Alan Frazer, Ph.D. - US grants

The goals are to explore the effect of antidepressants and other psychoactive compounds on the regulation of beta-adrenergic and serotoninergic responsiveness, with particular emphasis on receptor mechanisms. Emphasis will be given to the effects of beta-adrenergic agonists, when given repeatedly to rats, on central beta-responsiveness so as to provide a pre-clinical rationale for the evaluation of such drugs as antidepressants. Beta-agonists will be administered to the rats either systemically or by intracerebroventricular infusion, using Alzet minipumps. Beta-adrenergic responsiveness will be assessed by measuring the ability isoproterenol to raise the concentration of adenosine 3', 5'-monophosphate (cyclic AMP) in slices of cortex and cerebellum prepared from control or drug-treated rats. Highly selective beta-antagonists will be used so as to link the rise in cyclic AMP in the cortex to beta1 receptors and in the cerebellum to beta2 receptors. The molecular mechanisms responsible for agonist-induced changes in responsiveness will be studied by measuring agonist effects on the density of subtypes of the beta-receptor in different parts of the brain (using both in vitro ligand binding techniques and quantitative autoradiography) as well as the interaction of the beta receptor subtypes with the guanine nucleotide binding regulatory protein (N protein). Such studies will provide important information on agonist-induced regultion of central beta-adrenergic receptors and their linkage with the N protein in vivo. Similar methodology will be used to examine whether antidepressant-induced changes in central beta-responsiveness are influenced by the concurrent administration of thyroid hormone. Such data might provide an explanation for the observation that administration of triiodothyronine to depressed female patients hastens imipramine-induced recovery from depression. Studies with receptors for serotonin (5-HT) will focus on the 5-HT1 receptor with emphasis placed on the effect of antidepressant treatments and serotonin agonists on the subtypes of this receptor and its high affinity state. Receptor alterations could explain the effects of these drugs, when given repeatedly to rats, on central serotonin responsiveness. At present, there is no selective radioligand for the 5-HT1B receptor subtype. However, the piperidinyl derivative, RU 24969, is selective for this subtype, consequently, experiments are proposed to incorporate tritium into the molecule and then to assess whether it could be used as a radioligand selective for 5-HT1B receptors. Drug-induced effects on the high affinity state of the 5-HT1 receptor can be assessed by doing saturation experiments in the absence of GTP and analyzing the data using non-linear regression analysis with the aid of a computer.

Prior research strongly suggests that patients with affective disorders have abnormalities in the functioning of one or more neurobiological systems. At a conference convened by the Clinical Research Branch, NIMH, these findings were reviewed and some of the factors which were impeding movement towards a more complete and ingegrated view of the functioning of neurobiological system in patients with mania or depression were identified. As a result, the NIMH sponsored the development of a multiresearch center, collaborative approach to the study of the psychobiology of affective disorders. In this collaborative program, underway for several years, the major objectives have been the testing of a wide range of hypotheses which implicate neurochemistry in the etiology and maintenance of the affective disorders. In order to accomplish this, the focus has been upon: (a) the assessment of the functioning of several different types of biological systems in the same patient both before and during treatment; (b) obtaining a large number of patients and comparison subjects; and (c) the use across centers of standardized diagnostic categories and behavioral methodologies. During the preceding grant period the data have been consolidated and data analysis has begun. Preliminary results have been reported and a range of studies are in progress. The specific aim of this proposal is the continuation of the testing of earlier, as well as current, hypotheses related to the behavioral and physiological pathology, as well as to treatment responsivity which may characterize depressed patients and differentiate them from healthy subjects.

