Anna Dunaevsky - US grants

DESCRIPTION A major question in developmental neurobiology is how neuronal cells develop into their mature phenotypes. This proposal investigates the development of the Purkinje neuron in relation to one of its afferents, cerebellar granule cells. These developmental processes are examined in a noel culture system in which purified Purkinje cells undergo full dendritic differentiation when co-cultured with purified granule cells as well as in cerebellar slices. The culture system provides a unique opportunity to study the dynamics of Purkinje cell development nd its interactions with one of its presynaptic cells without the confounding influence of many other cell types that have been present in previous work. In the first set of experiments the dynamics of behavior of developing dendritic spines of the Purkinje cells will be characterize in culture and slices using time lapse microscopy. These experiments will provide a bioassay for testing the role of agents that could be provided by granule cells in the development of dendritic spines. As a first step, the role of BDNF in spine formation will be tested. In the second set of experiments ultrastructural and molecular analysis of Purkinje-granule cell synapse formation will be performed. The process of assembly of molecular components of a functional synapse will be monitored on both the structural (electron microscopy and immunocytochemistry) and functional level (synaptic vesicle recycling. These studies will provide much needed information about the process of development of an important central synapse.

[unreadable] DESCRIPTION (provided by applicant): The major goal of this R21 Exploratory/Developmental grant proposal is to develop a new model in which the morphological correlates of synaptic strengthening induced by learning can be studied. Learning a new forelimb reach and grasp task has been shown to leave a trace in the forelimb area of the primary motor cortex (MI): synapses are strengthened in layer II/III intracortical horizontal connections in the hemisphere controlling the trained forelimb. This proposal will test the hypothesis that changes in synaptic morphology and dynamics underlie this learning-induced synaptic strengthening in the adult MI. Layer II/III neurons in MI will be labeled by in vivo viral injections. The first aim will determine changes in spine density and morphology in slices from trained rats and correlate them to synaptic strength changes and long-term potentiation. The second aim will determine changes in dynamics of dendritic spines by two photon repeated In vivo imaging of MI neurons at different time points during skill learning and consolidation. This model will link a quantifiable behavior (forelimb reach and grasp task) with changes in synaptic strength, and changes in morphology and dynamics of synaptic structures. The strength and uniqueness of the proposed project is the combination of powerful two-photon live imaging techniques with a model for studying learning-induced changes in synaptic efficacy. If successful, this exploratory/developmental grant (R21) will be expanded to a five-year proposal to study the molecular basis of learning-induced morphological changes in synaptic strength and morphology. Information gained from these studies will be crucial in ultimately understanding the mechanisms of memory loss during pathological conditions and aging. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the molecular and cellular mechanisms that regulate the development of cerebellar neuronal circuits. Dendritic spines, sites of synaptic input on many projection neurons such as the cerebellar Purkinje cell are highly dynamic structures and their motility is developmentally regulated. The mechanisms that regulate spine dynamics over development are not known. Here, we will use multiphoton live imaging of neuronal structures in organotypic slices and in vivo in conjunction with electron microscopy to study the mechanisms of synaptic maintenance. Our central hypothesis is that ensheathment by glial processes critically regulates dendritic spine motility and synaptic stability. In the first aim, we will characterize the development of Bergmann glia processes using static and dynamic imaging approaches. In the second aim, we will test how spine dynamics are regulated by glial ensehathment by measuring spine motility in genetic models with reduced glial ensheathment. In the third aim, we will determine the role of EphA receptors and the ephrin ligands in glia-spine cross talk and regulation of spine dynamics. Finally, we will determine how synaptic maintenance is related to spine motility and is regulated by glial processes. Abnormal development of neuronal connections can be the cause of neurodevelopmental disorders in humans. Moreover, recently it has been demonstrated that abnormal glial-neuronal interactions during development might cause mental disorders in the adult. Therefore, understanding the cellular and molecular mechanisms of glial-neuronal interactions during synapse formation and maintenance has important health significance. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is the most common inherited form of an intellectual disability. Children with FXS have been found to have a developmental impairment in the performance of learned skilled limb movements. Motor skill learning is thought to require synaptic plasticity in the primary motor cortex (M1). To better understand how neuronal communication changes with motor learning, it is necessary to determine if learning can induce changes in number, morphology, efficacy, and molecular composition of synapses. FXS results from mutation that causes silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP). Here we will use the fmr1 KO mouse, a murine model for FXS, to study the mechanisms of learning in the primary motor cortex. Our goal is to understand how fmr1 contributes to regulation of synaptic plasticity in the motor cortex and thus elucidate the mechanisms of motor skill learning deficits in the fmr1 KO. We will combine behavioral, electrophysiological, pharmacological, 2-photon imaging and molecular approaches to characterize the changes that occur at synapses in M1 following the learning of a new motor skill in the fmr1 KO mouse. This work is expected to provide important knowledge to develop therapies for FXS and other neurodevelopmental disorders such as autism, a mission of the NIH.

