitoring a trainee progress were implemented in the most recent funding period; a new workshop focused on techniques with a focus on rigor/reproducibility will start in the Fall of 2017. This program combines the outstanding CHOP/Penn training environment, an exceptional cadre of trainees and mentors, and substantial institutional resources for research and training in neurodevelopmental disabilities to fuel a true ?bench to bedside? approach to translational research.

? DESCRIPTION (provided by applicant): Glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excessive activation of glutamate receptors leads to excitotoxicity, which in turn contributes to cell death observed after acute neurologic insults and in chronic neurodegenerative diseases. A family of Na+-dependent transporters controls extracellular Glu and prevents excitotoxic activation of Glu receptors. The GLT1/EAAT2 subtype of transporter mediates the bulk of this activity in the forebrain, and it is almost exclusively expressed in astrocytes. The levels of GLT1 are decreased in several neurologic diseases, including amyotrophic lateral sclerosis (ALS). To study transcriptional regulation of GLT1 we generated a BAC-GLT1-eGFP transgenic mouse that utilizes a large bacterial artificial chromosome (BAC) to express eGFP under the control of the full length GLT1 promoter. To better understand which region of the GLT1 promoter is necessary and/or sufficient for astroglial GLT1 expression, we generated a family of promoter reporter mice that utilize increasing amounts of the 5' non-coding region of the GLT1 gene (2.5, 6.7, 7.9, and 8.3 kilobases) to express tdTomato. When we crossed these mice with the BAC-GLT1-eGFP mice to produce dual reporter mice we made two exciting and unexpected observations. First, the promoter region between 7.9 and 8.3 kb is required for specific expression of tdTomato in astroglia. This region contains a domain that is evolutionarily conserved from rodents to humans, suggesting that this domain is critical for selective in vivo astroglia expression of GLT1. Second, although tdTomato is only found in eGFP-expressing astroglia, not all eGFP-expressing astroglia express tdTomato; the tdTomato/eGFP (double+) astrocytes are enriched in regions where GLT1 selectively decreases in ALS. This suggests that the 8.3 kb portion of the GLT1 promoter is only sufficient to induce expression of GLT1 in a defined subset of astrocytes, providing evidence for the existence of distinct subtypes of astrocytes. Based on these and other preliminary data, we propose two specific aims: 1) We will test the hypothesis that subtypes of astrocytes use different extrinsic stimuli (neurons and endothelia) to activate different intrinsic signals (Pax6 and Notch with associated promoter elements) to control subtype-specific expression of GLT1. We will determine if this differential control of GLT1 generalizes to proteins that are differentially expressed in these subpopulations of astrocytes. We will confirm that these subpopulations of astrocytes are found in humans. 2) We will test the hypothesis that the subtypes of astroglia identified by the 8.3 kb promoter reporter mice are selectively affected in mouse models of ALS. We will confirm pathologic changes using human tissue. Finally, we will test the hypothesis that the subtype of astroglia identified by the 8.3 kb promoter reporter mice selectively contributes to the known non-cell autonomous motor neuron degeneration.

(ANALYTICAL NEUROCHEMISTRY CORE (ANC): CORE D)  PROJECT SUMMARY Description: The Analytical Neurochemistry Core (ANC) supports these services: (a) Quantitation of amino acids and biogenic amines; (b) Kinetic studies of flux through metabolic pathways using stable isotopes as tracers and mass spectrometry to measure isotopic enrichment; (c) Qualitative and quantitative mass spectroscopic analyses of proteins/peptides and their modifications; and (d) Bioenergetic assays of mitochondrial function. Analyses are performed: (a) In vitro; (b) In pre-clinical animal models of intellectual and developmental disabilities (IDD); and (c) In vivo, in human patients. Users have access to a broad repertoire of analytical services, as well as expert advice with regard to experimental design and data interpretation. The core emphasizes all aspects of quality control, including assurance that studies are adequately powered and properly controlled. When feasible, analyses are performed in a blinded fashion. Relevance to IDDRC Mission: The overall theme of our Center is ? ?Genes, Brain and Behavior?. The primary focus of the ANC is ?Brain?, or the neurochemical changes which result from gene mutation and variation. These neurochemical aberrations may contribute to the behavioral anomalies that become clinical manifestations of IDD. The ANC enables users to identify and quantify changes in brain biochemistry. Such information deepens understanding of the IDD and affords development of biomarkers with which to gauge the efficacy of therapeutic interventions. Eligibility: These services are available both to approved users of the IDDRC at CHOP/UPenn and to users at other Centers in the Network.   