The goals of this project are to determine: (1) if different types of antidepressants alter the density and/or affinity of subtypes of either beta adrenoceptors (BARs) or 5-HT-1 receptors either throughout the brain or in localized areas; (2) how serotonergic nerves alter the ability of antidepressants to decrease the density of BARs; (3) factors involved in the regulation of central BARs in vivo; and (4) if alterations in central receptors for certain monamines occur in an animal model of depression. All experiments will use rats and receptors will be visualized and quantified using the technique of in vitro quantitative autoradiography. Many antidepressants decrease the density of BARs and this pharmacological effect has been speculated to be involved in their clinical effects. The experiments proposed will evaluate if a particular subtype of BAR is preferentially affected by antidepressants and whether changes are produced in common areas of the brain. In addition to affecting noradrenegic responsiveness, antidepressants change both electrophysiological and behavior responses elicited by serotonin (5-HT) agonists. The responses altered have been linked to subtypes of a receptor for 5-HT, termed the 5-HT-1 receptor. By using quantitative autoradiography, it will be possible to determine whether antidepressant-induced changes in these subtypes account for the alterations in responsiveness. In the experiments involving subtypes of BARs or 5-HT-1 receptors, rats will be given antidepressant of different types for 21 days. Antidepressants studied will be those that block selectively the uptake of norepinephrine or serotonin or inhibit selectively type A or type B monoamine oxidase. Serotonin neurons are involved in antidepressant-induced decreases in the density of BARs. To study this, the ability of either the antidepressant, desipramine, or the beta-agonist, isoproterenol (ISO), to decrease the density of BARs will be measured in intact rats or rats with lesions of central serotonergic neurons. Lesions will be made with the neurotoxin, 5,7-dihydroxytryptamine, given not only intraventricularly but also directly into areas containing noradrenegic cell bodies or nerve terminals. Lesions with a neurotoxin for catecholaminergic nerves, 6-hydroxydopamine, will be made in separate groups of rats to determine how central noradrenegic neurons influence the ability of ISO to decrease the density of beta-1 adrenoceptors. In these experiments, ISO will be given into the right lateral ventricle of rats through a permanently indwelling cannula connected to an Alzet minipump. This experiment will provide information about the feasibility of using beta agonists as antidepressants. The status pf central monoamine receptors will also be measured in rats exposed to uncontrolled shock, as this may be an animal model of depression.

serotonin receptor; receptor sensitivity; psychopharmacology; neuropharmacology; serotonin;

The serotonin transporter (SERT) is an integral membrane glycoprotein that is responsible for the reuptake of serotonin from the synapse. Drugs that inhibit the SERT are effective in several psychiatric disorders including depression. Recent studies have shown that the SERT can be regulated in vitro. Less is understood about its acute or chronic regulation in vivo. Different approaches have been used to address this issue, with inconclusive results. We have used in vivo chronoamperometry, a fast voltammetric technique, to demonstrate that there are areas in brain such as the CA3 region of the hippocampus, where the active clearance of exogenously ejected serotonin is primarily a function of SERT activity. This technique, then, is capable of generating rapid kinetic, quantitative measures of SERT function in vivo. In the proposed experiments, we will use this approach to study if long term administration of two different selective serotonin reuptake inhibitors (SSRIs), paroxetine and fluoxetine, produce a time-dependent subsensitivity of the SERT. The influence of treatment parameters such as route of administration, duration of administration, and drug-free washout period will be evaluated. Based on our preliminary data, we will explore if activation of terminal 5-HT1B autoreceptors alters the kinetics of SERT function in vivo. If so, then we will examine whether such a phenomenon impacts on any apparent regulatory effect of chronic SSRI treatment on SERT activity. In addition to examining the CA3 region, two other brain areas where the active clearance of 5-HT is due exclusively to the SERT will be studied. These experiments provide a new approach to study whether the SERT, a primary target for several types of antidepressants, is capable of being regulated either acutely or chronically in vivo.

