Carlos F. Valenzuela - US grants

DESCRIPTION: Alcohol abuse and alcoholism are serious health problems in the United States. However, the molecular bases of alcohol's actions and alcoholism are unknown. The overall goal of this proposal is to study the molecular mechanism(s) of action of alcohol at the neurotransmitter-gated ion channel level. The effects of ethanol on GABA-A and NMDA receptors, the major inhibitory and excitatory neurotransmitter-gated ion channels in the mammalian central nervous system, respectively, will be studied. The function of these ion channels is regulated by protein phosphorylation/dephosphorylation. This regulatory process is mediated by multiple kinases and phosphatases, and appears to be important for ethanol's actions. A combination of electrophysiological and biochemical techniques in cultured transfected cell lines is proposed to be used to study the role of protein phosphorylation in the ethanol-induced effects on these channels. Electropysiological experiments will determine the effect of ethanol on receptor function. Immunoprecipitation experiments in the same batch of cells will directly examine the phosphorylation state of GABA-A and NMDA receptor subunits in the presence of ethanol. If ethanol changes the phosphorylation state of receptor subunits, then phosphoamino acid and phosphopeptide analyses, and studies with kinase & phosphatase inhibitors, will determine which kinases or phosphatases are required for ethanol's actions. Correlation of electrophysiological and site-directed mutagenesis experiments will determine whether phosphorylation of specific amino acids in GABA-A AND NMDA receptor subunits is required for ethanol's actions. The findings of the proposed experiments will contribute toward a better understanding of the actions of ethanol in the central nervous system.

The brain is severely affected by prenatal alcohol exposure resulting in neurobehavioral deficits that have devastating effects later in life. Therefore, it is important to develop therapeutic measures that minimize these noxious effects of fetal alcohol exposure. Understanding the cellular and molecular mechanism of the prenatal effects of ethanol is a prerequisite to developing such treatments. Among the neuronal proteins affected by in utero ethanol exposure are the NMDA receptors (NMDA- Rs), which play important roles in neuronal development, plasticity and death. A number of studies indicate that prenatal ethanol exposure affects NMDA-Rs; however, the mechanism of this effect of ethanol has not yet been elucidated. Our hypothesis is that prenatal ethanol exposure alters the actions of neurosteroids, which are endogenous modulators of NMDA-Rs. The research design includes exposure of rats during pregnancy to a diet that results in blood alcohol concentrations near the legal intoxication limit in humans (approximately 0.08 g/dl). Aim #1 is to measure the effects of fetal ethanol exposure on the regulation of NMDA-Rs by different classes of neurosteroids. We will use electrophysiological techniques to study the actions of neurosteroids that have been shown to either potentiate or inhibit NMDA-R function. These studies will be performed in cultured hippocampal neurons from neonatal rats as well as hippocampal slices from adult rats. Aim #2 is to determine the mechanism of the prenatal ethanol-induced alterations o the modulatory actions of neurosteroids on NMDA-Rs. Subunit composition and protein phosphorylation are factors that could regulate the sensitivity of NMDA-Rs to neurosteroids. We will use Western blot, radioimmunohistochemistry and single-cell RT-PCR techniques to assess subunit composition. We will test the effect of inhibitors and/or activators o protein kinases and/or phosphatases on neurosteroid sensitivity of NMDA-Rs in cultured neurons and hippocampal slices. Aim #3 is to determine the effect of fetal alcohol exposure on the hippocampal levels of neurosteroids that regulate NMDA-R function. We will use radioimmunoassays to determine levels in hippocampi from embryos, and neonates as well as adult rats. Importantly, studies have shown that prenatal stress produced by fetal alcohol exposure results in a reduction in the responsiveness to the neurobehavioral effects of neurosteroids later in life. The experiments that we propose to do could increase our understanding of the mechanism of this ethanol-induced decrease in sensitivity to neurosteroids. Moreover, these experiments will increase our knowledge of the roles played by hippocampal NMDA-Rs in the pathogenesis of alcohol-related birth defects.