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Acute insults to the nervous system, such as stroke or traumatic brain injury, cause an increase in extracellular glutamate, excessive activation of glutamate receptors, and neuronal death through a process called excitotoxicity. Excitatory synaptic transmission is also an energy consuming process. In fact, increases in excitatory activity cause an increase in blood flow to meet energetic demands imposed by this excitatory activity. Compared to most other neurotransmitters, glutamate is relatively uniquely cleared into astrocytes rather than being directly recycled back into the nerve terminal. Two Na+-dependent glutamate transporters, GLT-1 and GLAST (also called EAAT2 and EAAT1), are almost exclusively expressed by astrocytes. In astrocytes, expression of GLT-1 and GLAST is enriched on fine processes near synapses. During our first funding cycle, we studied the co-compartmentalization of GLT-1 and GLAST with mitochondria. We demonstrated mitochondria are found throughout these processes, they are mobile, and the percentage of mobile mitochondria is regulated by neuronal activity. Furthermore, we demonstrated that inhibition of glutamate transport or inhibition of reversed operation of the Na+/Ca2+ exchanger increases the percentage of mobile mitochondria; we showed that these effects are accompanied by a decrease in basal Ca2+ in astrocyte processes. We developed several lines of evidence that strongly suggest that mitochondria shape spontaneous Ca2+ spikes (amplitude, duration, and spread) in astrocyte processes. We showed that oxygen glucose deprivation causes a loss of mitochondria from astrocytic processes. We showed that inhibition of glutamate transport or inhibition of the reversed operation of the Na+/Ca2+ exchanger blocks this loss of mitochondria. Our data suggest that the elevations in extracellular glutamate observed with acute insults, such as stroke, cause a loss of astrocytic mitochondria. The mechanism by which glutamate transporters cause this loss of mitochondria has not been defined, and it is not clear if this loss has pathologic consequences. In this renewal, we will define the mechanisms involved in this loss of mitochondria and determine if this loss contributes to the pathologic consequences of stroke. We will also determine if glial glutamate transport, reversed Na+/Ca2+ exchange, and mitochondria control the increase in blood flow observed with excitatory neuronal activity.

? DESCRIPTION (provided by applicant): This application seeks funding for the Intellectual and Developmental Disabilities Research Center (IDDRC) at the U of PA (Penn) and the Children's Hosp of Philadelphia (CHOP). Our IDDRC, now in its 25th year, is an inter- disciplinary program that is the chief agency at CHOP/Penn for the promulgation of research into the developmental disabilities. In the next cycle we build upon this foundation by proposing an innovative Center with 6 components: (1) A research project which uses magnetoencephalography (MEG) to develop a novel biomarker - an electronic signature - for non-verbal and minimally verbal children with autistic spectrum disorders; (2) A series of 6 research core facilities which will provide investigators with state-of-the-art facilities and expertise, including 2 new cores (Preclinical Models and Clinical Translational); (3) An educational program which will nurture a feeling of IDD Community by featuring monthly seminars, including the IDDRC Research Lecture, the Monthly Chalk Talks and the Autism Distinguished Lecture Series; (4) A career development initiative which will benefit dozens of exceptional young investigators who will receive support from the IDDRC New Program Development award (to be funded by the Philadelphia Foundation), the IDDRC-administered T32 Training Grant in Neurodevelopmental Disabilities and several private awards (aggregate value ~ $3 million over 5 years) given by local philanthropies to the IDDRC; (5) A research advocacy mission which involves an IDDRC- CHOP/Penn collaboration to create Centers of Excellence that encourage inter-disciplinary translational research into IDD; and (6) Participation in the larger IDDRC Network in order to realize the scientific and organizational goals of the IDD Branch of NICHD. The Center will support 78 federally-funded projects ($23.3 million/yr), of which 15 are from NICHD ($5.8 million/yr). The theme of our IDDRC is Genes, Brain, Behavior, a designation which reflects our ongoing effort to understand IDD in 3 inter-related domains: (a) The genetic anlage which causes a disability or modulates disease severity; (b) The anomalies of brain biochemistry and neurophysiology which accompany genetic changes; and (c) The phenomenological manifestations of these genetic/neurophysiologic alterations which we recognize as clinical manifestations of the IDD. Our rationale for this reductionist approach is that the dissection of a disability into isolated and simpler components affords a strategy with which to develop therapies that will prevent, attenuate or even reverse the devastating consequences of the disorder. Over the coming 5 years the Center will pursue this aim by deploying these tools: cutting-edge research cores, a focus on translational research, the enormous clinical resources of CHOP/Penn, nurturing a cadre of future leaders, and a strong emphasis on creative partnership with the IDDRC Network.