The main goal of this proposal is to study the functional consequences and mechanisms underlying antidepressant-induced down-regulation of the serotonin transporter (SERT) and norepinephrine transporter (NET). These proteins are the key cellular targets for drugs used in the treatment of depression. Our general hypothesis is that the time-dependent antidepressant-induced decrease in these transporters may be one of the crucial determinants of both why and when serotonergic and noradrenergic transmission is markedly enhanced, with consequent behavioral improvement. To study this, both time course and mechanistic studies will be carried out. Time course studies will correlate the time-dependent sertraline-induced changes in SERT density (as measured by quantitative autoradiography) with changes in the chronoamperometric signal caused by local application of 5-HT in brain. This will be done after cessation of treatment also. Non-linear regression techniques will be applied to the electrochemical signal to obtain Vmax and KT values for 5-HT clearance by the SERT. The time course of down-regulation of the NET caused not only by DMI but also reboxetine and venlafaxine, at doses producing steady-state serum levels at the low and high ends of its therapeutic range, will be examined, as will the time course of the return of NET binding sites after cessation of treatment. At times when NET binding sites are down-regulated, function of the NET will be assessed by measuring the uptake of 3H-NE in synaptosomes. Different approaches will be used to examine the mechanisms involved in these phenomena. We will study the time course of changes in mRNA for the SERT and its binding sites and mRNA for the NET and its binding sites during treatment of rats with antidepressants and after their cessation. We will study if chronic treatment of rats with uptake inhibitors either alters the distribution or decreases SERT or NET protein in purified membrane fractions obtained from brain. Purified membrane fractions will be obtained by ultracentrifugation of membranes layered on a sucrose gradient and the amount of SERT or NET protein in these fractions measured by Western blot analysis with specific antibodies. Knockout mice will be used to determine influences of PKC epsilon on antidepressant-induced down-regulation of the SERT and NET. Mice lacking 5-HT1B receptors will be used to see if they exhibit altered sertraline-induced down-regulation of the SERT. These experiments involve multiple approaches to study an effect of specific antidepressant drugs that may be very important in sustaining the enhancement of monoaminergic transmission that underlies clinical improvement

The overall goal of this proposal is to increase the number of underrepresented minority students (URMs) into doctoral programs relevant to mental health research in the USA. The majority of such students will likely enter the doctoral program in pharmacology at the University of Texas Health Science Center at San Antonio (UTHSCSA) or neurobiology at the University of Texas San Antonio (UTSA). Data compiled by the National Research Council indicate that only 4.7% of new Ph.D.s in the biomedical sciences were awarded to members of URM groups (African Americans, Hispanics, Native American and Mainland Puerto Ricans), and that only 4.2% of biomedical research scientists in the workforce are URMs. Faculty contacts have been development at six primarily undergraduate universities in South Texas that have a total enrollment of 36,150 URMs, primarily Hispanic. Students selected jointly by these faculty as well as the Executive Committee of our program, termed the South Texas Graduate Diversification Program (STGDP), will enter its education program. The STGDP is meant to give undergraduate students a true taste of what being a graduate student in the biomedical sciences entails. They will have research responsibilities, attend seminars, actively participate in student journal clubs and take formal courses geared specifically towards undergraduate students. Social activities will also be planned for the undergraduates and will promote an environment in which the students will feel involved in the departments at UTHSCSA or UTSA. The research component will initially take place during a ten week period over the summer. This will occur in one of the laboratories of the 20 faculty (both from UTHSCSA and UTSA) in the program. During this time, the students will be required to take two courses. One deals with research methodologies of relevance to mental health. The other is a course specifically designed for the STGDP, "Biological Bases of Brain Function". They will also attend a weekly seminar series presenting topics such as Ethical Issues in Biomedical Research and Career Options in Biomedical Research. Promising students will be strongly encouraged to continue their affiliation with the STGDP, either during the academic year or for a second summer session. If this occurs during the academic year, the students will carry out research for at least 16-18 hours per week while taking courses either at their home universities or at UTSA. Any student carrying out research during a second summer session will also take a specially-developed course, "Neuropsychopharmacology". Funds are requested in the first year for 11 students (9 during the summer and 2 for a semester) and twice that number thereafter. The purpose of the STGDP is not only to give these students "research experience , but also, and more importantly, to allow them to gain confidence in their research abilities. The experience will show them what life is like as a graduate student and as an academic scientist. Talented URMs are available who are capable of having successful careers in mental health research. Our program is designed to recruit these students and demonstrate to them that the can be successful.