DESCRIPTION (provided by applicant): Ethanol at concentrations greater than or qual too 0.08 g/dl significantly impairs motor skills and this effect is responsible for the majority of traffic accident-related deaths in the United States. Importantly, individuals with low sensitivity to this action of ethanol are more likely to develop alcoholism. In addition, chronic alcoholism and fetal alcohol syndrome are associated with significant decreases in the number of cerebellar neurons. Therefore, understanding the mechanism of action of ethanol in the cerebellum is an area of tremendous interest. Neurons of the cerebellar cortex play a central role in the control of motor functions. These neurons form a basic circuit unit and Purkinje neurons are the main output of this circuit. Purkinje neurons receive excitatory inputs from the spinal cord and brain stem via mossy fibers and from the inferior olive via climbing fibers. These neurons also receive inhibitory input from molecular layer interneurons. Excitatory input from the mossy fibers is relayed to Purkinje neurons by the cerebellar granule cells and these cells are under the inhibitory control of Golgi interneurons. Preliminary data indicate that acute ethanol exposure increases GABAergic tone at Golgi interneuron-to-granule cell synapses and at molecular layer interneuron-to-Purkinje neuron synapses. Moreover, it also inhibits excitatory input at climbing fiber-to-Purkinje neuron synapses. We hypothesize that ethanol depresses synaptic transmission in the mature cerebellar cortex by presynaptically modulating neurotransmitter release at major synapses within the basic circuit unit. We propose to use the acute cerebellar slice preparation and patch-clamp electrophysiological techniques to test this hypothesis. Specific Aim #1 is to investigate the acute effect of ethanol on neurons of the granule cell layer. We will assess the effect of ethanol on GABAergic transmission at granule cells and its impact on the excitability of these cells. We will also evaluate the effects of ethanol on Golgi cell excitability. Specific Aim #2 is to investigate the acute effects of ethanol on neurons of the Purkinje and molecular layers. We will measure the effects of ethanol on Purkinje cell firing in response to stimulation of the mossy fiber granule cell pathway or the parallel fiber pathway. We will assess the effect of ethanol on the frequency of mIPSCs evoked by action potential-independent GABA release at molecular layer interneuron-to-Purkinje cell synapses. Finally, we will evaluate the mechanism by which ethanol affects the complex spike evoked in Purkinje cells by stimulation of climbing fibers. Together, these experiments will provide a comprehensive view of the acute effects of ethanol on mature cerebellar synapses of the cerebellar cortex. This type of comprehensive study has not been performed before and it will significantly increase our understanding of the mechanism of action of ethanol in the central nervous system.

DESCRIPTION (provided by applicant): Fetal alcohol spectrum disorder (FASD) is a prevalent disorder characterized by learning and memory deficits that are likely a consequence of alterations in synapse formation, refinement and/or maintenance. During the previous funding period, we showed that 3rd trimester-equivalent ethanol (EtOH) exposure alters activity- dependent plasticity mechanisms that are essential for synapse maturation. In the most recent of these studies, we demonstrated that EtOH potently inhibits a form of synaptic plasticity that depends on local, retrograde release of BDNF from CA3 pyramidal neuron dendrites that is triggered by activation of L-type voltage-gated Ca2+ channels (L-VGCCs). Our hypothesis is that chronic EtOH exposure during the 3rd trimester equivalent persistently inhibits L-VGCCs, leading to a decrease in synaptic plasticity dependent on local retrograde BDNF release and ultimately causing delayed maturation of CA3 pyramidal neuron synapses. Aim #1 is to test the hypothesis that chronic EtOH causes persistent L-VGCC inhibition by inducing channel degradation via depletion of internal Ca2+ stores and STIM1 binding to 11 subunits. Using slice electrophysiological and Ca2+ imaging techniques, we will assess whether EtOH exposure causes persistent functional inhibition of L-VGCCs. We will also determine whether EtOH exposure decreases plasma membrane expression of L-VGCC subunits using a surface biotinylation assay. Using Ca2+ imaging, co- immunoprecipitation and immunhistochemical techniques, we will investigate if these effects are a consequence of depletion of internal Ca2+ stores and STIM1 binding to CaV11.2/1.3. Aim #2 is to test the hypothesis that chronic EtOH inhibits L-VGCC/BDNF-dependent plasticity at mossy fiber-CA3 pyramidal neuron synapses. At these synapses, L-VGCC-dependent retrograde release of BDNF induces spike timing- dependent long-term potentiation and we will investigate if this form of synaptic plasticity is inhibited by EtOH exposure using slice electrophysiological techniques. Aim #3 is to test the hypothesis that EtOH-induced inhibition of L-VGCC-dependent retrograde release of BDNF impairs the maturation of interneuron- and MF- CA3 pyramidal neuron synapses using slice electrophysiological and immunohistochemical techniques. We will also use a novel in vivo neonatal electroporation/shRNA experimental paradigm to determine if selective downregulation of L-VGCC or BDNF expression in CA3 pyramidal neurons mimics the effect of chronic EtOH on development of these synapses. Collectively, the proposed studies will define L-VGCC/BDNF dysfunction as a key element in the pathophysiology of FASD, forming the basis for the rational development of therapeutic interventions against this prevalent disorder. Results will also provide strong evidence supporting the recommendation that even light drinking during the 3rd trimester could adversely affect fetal brain development.