This proposal requests partial support for the 4th biennial conference, Brain in Flux: Genetic, Physiologic, and Therapeutic Perspectives on Transporters in the Nervous System, a satellite to the joint International Society of Neurochemistry and the American Society for Neurochemistry biennial conference to be held in Montreal Canada. Brain in Flux will be convened at Le Buchelon Eco Resort located in Saint-Paulin (Quebec), Canada from Aug 9-12, 2019. This meeting is designed to create a stimulating event for leading researchers, junior investigators, and trainees who study brain membrane transport proteins by fostering the rich exchange of cutting-edge scientific and technical knowledge as well as supporting the professional development of and networking opportunities for junior scholars and trainees. Our goal is to convene a small, diverse group (~75-100) of geographically diverse researchers with meaningful representation at all academic ranks. Of note, one-half of the integrated topic session speakers will be chosen from junior faculty, post-doctoral and pre-doctoral abstract pools. The following themes, which complement the speakers already invited and committed, are envisioned: 1) Vesicular transport: from cells to circuits; 2) From biophysical structure to function; 3) Amino acid transporters; 4) Sexual dimorphism in transporter expression and function; 5) Transporters in information processing; 6) Contribution of transporters to astrocyte-neuron interactions and 7) Transporters in neurological disease and as therapeutic targets. Ample discussion time, two evening poster sessions and afternoon free time provide opportunity for formal and informal discussion, networking and for the formation of new collaborations. Historically Brain in Flux has demonstrated a strong record of accomplishment with respect to gender equity among its participants; however, conference organizers are keenly aware of the barriers to participation at conferences and in science faced by persons from underrepresented populations, or with disabilities. Thus, we will be making concerted efforts to promote diversity and inclusion by using a combination of existing and new recruiting and support initiatives. Representation goals include equity in woman's numbers; 50% or less tenured or equivalent, 25% early stage untenured or equivalent, and 20% groups racial and ethnically underrepresented or otherwise disadvantaged in pursuing the sciences, including persons with disabilities. Active engagement of the program committee as well as targeted outreach efforts will help us to hit these targets. Funds requested will support the participation and professional development of trainees, postdoctoral scholars and early stage investigators, including those from groups under-represented in science, allowing them the opportunity to present, discuss, and network with other scientists studying brain transporters from diverse disciplinary and technical perspectives. Career development activities offered include formal presentations on rigor and reproducibility in science as well as implicit bias and informal lunch-time discussions on topics such as work-life balance, strategies to advance one's career, or creating peer mentor supports.