[unreadable] DESCRIPTION (provided by applicant): Chronic intermittent ethanol (CIE) exposure produces longer-lasting molecular adaptations including up-regulation of NR2B gene expression and persistence, which is viewed as an important neurobiological basis for the development of ethanol dependence. However, the exact mechanisms underlying this long lasting phenomenon are still unclear. DNA methylation is one of many epigenetic modifications that can alter gene expression, thus, it represents as an ideal candidate mechanism for CIE-induced long-term effects. Therefore, we hypothesize that CIE-induced DNA demethylation of specific CpG sites in the NR2B gene 5' region may increase accessibility of transcription factors to DNA in 5' region of NR2B gene. Repeated ethanol exposures may promote intracellular signaling mechanisms, which regulate DNA methylation and lead to the long lasting plastic changes in NR2B gene expression. These may contribute to the development of alcohol dependence. To address this hypothesis, we plan to use an existing primary cortical neuronal culture CIE model in our laboratory combining with an animal CIE model to a) examine CIE-induced changes in DNA methylation state by mapping individual CpG dinucleotide of the NR2B gene 5' region, which are responsive to long lasting up-regulation of NR2B gene transcription by using bisulfite genomic sequencing, and also examine the correlation of this changes to the development of ethanol-dependence in mice CIE model; b) determine how CIE-induced changes in methylation state of specific CpG sites near the CREB and AP-1 binding sites may impact transcription factors CREB and AP-1 binding complex formation via examining the alterations in CREB/AP-1-DNA binding affinity by using in vitro methylation, transfection, EMSA, site-direct mutation and ChIP; and c) identify the role of relative signaling pathways mediating CIE to DNA methyltransferase activity as well as NR2B gene transcription, including traditional signaling pathways cAMP/PKA and MAPK/ERK and novel signaling proteins involving in modifications of DNA methylation and persistent expression of the NR2B gene by using specific inhibitors, western blot, ELISA and 2-D gel analysis. The results from this proposal will provide new and valuable insight of molecular mechanisms of ethanol regulating NR2B gene expression through epigenetic modifications, which are expected to serve as a basis for possible intervention of alcohol dependence and alcohol withdrawal syndrome. [unreadable] [unreadable] [unreadable]

The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter in brain, is an important site of action of ethanol. Chronic ethanol treatment in vivo and in vitro upregulates the NMDA receptor number and function, with a concomitant increase in R1 and R2B polypeptide levels in vitro. An upregulation of R2B polypeptide levels following chronic ethanol treatment in vivo is due to an increase in R2B mRNA levels by ~ 40% in cerebral cortex and by ~ 30% in hippocampus. Similar increase in R2B mRNA levels is seen to occur in vitro in cultured fetal cortical neurons. The molecular mechanism underlying an increase in R2B mRNA levels in response to chronic ethanol treatment is an increase in NMDA R2B gene transcription rate (Kumari and Ticku, 1998). The importance of NMDA R2B receptor subunit in alcohol mediated changes in the brain lies in the fact that ethanol's effect on R2B subunit is seen to occur both in adult and fetal tissue and the intensity of alcohol's effect is same in both instances. Long term plans of this project are to (i) identify if alternative promoters are utilized in adult and fetal tissues; (ii) examine the role of methylation in R2B gene expression; (iii) identify ethanol-responsive c/s-controlling regulatory elements in the promoter region of the R2B gene by deoxyribonuclease I hypersensitive analysis; (iv) identify "minimal c/s-acting DNA sequences" that are sufficient to show response to ethanol's action by deletion transfection analysis; (v) identify nuclear protein factor(s) that may interact with minimal c/s- acting DNA sequences to alter R2B gene expression and to prec/sely map how many nucleotides within minimal c/s-acting DNA sequences are sufficient for the binding of nuclear factors identified above, and lastly (vi) determine if ethanol mediated increase in intracellular calcium activates specific signal pathways that lead to phosphorylation of cyclic AMP response element binding protein which in turn, binds to cyclic AMP response element in the 5' flanking region of the R2B gene resulting in an increase in R2B gene transcription rate. We propose to utilize mouse fetal cortical neurons to achieve these goals as during the first 7 days in culture, fetal cortical neurons express mainly R2B subunit. A more through understanding of the pertinent molecular mechanisms through which ethanol alters rate of NMDA R2B gene transcription may permit the design of novel therapeutic approaches to alcohol-related diseases.

