Paul D. Drew - US grants

9982871
Drew

Major histocompatibilities (MHC) class I molecules bind virus and tumor derived peptides intracellularly and transport these peptides to the cell surface. Cells must express the MHC class I molecules in order to be identified and killed by immune cells termed T-cells. MHC class I molecules are constituitively found on almost all cells, except cells in the central nervous system (CNS). Within the CNS, inflammatory stimuli induce MHC class I surface expression on glia, but not neurons. This may protect neurons from destruction by the immune system. Recent studies have suggested that neurons regulate MHC class I expression in both neurons and glia. The current study is designed to determine the mechanisms underlying neuronal regulation of MHC class I expression on neurons and glia through cell-cell contact and soluble mediators. The studies will be achieved by applying standard immunocytochemical and molecular biology techniques to purified and mixed neuronal-glial cultures of rat embryonic hippocampal neurons, postnatal cerebella granule cell neurons, and postnatal astrocytes.

These studies will provide important information concerning the mechanisms by which neurons avoid immuno-surveillance, potentially resulting in persistent viral infections. In addition, the studies will provide new insights into the unique role that neurons play regulating immune function in the CNS.

DESCRIPTION (provided by applicant): The University of Arkansas for Medical Sciences (UAMS) seeks an NINDS Institutional Center Core Grant to Support Neuroscience Research. Neuroscience research is a major contributor to the recent growth of extramurally supported funding at UAMS, with a significant number of principal investigators (PIs) funded by the NINDS. The leadership of UAMS recognizes that having a greater number of NINDS-funded investigators will benefit the entire campus. One mechanism to achieve this increase is to establish a centrally located resource for all neuroscientists at UAMS, providing access to sophisticated equipment, trained technical staff, and the research expertise of established NINDS funded investigators. The long-term goal of this Neuroscience Center is to establish a facility that will foster interactions between members of the UAMS neuroscience community, leading to an increase in competitive research grants and the development of thematic program research. It is expected that creation of this center will serve as a recruiting tool to attract new faculty members with research interests in neuroscience, further expanding neuroscience research at UAMS. To achieve these goals the present proposal focuses on the development of three Cores: 1) Animal Surgery and Animal Model Development, 2) Histology and Image Analysis, and 3) Biochemistry, Cell and Molecular Biology. Four Specific Aims address the manner in which the proposed Core facilities will meet the present and future demands of NINDS-funded investigators, thereby ensuring continued productivity and expansion of research directions. Aim 1 is to provide a centralized facility for the three proposed Cores to enhance the research productivity of NINDS-funded investigators so they remain competitive in their research. Aim 2 is to encourage new interactions between NINDS-funded investigators by creating an environment conducive to conducting interdisciplinary neuroscience research. PIs with qualifying projects will have regular opportunities to share ideas and build collaborative relationships when a common Core facility is available. Aim 3 is to enhance the research expertise of neuroscientists at UAMS by providing specialized research resources currently unavailable to most investigators on campus. The application of new research techniques will enhance the present and future research objectives of funded researchers. Aim 4 is to increase the number of NINDS-funded investigators at UAMS by encouraging the interaction of neuroscientists currently without NINDS-funding with PIs conducting qualifying research. As neuroscience researchers continue working together to build strength in critical areas, the NINDS will benefit by working with institutions such as UAMS to develop state of the art, contiguous Core facilities that will support growth and integration of the campus-wide neuroscience community.