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a diverse disorder that is likely to be caused by a combination of genetic alterations and environmental insult during early development. Studies demonstrate an association between maternal immune activation (MIA) during pregnancy and an increased risk for ASD. Recent development of mouse models for prenatal exposure to maternal immune activation (MIA) show that the challenged offspring demonstrate impaired behaviors relevant to ASD as well as exhibit altered levels of immune proteins. A significant gap exists in understanding how altered immune state interacts with ASD risk genes to result in impaired behaviors. Here we will test the hypothesis altered regulation of MHC1 proteins is a mechanism of convergence for MIA and ASD genetic risk factors. Specifically, we will examine how mutations in mecp2, a gene responsible for the Rett syndrome and also associated with non-Rett ASD cases, interact with MIA. We will, combine behavioral, molecular, electrophysiological and synaptic in vivo imaging analyses in relevant brain circuits to determine how altered Mecp2 levels and MIA converge to results in altered behavior and neuropathology.

? DESCRIPTION (provided by applicant): Astrocytes, non-neuronal cells in the brain, are in intimate structural relationship with synaptic contacts and are emerging as active players in the development, function and plasticity of synapses. Impairments in astrocytes are increasingly implicated in multiple brain disorders with cognitive impairments including autism and schizophrenia and therefore might be contributing to the synaptic impairments in these neurodevelopmental disorders. The goal of this proposal is to fill the gap on the role of astrocyti signaling in molecular and structural synaptic remodeling during development and learning. Here, we will test the hypothesis that activity in astrocyte processes surrounding dendritic spines leads to maturation and stabilization of dendritic spines during development and learning. To test the hypothesis, we will combine in vivo imaging of dendritic spines with astrocytic calcium during development and learning in conditions with altered astrocytic calcium signaling.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and one of the main identified genetic causes of autism spectrum disorder. Astrocytes, a type of glial cells, perform multiple tasks in the brain and have been implicated in regulating synaptic development, function and plasticity. Yet the role astrocytes play in neurodevelopmental disorders is not well understood. In this proposal, we aim to identify the contribution of astroglial Fragile X Mental Retardation Protein (FMRP) to altered astrocyte Ca2+ signaling, synaptic deficits and cortical plasticity impairments associated with FXS. We approach this through three aims which utilize mouse genetics, in vivo imaging of Ca2+ signaling and synaptic protein trafficking, cell and molecular techniques and mouse behavior. In Aim 1, we will study the role of FMRP in regulating astrocytic Ca2+ signaling. We will perform in vivo imaging of astrocytic Ca2+ signaling in awake fmr1 KO mice and in transgenic mice in which FMRP is selectively deleted or exclusively expressed in astrocytes. In Aim 2, we will examine mechanisms of altered astrocytic Ca2+ signaling in FXS with cell, molecular and pharmacologic experiments. In Aim3, we will examine if loss of FMRP in astrocytes results in basal and learning-induced alterations in functional and structural synaptic plasticity by electrophysiology and in vivo imaging of tagged AMPAR trafficking in dendritic spines, in astrocyte-specific FMRP knockout mice. This work is expected to provide important knowledge about the role of astrocytes in FXS and has the potential to identify novel therapeutic targets for FXS.