DESCRIPTION (provided by applicant): Almost 30% of patients with major depressive disorder have a chronic illness that is treatment refractory. Vagus nerve stimulation (VNS) has recently been approved by the FDA for treatment refractory depression. However, there have been few pre-clinical studies addressing the central actions of VNS that may be relevant for its antidepressant effect. We completed recently some preliminary studies in which VNS was administered either acutely or chronically to rats using clinically-relevant stimulation parameters. Both functional neuroanatomical and behavioral effects were observed. The goal of this proposal is to expand and amplify these observations so as to evaluate more comprehensively effects produced by VNS in brain and the mechanisms underlying such effects. Our overall hypothesis is that VNS produces its beneficial clinical effects by activation of multiple neurotransmitter/neuromodulator systems in brain, in particular serotonin or norepinephrine and/or brain- derived neurotrophic factor containing neurons (through phosphorylation of TrkB receptors). To test these ideas, we will: (1) initially optimize stimulation parameters, using both acute and chronic (3 week) VNS by determining behavioral effects it produces in the FST and compare its effects to those produced by the selective noradrenergic reuptake inhibitor, desipramine, and the selective serotonin reuptake inhibitor, sertraline, (2) use the chronic VNS protocol that has the best effect in the FST to measure its effects on c-Fos, FosB, Egr-1, activation of TrkB, 21-adrenoceptors, and somatodendritic 5-HT autoreceptor sensitivity and compare results to those caused by chronic treatment with desipramine or sertraline, and (3) test the role of norepinephrine and serotonin in the antidepressant-like effect of chronic VNS, desipramine and sertraline. Immunohistochemistry will be used to measure c-Fos, FosB, Egr-1, and phosphorylated TrkB. Quantitative autoradiography will be used to measure 21- adrenoreceptors. DPAT-induced changes in extracellular 5-HT in the striatum will be used as an index of autoreceptor sensitivity. The results could provide important, new information regarding the central nervous system effects of VNS that are important for the treatment of depression.

DESCRIPTION (provided by applicant): The proportion of underrepresented minorities (URMs) earning doctorate degrees in the basic biomedical sciences has increased modestly over the past few decades. Our proposed Program, the South Texas Advanced Research Training Undergraduate Program (START-UP) is a response to a Funding Opportunity Announcement from the Blueprint Program for Enhancing Neuroscience Diversity through Undergraduate Research Education Experiences (BP-ENDURE). As such, the overall goal of our Program is to encourage and prepare junior and senior undergraduate URMs from the San Antonio and South Texas Region to enter doctoral programs in neuroscience, to complete them successfully, and become well-trained and competitive neuroscientists. To accomplish this, a comprehensive program is proposed for the URM students accepted into the program, involving extensive research experiences in the laboratories of successful neuroscientists, and opportunities to develop and improve their writing, speaking, and time management skills. Students will be recruited into START-UP from five partner institutions in San Antonio, namely Our Lady of the Lake University, St. Mary's University, Trinity University, University of the Incarnate Word, and the University of Texas, San Antonio. Collectively these schools have 24,527 undergraduates who are URMs (based on ethnicity), of whom 2,947 are Science Majors. Also, these schools have a high number of students from low income families, many of whom are the first in their families to attend college. Faculty contacts have been established at each school to assist us recruit suitable students into START-UP. Thirty-one training faculty have been identified (including three from UTSA), who are appropriate to mentor these students in their laboratories. The students will participate in laboratory research for an average of 12 hours per week during the two academic semesters, and 40 hours/week during a 10-week intensive summer research exposure. Students will also have an opportunity to work in one of seven major neuroscience programs at institutions outside of San Antonio during the summer. In addition to their laboratory research, the students will also attend seminars and journal clubs, research retreats, and have exposure to neuroscientists from other institutions. The students will all receive instruction on the responsible conduct of research. The Co-Directors of the Program are Drs. Alan Frazer and David Weiss, experienced scientists and administrators, who have run programs similar to START-UP previously. They will be members of an Executive Committee that will oversee all aspects of the Program. There is a formal evaluation plan for the Program, as well as an outcomes assessment process. Further, a plan is described to disseminate nationally all materials developed for the design and implementation of START-UP.