(RESEARCH PLAN- OVERALL) PROJECT SUMMARY With this application, we seek funding for the Intellectual and Developmental Disabilities Research Center (IDDRC) at the Children?s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (Penn), which has been continuously funded for the past 30 years. Our IDDRC supports an interdisciplinary program and is the chief agency at CHOP/Penn for the promulgation of research into the Intellectual and Developmental Disabilities (IDDs). Our mission, to identify the pathogenesis of and develop therapies for individuals with IDDs, is pursued through three aims. (Aim 1) Lead a cutting-edge IDD research agenda. We will support five research cores that harness innovations in genetics and neuroscience to identify the causes of IDDs, to determine how gene variants alter brain structure, circuitry, and behavioral outputs (cognitive, motor, sensory, social, affective), and to utilize this information to develop biomarkers and new treatments for IDDs. Our cores deploy complementary state-of- the-art technologies, focusing on studies performed in two species (mouse & human), making it easier for center members to perform more impactful research. Cores emphasize research along the developmental spectrum. These strategies ensure that the advances will have a translational impact. The cores provide cost-effective support for 61 world-class center members, who are funded by 78 grants totaling $29.1 million annually to study the pathogenesis of IDDs, to identify new biomarkers of IDDs, and to develop novel interventions (pharmacologic and genetic). In addition, we will support an innovative research project that uses center cores to determine if magnetoencephalography (MEG) measures of auditory processing in infants at genetic risk for IDD can be used to predict cognitive and language outcome. Our cores focus on rigorous and reproducible research practices, including sound experimental design for hypothesis testing, well-justified sample sizes, and robust data analytics. (Aim 2) Lead a multi-disciplinary career development program to support trainees and early-stage faculty. Our trainees are diverse and have PhDs, MDs, and MD/PhDs with backgrounds in genetics, neuroscience, and related disciplines. They receive support from IDDRC-administered programs: a NINDS-funded T32 Training Grant in Neurodevelopmental Disabilities, a CHOP Research institute-funded supplement program for clinical research fellows, and a CHOP-institute funded New Program Development award for Assistant Professors. They obtain multidisciplinary training that helps them become future leaders in IDD research. (Aim 3) Support Networking/Collaboration, Advocacy, and the Dissemination of IDD Research findings. The Center leadership will enable networking to support collaborative initiatives, both within the CHOP/Penn IDDRC community and between IDDRCs. Center leadership will advocate both internally and externally to advance an IDD research agenda. Finally, the Center will lead an effort to disseminate research advances to patients, their families, to government officials, and to other scientists. With this comprehensive approach, the IDDRC at CHOP and Penn will achieve our goal of advancing patient-centered innovative treatments for individuals with IDDs.

(CORE A: ADMINISTRATIVE CORE) PROJECT SUMMARY This Administrative Core will provide oversight and management of the Intellectual and Developmental Disabilities Research Center at the Children?s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (Penn). The center has three specific aims. First, we will lead an innovative, translational research agenda by supporting cores that continually evolve to provide cutting-edge technologies and meet the needs of center members. The center also supports a new research project that uses most of these cores to determine if magnetoencephalography (MEG) can be used to predict general cognitive ability and language-specific ability in children with a genetic risk for intellectual disability. Second, we will lead a career development initiative that supports multidisciplinary training. Third, we will promote interactions and collaboration, advocate for IDD research and its growth at CHOP/Penn and beyond, and we will ensure that advances in IDD research are rapidly disseminated to a wide variety of scientific and lay audiences with a focus on families of children with IDDs. The Administrative Core oversees management, strategy and vision for the IDDRC. It will: (1) Provide oversight of the cores including review and approval of new users, review of utilization, and survey user satisfaction; (2) Provide oversight of the research project, including regular progress meetings; (3) Provide oversight of core budget and monitoring their of cost-effectiveness; (4) Manage all training related activities, including organizing seminar series; (5) Maintain websites for the IDDRC and our training programs; (6) Coordinate meetings with internal, external, and patient advisory groups; (7) Manage all communication with NICHD; (8) Foster collaborations with other IDDRCs across the network; (9) Lead institutional, regional, and national advocacy; (10) Disseminate research findings to academic and community/family organizations and stakeholders; and (11) Organize activities that encourage networking and collaboration between cores, among center members, and with other centers and resources at CHOP/Penn. The center has three guiding principles. First, we seek to foster community, to encourage collaborative, inter- disciplinary research and to attract the next generation of scientists to the field. Second, solicit advice from our advisory committees to help the center continually evolve so that we meet the needs of our users and our community of constituents. Finally, we emphasize support of innovative, translational research that will define the etiology/pathophysiology of IDDs and lead to new interventions for children and adults with neurodevelopmental disabilities. Collectively, our activities are sharply focused on supporting research that has the potential to greatly impact the lives of those with an IDD.