Abstract/Summary: Overall Component This application for a Center of Biomedical Research Excellence (COBRE) would initiate a formal Center to lead, support, and expand neuroimaging and clinical cognitive neuroscience research in Nebraska, with an emphasis on lifespan development. It is an opportune moment for human neuroscience research in the Omaha community, regionally, and across the world, with many new research tools and government initiatives intended to illuminate the next frontier of biomedicine. The proposed Cognitive Neuroscience of Development and Aging (CONDA) Center will provide critical resources and support to faculty from four institutions: the University of Nebraska Medical Center (UNMC), the Boys Town National Research Hospital, the University of Nebraska ? Omaha, and Creighton University. The Administrative Core of the CONDA Center will be housed at UNMC, which is less than two miles from each of the other institutions, and will be comprised of Training, Evaluation, Engagement, Administration, and Mentoring areas (i.e., the TEEAM Core). The new CONDA Center will also include a state- of-the-art Neuroimaging Acquisition and Analysis Core, as well as extensive support for four primary COBRE research projects led by a promising group of NIH-defined early-stage investigators. Upon initiation of the Center, the TEEAM Core would immediately implement a series of new programs that are designed to develop human cognitive neuroscience on the CONDA Campus and across the region. The TEEAM Core would also rapidly implement a comprehensive research support structure that includes the Neuroimaging Core facility, training opportunities, pilot projects and mini-grants programs, a new seminar series, postdoctoral fellowships, intern- ships for growing temporary and long-term laboratory staffing, and a participant registry to enhance recruitment. In parallel, the TEEAM Core would promote the successful launch of the primary COBRE research projects and, through a mentoring network approach, implement a career development and evaluation support system to monitor progress and ensure Junior PIs and their mentoring teams reach critical milestones. As per the new research core, the Neuroimaging Acquisition and Analysis Core facility would be the only core dedicated to human brain research in the state of Nebraska, and would provide regional scientists with state-of-the-art tools for neuroimaging and neuromodulation, including research-dedicated MRI and MEG systems. The new Center would also receive exceptional institutional support, including financial support for CONDA programs, major equipment expenses, and Core staffing. The overall CONDA team includes an established group of PIs in neuro- imaging, brain dynamics, aging, and brain and cognitive development, as well as a strong cohort of emerging junior investigators using innovative cognitive and affective neuroscience methods to address major questions in human neuroscience across the lifespan. These junior scientists were all hired in the past three years, and this new wealth of concentrated local expertise has primed us for a trajectory of accelerated development of neuro- science and neuroimaging toward national prominence, an aim for which the new CONDA Center will be integral.

This application for a Center of Biomedical Research Excellence (COBRE) would initiate a formal Center to lead, support, and expand neuroimaging and clinical cognitive neuroscience research in Nebraska, with an emphasis on lifespan development. It is an opportune moment for human neuroscience research in the Omaha community, regionally, and across the world, with many new research tools and government initiatives intended to illuminate the next frontier of biomedicine. The proposed Cognitive Neuroscience of Development and Aging (CONDA) Center will provide critical resources and support to faculty from four institutions: the University of Nebraska Medical Center (UNMC), the Boys Town National Research Hospital, the University of Nebraska ? Omaha, and Creighton University. The Administrative Core of the CONDA Center will be housed at UNMC, which is less than two miles from each of the other institutions, and will be comprised of Training, Evaluation, Engagement, Administration, and Mentoring areas (i.e., the TEEAM Core). The new CONDA Center will also include a state- of-the-art Neuroimaging Acquisition and Analysis Core, as well as extensive support for four primary COBRE research projects led by a promising group of NIH-defined early-stage investigators. Upon initiation of the Center, the TEEAM Core would immediately implement a series of new programs that are designed to develop human cognitive neuroscience on the CONDA Campus and across the region. The TEEAM Core would also rapidly implement a comprehensive research support structure that includes the Neuroimaging Core facility, training opportunities, pilot projects and mini-grants programs, a new seminar series, postdoctoral fellowships, intern- ships for growing temporary and long-term laboratory staffing, and a participant registry to enhance recruitment. In parallel, the TEEAM Core would promote the successful launch of the primary COBRE research projects and, through a mentoring network approach, implement a career development and evaluation support system to monitor progress and ensure Junior PIs and their mentoring teams reach critical milestones. As per the new research core, the Neuroimaging Acquisition and Analysis Core facility would be the only core dedicated to human brain research in the state of Nebraska, and would provide regional scientists with state-of-the-art tools for neuroimaging and neuromodulation, including research-dedicated MRI and MEG systems. The new Center would also receive exceptional institutional support, including financial support for CONDA programs, major equipment expenses, and Core staffing. The overall CONDA team includes an established group of PIs in neuro- imaging, brain dynamics, aging, and brain and cognitive development, as well as a strong cohort of emerging junior investigators using innovative cognitive and affective neuroscience methods to address major questions in human neuroscience across the lifespan.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and one of the main identified genetic causes of autism spectrum disorder. Our understanding of neuropathophysiology of FXS is mainly gained from studies in the fmr1 null mouse model. However, these studies don?t always translate to the human condition due to species and possibly the mode of gene silencing in FXS. These limitations have been recently circumvented by the advent of human induced pluripotent stem cells (hiPSCs), and the generation of FXS patient-derived hiPSCs, which presents an opportunity for studying the pathogenesis of FXS with unlimited human brain cells. Although astrocytes, non-neuronal cells in the brain, have extensive roles in normal brain function, are increasingly implicated in multiple brain disorders and express FMRP into adulthood, a comprehensive understanding of the role of astrocytic FMRP in the pathogenesis of FXS is still lacking. We will therefore develop a human cellular model of FXS by differentiating astrocytes from control and FXS induced hiPSCs. In order to determine how FXS astrocytes are impaired and how they impact formation and function of neural circuits, we propose to create a humanized chimeric mouse model with hiPSCs. In Aim1 we will examine how FXS astrocyte morphology, Ca2+ signaling and gene expression are impaired in vivo. In Aim 2, we will determine the effect of FXS hIPSC-astrocytes on neuronal structure and function. This work is expected to provide important knowledge about the role of human specific astrocytes in FXS and has the potential to identify novel therapeutic targets for FXS.