DESCRIPTION (provided by applicant): The vulnerability of some women to develop major depressive disorder (MDD), which occurs more frequently in women than in men, is associated with hormonal fluctuations (monthly, pregnancy, postpartum, and menopause). We have shown recently, using the technique of in vivo chronoamperometry, that acute administration of ovarian hormones (estradiol benzoate (EB) or progesterone (P)) impacts the ability of selective serotonin reuptake inhibitors (SSRIs) to alter the function of what is widely considered their initial cellular target in brain - the serotonin transporter (SERT). In addition, EB but not P, when given acutely, blocked SERT function as demonstrated by a diminished clearance of exogenously applied serotonin. Thus, EB has two actions. On its own, it slows the clearance of 5-HT, an effect similar to that caused by SSRIs. One might speculate that estrogen is antidepressant based on such an effect. However, we also find estrogen to interfere with the ability of SSRIs to slow 5-HT clearance. Such an effect might be expected to compromise the antidepressant effects of SSRIs. Estrogen, then, seems to have two quite distinct effects. By contrast, progesterone only seems to inhibit the effect of SSRIs on 5-HT clearance. There appears to be some difference in the mechanism(s) mediating these two effects of estradiol. Evidence was obtained that these effects of estradiol are mediated via activation of membrane as well as nuclear estrogen receptors (ER), indicating a genomic as well as a non-genomic component to these effects. By contrast, the effect of progesterone seems to be mediated primarily by nuclear receptors. A principal goal of this proposal is to extend these studies using other measures, either neurochemical (in vivo microdialysis) or behavioral (forced swimming test (FST)), to see if additional types of evidence can be obtained showing that treatment with female sex hormones either influences SERT function and/or interferes with the ability of SSRIs to inhibit the SERT. In addition, to examine possible mechanisms underlying hormonal effects, experiments are planned to study the plasma membrane distribution of the SERT using biotinylation studies; to examine the role of specific ER subtypes; and to study possible involvement of brain-derived neurotrophic factor (BDNF), as we found its administration to mimic the effect of EB and P on the ability of SSRIs to inhibit the SERT. Although the primary focus is the hormone/SSRI interaction, some studies focus on the inhibitory effect of estrogen alone. Finally, in a rat model of hormone-induced pseudopregnancy and subsequent hormone withdrawal, we will study the effects of high hormone levels administered chronically and their withdrawal in the FST and on the phosphorylated state of TrkB as well as their influence on SSRI/SERT interactions.