Project Summary This is a competitive renewal application for an Alcohol Training Program in Neurosciences at the University of New Mexico (UNM-ARTN). This program is currently ending Year 14 of support. It has had a tremendously positive impact on graduate education at our institution and has increased visibility of alcohol research across campus. The program provides multidisciplinary training that includes molecular biological, biochemical, electrophysiological, imaging, and behavioral techniques. The focus of the UNM-ARTN program is to provide training in alcohol neuroscience research. Our group has strengths in three areas of fetal alcohol spectrum disorder research: 1. Basic mechanisms involved in ethanol neuroteratogenesis; 2. Biomarkers of prenatal alcohol exposure; and 3. Therapeutic interventions to ameliorate FASD-related cognitive deficits. A total of 12 faculty members from the Departments of Neuroscience and Psychology will be directly involved in the program as core faculty. These faculty members are well-funded and productive, and have extensive collaborative interactions in terms of research grants, publications, and mentoring of graduate students. Students will be from the Biomedical Sciences Graduate Program of the Health Sciences Center and the Cognition, Brain and Behavior Program of the Psychology Department. The program will support 4 Ph.D. students per year. The program will be overseen by Dr. Valenzuela, a steering committee composed of members of the training faculty, and an external advisory committee. Our trainees have been very successful in terms of obtaining individual predoctoral fellowships, presenting at scientific meetings, and publishing in well-respected journals. Our goal is to continue to provide high quality graduate students with the necessary training to prepare them for a successful future career in alcohol research.

Absolute ethanol; Acute; Affect; Affinity; Alcohol, Ethyl; Alcohols; Ammon Horn; Animals; Area; Arts; Aspiration, Respiratory; Assay; Attention; Autism-Dementia-Ataxia-Loss of Purposeful Hand Use Syndrome; BDNF; BDNF gene; Binding; Binding (Molecular Function); Binding Proteins; Bioassay; Biologic Assays; Biological Assay; Blotting, Western; Brain-Derived Neurotrophic Factor; Breathing; CHIP assay; Cerebroatrophic Hyperammonemia; ChIP (chromatin immunoprecipitation); Chemical Class, Alcohol; Comb animal structure; Combs; Common Rat Strains; Complement; Complement Proteins; Condition; Cornu Ammonis; DNA; DNA Chips; DNA Methylation; DNA Microarray; DNA Microarray Chip; DNA Microarray format; DNA Microchips; Data; Deoxyribonucleic Acid; Development; Dysfunction; ETOH; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Ethanol; Exposure to; FASD; Family member; Fetal Alcohol Spectrum Disorder; Functional disorder; Funding; Future; Gene Expression; Gene Targeting; Gene Transcription; Genes; Genetic; Genetic Alteration; Genetic Change; Genetic Transcription; Genetic defect; Grain Alcohol; Grant; Hippocampus; Hippocampus (Brain); Histones; In Vitro; Inhalation; Inhaling; Inspiration, Respiratory; Laboratories; Last Trimester; Ligand Binding Protein; Link; Literature; MGC34632; Mammals, Rats; MeCP-2 protein; MeCP2; MeCP2 protein; Mediating; Mental Retardation; Methods and Techniques; Methods, Other; Methyl CpG Binding Protein 2; Methyl CpG binding protein (MeCP2); Methyl CpG binding protein MeCP2; Methyl-CpG binding protein 2; Methyl-CpG-Binding Protein 2; Methyl-DNA binding protein MECP2; Methylcarbinol; Modification; Molecular Interaction; Mutation; Neonatal; Nerve Cells; Nerve Unit; Neural Cell; Neural Development; Neurobiology; Neurocyte; Neurodevelopmental Disorder; Neurological Development Disorder; Neurons; Numbers; Overexpression; Personal Satisfaction; Physiologic; Physiological; Physiopathology; Pilot Projects; Pregnancy Trimester, Third; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Protein Binding; Protein Family; Protein Overexpression; Proteins; RNA Expression; Rat; Rattus; Regulation; Reporting; Research; Rett Disorder; Rett Syndrome; Rett syndrome (RS, RTS); Role; Secondary to; Slice; Syndrome; Targetings, Gene; Techniques; Testing; Third Pregnancy Trimester; Transcription; Transcription, Genetic; Trimester, Third; Western Blotting; Western Blottings; Western Immunoblotting; Work; alcohol effect; alcohol exposed; alcohol exposure; base; chromatin immunoprecipitation; chromatin remodeling; ethanol effect; ethanol exposed; ethanol exposure; experience; experiment; experimental research; experimental study; exposed to alcohol; exposure to alcohol; fetal; gene product; genome mutation; hippocampal; in vivo; in vivo Model; infancy; infantile; inspiration; methyl-CpG (cytosine-guanine dinucleotide) binding protein 2; neonatal exposure; neurobiological; neurodevelopment; neuronal; overexpress; pathophysiology; pilot study; protein blotting; research study; social role; well-being