DESCRIPTION (provided by applicant): Fetal alcohol syndrome and related disorders occur in approximately 1% of live births in the United States and represent the most common cause of mental retardation. Fetal alcohol syndrome is commonly associated with significant lifetime disability, and thus the prevention and treatment of this syndrome is greatly needed. Normally, microglia protect neurons through the production of neurotrophic factors. However, microglia are resident CNS macrophages which play a critical role in the innate immune response to pathogens. Ethanol causes activation of microglia which may contribute to neuropathogenesis. Toll-like receptors (TLRs) respond to pathogens and endogenous "danger signals" produced following trauma or tissue injury. The mechanisms by which alcohol modulates microglia-neuronal interactions and neuropathogenesis in animal models of fetal alcohol syndrome have not been elucidated. The general hypothesis of the proposed studies is that TLRs expressed on microglia play a critical role in modulating ethanol induced neurodegeneration in the developing nervous system. The Specific Aims of the proposed research are as follows: f Specific Aim 1: Determine the mechanisms by which microglial TLRs modulate response to alcohol and neurodegeneration. The effects of alcohol on TLR signaling pathways in vitro will be examined in primary microglia. The role of specific TLR signaling molecules will be assessed in these studies utilizing microglia derived from animals in which these molecules have been knocked out. Co-culture paradigms will be used to assess the role of TLRs in modulating alcohol effects on neuron viability. f Specific Aim 2: Determine the effects of TLRs in modulating neurodegeneration in vivo in animal models of fetal alcohol syndrome. The effects of ethanol on specific TLR signaling pathways will be evaluated in studies utilizing animals in which key TLR signaling molecules have been knocked out. We have demonstrated that specific nuclear receptor agonists alter TLR signaling in microglia. We will evaluate the therapeutic potential of these agonists in modulating alcohol induced neurodegeneration in our animal model of fetal alcohol syndrome. The proposed studies will determine the mechanisms by which TLRs expressed on microglia modulate alcohol induced neurodegeneration in the developing nervous system. These studies have important implications concerning the treatment of fetal alcohol syndrome. PUBLIC HEALTH RELEVANCE: Fetal alcohol syndrome and related disorders occur in approximately 1% of live births in the United States and represent the most common non-genetic cause of mental retardation. Fetal alcohol syndrome is commonly associated with significant lifetime disability, and thus the prevention and treatment of this syndrome is greatly needed. The proposed studies are designed to evaluate the role of "danger signals" produced following alcohol exposure to the developing brain, with the goal of developing future therapies for fetal alcohol syndrome.

DESCRIPTION (provided by applicant): Functional Roles of Neuroimmune Factors in Mediating Behavior. Ethanol abuse at all life-stages can result in significant disability, and alcoholism has tremendous personal and societal consequences. Ethanol abuse at each of these life-stages is associated with inflammation in the central nervous system (CNS). This may contribute to neurodegeneration and impaired neurological function associated with excessive alcohol consumption. In addition, intriguing recent studies demonstrate that pro- inflammatory molecules including cytokines and chemokines modulate addiction to alcohol, which has resulted in a paradigm shift linking immune response to addictive behavior. The proposed studies will address a critical gap in our knowledge concerning the role of neuroinflammation in mediating ethanol-induced neurodegeneration throughout life. The general hypothesis of the proposed studies is that toll-like receptors (TLRs) expressed by resident CNS microglia respond to "danger signals" produced following ethanol exposure in the CNS resulting in the production of pro-inflammatory cytokines and chemokines that both modulate neurodegeneration as well as alcohol addiction. This will be tested in parallel in mice in vivo and in vitro in primary cultures, utilizing animals with targeted deletion of key TLR signaling molecules. f Specific Aim 1: Determine the mechanisms by which microglial TLRs modulate response to ethanol and neurodegeneration. f Specific Aim 2: Determine the effects of TLRs in modulating ethanol-induced neurodegeneration in vivo at different life-stages. f Specific Aim 3: Determine the effects of TLRs in modulating ethanol-induced neuroinflammation in vivo at different life-stages. PUBLIC HEALTH RELEVANCE: The proposed studies will determine the mechanisms by which TLRs expressed by microglia modulate ethanol induced neurodegeneration in the nervous system at different life-stages. These studies may have important implications concerning treatments designed to protect the brain from toxic effects of ethanol as well as

? DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorders (FASD) produces neurologic sequelae that persist throughout life and there is no treatment. The goal of our research is to understand the mechanisms that lead to the neurologic deficits of FASD and identify pharmaceutical interventions to prevent or treat these deficits. Damage to the cerebellum is common in FASD and contributes to deficits in motor function. Studies by us and others in rodent models of FASD reveal that fetal alcohol exposure produces loss of neurons in the cerebellum and deficits in motor function. Importantly, our studies in FASD models also reveal that alcohol induces neuroinflammation in the cerebellum. Furthermore, our studies indicate that treatment with anti-inflammatory PPAR-? agonists protect against alcohol-induced neuroinflammation and neuron loss. We hypothesize that ethanol activates specific immune signaling pathways resulting in neuroinflammation in the developing cerebellum which contributes to neuron loss and long-term behavioral deficits associated with FASD. As a corollary, ethanol-induced neuroinflammation, neuron loss, and behavioral deficits in the developing cerebellum can be blocked by anti- inflammatory agents including PPAR-? agonists. We will test this hypothesis using our well-established neonatal mouse model of FASD. Aim 1: Determine the molecular mechanisms of ethanol-induced neuroinflammation and PPAR-? agonist protection in the developing cerebellum. The role of TLR4 and CX3CL1-CX3CR1 signaling in ethanol-induced neuroinflammation and neuron loss will be probed using mice in which critical molecules in these signaling pathways are genetically knocked out. Furthermore, the mechanisms of PPAR-? agonist protection against ethanol will be explored by investigating agonist impact on neuroinflammation, neuron loss, TLR4 and downstream signaling, and CX3CL1-CX3CR1 signaling. Aim 2: Determine whether treatment with PPAR-? agonists will prevent ethanol-induced long-term motor function deficits in FASD and whether ethanol-induced neuroinflammation contributes to these deficits. The ability of PPAR-? agonists to protect against long-term ethanol-induced motor coordination, balance, and gait dysfunction will be assessed with CatWalk, beam walk, and rotarod behavioral analyses. Furthermore, we will determine if TLR-4 or CX3CL1-CX3CR1 signaling pathways play a critical role in ethanol-induced motor deficits using mice in which critical molecules in these signaling pathways are knocked out. Impact: This study will provide proof-of-principle that anti-neuroinflammatory agents including PPAR-? agonists can exert protective effects against neuroinflammation, neurodegeneration, and long-term behavioral deficits in FASD. This study will also define molecular mechanisms by which ethanol induces and PPAR-? agonists suppress neuroinflammation, neurodegeneration, and long-term behavioral deficits. These findings will foster development of new anti-inflammatory strategies for intervention in FASD.

PROJECT SUMMARY Fetal alcohol spectrum disorders (FASD) and associated neurologic sequelae occur in 1-5 of 100 births in the U.S. There is no cure. The overarching goal of our research is to understand the molecular mechanisms that lead to neurologic deficits of FASD and, with this knowledge, identify pharmaceutical interventions. Studies in FASD animal models show that alcohol (ethanol) causes loss of hippocampal neurons, impaired synaptic plasticity in hippocampal neurons, and deficits in learning and memory. Our studies in the neonatal mouse model of FASD demonstrate that ethanol induces neuroinflammation in the developing hippocampus including microglial activation and production of pro-inflammatory cytokines and chemokines. Ethanol induction of neuroinflammation in the hippocampus is particularly important to FASD since recent studies demonstrate neuroinflammation during brain development can produce life-long cognitive disorders. The studies proposed here will investigate whether ethanol-induced inflammation is linked to the long-term cognitive deficits in FASD. Further, our studies further demonstrate that treatment with anti-inflammatory peroxisome proliferator activated receptor-? (PPAR-?) agonists protect against ethanol-induced microglial activation and expression of inflammatory molecules in the developing hippocampus. Collectively, this evidence suggests neuroinflammation in the developing hippocampus may contribute to long-term cognitive deficits in FASD. However, critical gaps in knowledge must be addressed before this information can be used toward the treatment of FASD. The proposed studies will test the hypothesis that ethanol induction of neuroinflammation contributes to long-term learning and memory deficits associated with FASD, and anti-inflammatory agents, including PPAR-? agonists, can block these ethanol effects. We will test this hypothesis using our well- established neonatal mouse model of FASD, which represents human third-trimester fetal alcohol exposure. Aim 1 will determine the mechanisms of ethanol-induced neuroinflammation in the developing hippocampus. The role of (A) TLR4 and downstream signaling, (B) NLRP3 inflammasome activation, and (C) CX3CL1? CX3CR1 signaling in ethanol-induced neuroinflammation and neuron loss will be investigated using transgenic mice with genetic knockout of key molecules in these signaling pathways. Aim 2 will determine if ethanol- induced neuroinflammation contributes to long-term impairment of hippocampal synaptic plasticity and long- term learning and memory deficits in FASD, and if suppression of neuroinflammation prevents these deficits. (A) Establish whether TLR4 and downstream signaling, NLRP3 inflammasome activation, and/or CX3CL1? CX3CR1 signaling play a critical role in long-term synaptic plasticity and learning and memory deficits using transgenic mice with genetic knockout of key molecules in these signaling pathways. (B) Evaluate whether anti-inflammatory PPAR-? agonists protect against ethanol impairment of synaptic plasticity and cognitive deficits.