ABSTRACT Almost 30% of patients with major depressive disorder have a chronic illness that is treatment refractory. Vagus nerve stimulation (VNS) has recently been approved by the FDA for treatment refractory depression. However, there have been few pre-clinical studies addressing the central actions of VNS that may be relevent for its antidepressant effect. We completed recently some preliminary studies in which VNS was administred either acutely or chronically to non-anesthetized rats using clinically-relevant stimulation parameters. Both functional neuroanatomical and behavioral effects were observed. The goal of this proposal is to expand and amplify these observations so as to evaluate more comprehensively effects produced by VNS in brain and the mechanisms underlying such effects. Our overall hypothesis is that VNS produces its beneficial clinical effects, as measured by reduced immobility in the forced swim test (FST) in rats, by activation of multiple neurotransmitter/neuromodulator systems in brain, in particular serotonin or norepinephrine and/or brain- derived neurotrophic factor containing neurons (through phosphorylation of TrkB receptors). To test these ideas, we will: (1) initially optimize stimulation parameters, using both acute and chronic (3 week) VNS by determining behavioral effects it produces in the FST and compare its effects to those produced by the selective noradrenergic reuptake inhibitor, desipramine, and the selective serotonin reuptake inhibitor, sertraline, (2) use the chronic VNS protocol that has the best effect in the FST to measure its effects on c-Fos, ¿FosB, Egr-1, activation of TrkB, ¿1-adrenoceptors, and somatodendritic 5-HT autoreceptor sensitivity and compare results to those caused by chronic treatment with desipramine or sertraline, and (3) test the role of norepinephrine and serotonin in the antidepressant-like effect of chronic VNS, desipramine and sertraline by lesioning rats with either 6-hydroxydopamine or 5,7- dihyroxytryptamine. Immunohistochemistry will be used to measure c-Fos, ¿FosB, Egr-1, and phosphorylated TrkB. Quantitative autoradiography will be used to measure ¿1-adrenoreceptors. DPAT-induced changes in extracellular 5-HT in the striatum will be used as an index of autoreceptor sensitivity. The results could provide important, new information regarding the central nervous system effects of VNS that are important for the treatment of depression. PROJECT NARRATIVE About 30% of patients with major depressive disorder are resistant to standard antidepressant therapy. Vagal nerve stimulation (VNS) was approved recently for such refractory patients; it is moderately effective, but how it works is unknown. Our studies will evaluate comprehensively effects produced by VNS in the brain of rats and the mechanisms underlying such effects, so as to provide data to allow VNS to become even more effective.

DESCRIPTION (provided by applicant): NMDA receptor up-regulation is a major neuroadaptive process that causes excitatory syndrome upon withdrawal from chronic ethanol exposure. The mechanisms of neuroadaptation of NMDA receptors during withdrawal remain unclear, but several lines of evidence suggest that changed gene expression plays important roles. MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs which are involved in the fine tuning of gene expression in various biological processes. We have recently shown a distinct expression pattern of miRNA in response to chronic intermittent ethanol exposure, including the up- regulation of miR-152, -150, and -126 in mice VTA DA neuron. This proposed study is designed to study the role of miRNA in the DNA methylation underlying neuroadaptive up-regulation of NR2B gene during alcohol withdrawal. Therefore, we hypothesize that alcohol withdrawal-induced up-regulation of miR-152, -150 and - 126 modulates DNA methylation by down-regulating MeCP2 and are subsequently involved in neuroadaptive up-regulation of the NR2B gene. Specific Aim 1: Identify and validate that miR-152, -150 and -126 target MeCP2 and regulate DNA methylation of the NR2B gene in vitro. We will confirm these miRNAs (1) biologically interact with the 3' UTR of MeCP2 mRNA using a lentiviral delivery system to introduce these miRNAs into cultured neurons; (2) functionally inhibit the target effort level (repression) using an in vitro reporter gene assay; and (3) determie the regulatory role of these miRNAs on DNA methylation of the NR2B gene using bisulfate pyrosequencing. Specific Aim 2: To investigate whether the miRNAs are involved in the regulation of ethanol withdrawal-induced adaptation in ethanol dependence mice. TH- and GAD67-GFP transgenic mice will be exposed to alcohol with a CIE regimen followed by 5 days withdrawal. The DAergic or GABAergic neurons in the VTA of midbrain dopamine neurons in the mice will be obtained using laser capture microdissection and used for the analysis of the differential expression of these miRNAs, mRNA of MeCP2 and NR2B genes as well as DNA methylation of the NR2B promoter. The proposed study will determine a novel mechanism that miRNAs contribute to the epigenetic regulation of the NR2B gene during ethanol withdrawal of mice. For the first time, single neuronal populations obtained by laser capture microdissection will be used to study epigenetic mechanism on alcohol withdrawal-related neuroadaptation.