The overall goal of the Pilot Project Core of the New Mexico Alcohol Research Center (NMARC) is to foster cross-disciplinary FASD research across campus and promote growth of the Center. The Core will provide a critical opportunity to develop new basic, translational and clinical fetal alcohol research projects that complement and broaden the NMARC's research program portfolio. Selected projects must fit within the context of the Center's three strategic objectives as well as build on the overall integration and sustainability of the Center's research activities. Pilot projects will be funded for up to 2 years. The goal of each pilot project is to obtain sufficient preliminary data to develop competitive research grant applications and aid in cultivating new investigators that will integrate into the center; pilot project funding from our P20 developmental center was instrumental for the success of several NIH grant applications by UNM investigators. This Core will primarily support new investigators that have yet to obtain substantial independent research support. A year prior to the end of the Pilot Projects that are funded initially, a call for additional Pilot Project proposals will be widely advertised within the scientific community at UNM and the Mind Research Network. These proposals will be evaluated by extramural reviewers for scientific merit and relevance to the strategic objectives of the Center. Subsequently, projects will be reviewed by the Steering Committee and recommendations for funding support will be made by the NMARC Program Advisory Committee. Funding for these newly approved projects would begin at the start of the third project year. Each project will have an Advisory Team composed of senior alcohol researchers with complementary scientific expertise to that of the pilot project PI. The Center Director and NMARC Steering Committee will monitor progress of all pilot projects. The Pilot Project Core will support three projects during the first two years. Pilot Project 1, co-directed by Drs. Steffen Brown and Ludmila Bakhireva is a human 3D ultrasound imaging/Doppler velocimetry study that will assess the impact of prenatal ethanol exposure on brain structure and fetal hemodynamics. Results of this study will test the utility of these imaging techniques in the early diagnosis of FASD. Pilot Project 2, directed by Dr. Jason Weick will examine the effect of ethanol on cellular composition and network behavior in developing cortical neurons derived from human pluripotent stem cells. Pilot Project 3, directed by Dr. Erin Milligan, will examine whether fetal alcohol exposure increases spinal cytokines and glial (astrocytes and microglia) reactivity, and leukocyte extravasation in the adult leading to abnormal neural-immune interactions and neuropathic pain.

The overall goal of the Pilot Project Core of the New Mexico Alcohol Research Center (NMARC) is to foster cross-disciplinary FASD research across campus and promote growth of the Center. The Core will provide a critical opportunity to develop new basic, translational and clinical fetal alcohol research projects that complement and broaden the NMARC's research program portfolio. Selected projects must fit within the context of the Center's three strategic objectives as well as build on the overall integration and sustainability of the Center's research activities. Pilot projects will be funded for up to 2 years. The goal of each pilot project is to obtain sufficient preliminary data to develop competitive research grant applications and cultivate new investigators that will form part of the center. Support from the NMARC Pilot Project Core has played a crucial role in the success of R21 and/or R01 applications submitted by center investigators. Importantly, our Pilot Project Core has opened new and important research areas in the FASD field (e.g., impact of PAE on place cell, grid cell, and head direction cell function; effect of PAE on pain processing; interaction between placental dysfunction and PAE) and has brought novel approaches into the field (e.g., applied behavioral analysis in FASD management; circular RNA studies). The Core will primarily support new investigators that have yet to obtain substantial independent research support. A year prior to the end of the Pilot Projects that are initially funded, a call for additional Pilot Project proposals will be widely advertised within the scientific community at UNM, the Mind Research Network, and neighboring institutions. Each project will have an Advisory Team composed of senior alcohol researchers with complementary scientific expertise to that of the pilot project PI. The Center Director and NMARC Steering Committee will monitor progress of all pilot projects. Pilot project progress will be reviewed tri-annually by the NMARC Steering Committee and annually by the Program Advisory Committee. The Pilot Project Core goals have been successfully accomplished during the first 3½ years of the first five-year phase of the NMARC P50 award. We recruited several new investigators from diverse fields into the FASD research field. Two R21 and one R01 grants were obtained using preliminary data collected under the support of the Pilot Project Core. A total of 7 peer-reviewed papers have been published or submitted by Pilot Project Core investigators. Support was preferentially provided to junior faculty members. The Pilot Project Core will support two projects during the first two years. Project 6A will test the hypothesis that miR-150 inhibits Vezf1 to alter angiogenesis in the developing cortices of mice prenatally exposed to alcohol (P.I. Amy Gardiner, Ph.D., Research Assistant Professor, Dept of Neurosciences). Project 6B will investigate the effects of prenatal ethanol exposure on astrocyte and oligodendrocyte development in vivo (P.I. Tou Yia Vue, Ph.D., Assistant Professor, Dept of Neurosciences).