PROJECT SUMMARY Fetal alcohol spectrum disorders (FASD) and associated neurologic sequelae occur in almost 5% of children in the U.S. ? the deficits are life-long ? and there is no cure. The goal of the proposed studies is to define the dynamic roles of microglia and astrocytes in ethanol-induced neuroinflammation and neuropathology in FASD. Ethanol-induced neuroinflammation in the developing CNS is believed to contribute to the long-term sequelae associated with FASD. However, critical gaps in knowledge remain concerning the dynamic interactions between microglia, astrocytes and neurons which mediate the transition from normal CNS development to FASD neuropathology. Furthermore, the cellular and molecular mechanisms mediating transition to and progression of the neuropathology have not been elucidated, which prevents targeted treatment. The proposed studies directly address these critical gaps in knowledge. Hypothesis: Alcohol exposure in the developing CNS elicits neuroimmune activation of microglia and astrocytes which disrupts normal dynamic interactions between these glial cells and neurons during onset and progression of FASD, producing neuropathology. Further, elucidating molecular mechanisms (currently unexplored) that regulate ethanol-induced neuroinflammation, neuron loss, and alterations in normal glia-neuron interactions will reveal therapeutic targets for FASD. Aim 1. Determine the dynamic role of microglia and astrocytes in the transition from normal cerebellar development to ethanol-induced neuropathology and progression of FASD. (SubAim A) Evaluate changes in cell-cell interactions between microglia, astrocytes, and neurons visualized at the level of glial processes and neuronal dendrites, spines, and somas. This will be done with high resolution 3D reconstructions. Define and compare the temporal pattern of neuroinflammation and neuron loss following ethanol exposure. Neuroinflammation will be assessed by glial activation and by directed qRT-PCR arrays and multiplex protein arrays of immune molecules. Purkinje neuron loss will be assessed by unbiased stereology. (SubAim B) Probe dynamic changes in the transcriptome using unbiased RNA-Seq and bioinformatic analyses. Analyses will be performed on the whole cerebellum as well as purified glia and neurons. (SubAim C) Examine the role of specific cell types in ethanol-induced neuroimmune signaling and neuropathology. This will be achieved through cell-type specific knockout of Toll-like Receptor (TLR)4 in astrocytes or microglia. Aim 2. Quantify ethanol-induced changes in dynamic microglia-neuron and astrocyte-neuron interactions in vivo in the transition from normal cerebellar development to ethanol-induced neuropathology and progression of FASD. (SubAim A) Establish whether ethanol alters microglia interactions with dendrites and spines. (SubAim B) Determine whether ethanol alters astrocyte signaling in response to neuronal activity and interactions with neurons. Studies in Aim 2 will use in vivo time-lapse two-photon imaging and immuno-electron microscopy.