Obesity is a major risk factor for type II diabetes and metabolic syndromes. Increased understanding of body weight regulation may lead to effective strategies to combat obesity and diabetes. Hypothalamic neurons, including anorexigenic pro-opiomelanocortin (POMC) neurons and orexigenic Agouti-related peptide (AgRP) neurons, integrate multiple metabolic cues (e.g. leptin and insulin) to provide a coordinated control of energy and glucose homeostasis. We found that an adaptor protein, growth factor receptor-bound protein 10 (Grb10), is abundantly expressed in the hypothalamus, and its expression is elevated by HFD feeding. Further, Grb10 inhibits both leptin and insulin actions in neurons. Importantly, deletion of Grb10 in hypothalamic neurons leads to profound lean phenotypes in mice. Based on these, we hypothesized that Grb10 promotes body weight gain by negative regulation of leptin and insulin signaling in hypothalamic neurons. The first objective will focus on anorexigenic POMC neurons. We will generate two opposite genetic mouse models: one with Grb10 deleted in mature POMC neurons and the other with Grb10 overexpressed in mature POMC neurons. We will use these loss- and gain-of-function models to determine how Grb10 in POMC neurons regulates energy and glucose balance, modulates leptin and/or insulin signaling pathways, and controls firing activities and gene expression. The second objective will focus on orexigenic AgRP neurons. We will use the similar approaches to delete or overexpress Grb10 in mature AgRP neurons. We will use these loss- and gain-of-function models to determine the physiological role of Grb10 in AgRP neurons in the regulation of energy/glucose balance. Further, we will explore the cellular and molecular mechanisms by which Grb10 modulates leptin/insulin-induced signaling pathways and regulates firing activity and gene transcription of AgRP neurons. The third objective is to use in vitro approaches to determine the molecular mechanisms for Grb10 to inhibit leptin signaling. To this end, we will first map the interacting regions between Grb10 and the leptin receptor molecules, and then determine if such interaction provides a mechanism for Grb10 to inhibit leptin signaling. These studies could lead to important advances in our understandings regarding the central regulation of energy/glucose homeostasis. We may also provide mechanistic insights on the fundamental biology for leptin/insulin signaling in the brain. Finally, the proposed studies may carry translational impact on human health, as we may identify brain Grb10 as a rational target for potential anti-obesity therapy.

Abstract Mitochondria in hepatocytes play a major role in maintaining whole-body energy metabolism and normal function of the liver. Impaired mitochondrial function is closely associated with various metabolic diseases such as obesity, insulin resistance, and hepatosteatosis. However, the precise underlying mechanisms remain to be fully elucidated. Filling this major gap of knowledge will yield new information on the mechanisms underlying mitochondrial dysfunction-associated liver diseases, insulin resistance, and type 2 diabetes. Our current study focuses on the functional roles and mechanisms of action of the disulfide-bond-A oxidoreductase-like protein (DsbA-L). We recently found that DsbA-L expression is significantly reduced in the liver of obese human subjects and diet-induced obese mice. In addition, loss- and gain-of-function studies reveal that DsbA-L is a key regulator of mitochondrial integrity and function and its deficiency in the liver plays an important role in obesity-induced hepatosteatosis and metabolic dysfunction. In the current study, we will use molecular and cellular approaches as well as knockout animal models to elucidate the mechanisms regulating mitochondrial integrity and function under physiological and pathophysiological conditions. This research should shed new light on the link between obesity, mitochondrial impairment, and liver dysfunction and further our understanding of the mechanisms underlying obesity-induced insulin resistance and metabolic diseases. Thus, our proposed studies should provide valuable information on the biology of DsbA-L potentially being useful as targets of anti-obesity and anti-insulin resistance therapeutics.