Abstract/Summary: Administrative (TEEAM) Core The Cognitive Neuroscience of Development and Aging (CONDA) Center will be a NIGMS-supported COBRE located in Omaha, NE, with a focus on human neuroscience and neuroimaging research across the lifespan. The Administrative Core will be essential to the development, support, and evaluation of all major components of this Center. Given these responsibilities, the Administrative Core will be known as the Training, Evaluation, Engagement, Administration, and Mentoring (TEEAM) Core. The Core will be directed by Dr. Tony Wilson of the University of Nebraska Medical Center, with the assistance of a Master?s-level Scientific Administrator. Upon establishment of the CONDA Center, the TEEAM Core will launch a series of new and integrated programs, each aimed at supporting one or more of its requisite areas. Specifically, the Training area will be served by a series of annual neuroimaging and neuromodulation workshops, as well as a postdoctoral fellow- ship program. The Evaluation area will be served by research development forums and will use other tools for assessing progress and milestone achievement in the junior COBRE PIs. The community Engagement area will support two outreach programs, NeuroWow and BrainFit, to engage youth and senior citizens, respectively, through organized events that help educate the public, promote STEM interest and careers in the Omaha area, and increase the public?s pride in regional science. The youth-oriented NeuroWow program has already been active for several years and will be expanded through additional support from the Center, while the BrainFit program will be the first of its kind in the region. The Administration area will oversee a Summer Scholars program that offers 10-week research internships in the Center to promising undergraduates, administrate a Pilot Projects program for faculty and a Mini-Grants program for postdoctoral fellows and graduate students that together will award about $180,000 annually, and invest financial and material resources into building a participant registry. The Administration area will also host the monthly CONDA Seminar Series, which will bring internationally-known cognitive neuroscientists to Omaha to present their latest findings and network with local scientists, potentially fostering new collaborations with Center members. Finally, the Mentoring area will use a network approach comprised of both academic and technical mentors to help ensure a successful transition of the junior COBRE PIs to independent investigators, guide postdoctoral fellows toward faculty positions, and assist junior and senior PIs who wish to enter new research areas relevant to the Center. Through these targeted programs and a strong network of established mentors and advisors, the TEAAM Core will support four junior COBRE research projects led by NIH-defined early-stage investigators (ESI), a state-of-the-art Neuroimaging Acquisition and Analysis Core Facility, and a collection of existing extramurally-funded research projects led by a team of senior investigators interested in human developmental and aging neuroscience.