Gregory A. Ordway, Ph.D. - US grants

GABA receptor; beta adrenergic receptor; antidepressants; neuropharmacology; stimulant /agonist; receptor sensitivity; isoproterenol; messenger RNA; locus coeruleus; desipramine; serotonin; dorsal raphe nucleus; neurotransmitter metabolism; norepinephrine; receptor expression; cyclic AMP; in situ hybridization; laboratory rat; immunocytochemistry; brain mapping;

Converging evidence from years of intensive research has implicated that a disorder of central norepinephrine (NE) exists in depression. However, the basic neurobiology of depression has not yet been elucidated. The goal of this research proposal is to study the noradrenergic system in brain tissue from victims of suicide retrospectively identified as depressed. The components of the noradrenergic system studied here include that which responds to changes in noradrenergic neuronal activity (tyrosine hydroxylase mRNA), receptors which modulate NE release (alpha-2 adrenoceptors), receptors which are regulated by the concentrations of brain NE (alpha-2 adrenoceptors, beta adrenoceptors), neuropeptides which are colocalized with NE which modulate NE release (neuropeptide Y, galanin), and NE itself. The proposed studies are unique in that they will investigate noradrenergic chemistry in discrete brain regions, particularly the limbic system and the locus coeruleus, the principal source of NE in the brain. Furthermore, extensive psychological histories of the suicide completers will be obtained and used to determine if diagnoses of unipolar depression or bipolar affective disorder are correlated with alterations in noradrenergic chemistry. It is postulated here that an aberrant brain noradrenergic system in depression would manifest altered levels of mRNA encoding tyrosine hydroxylase and/or noradrenergic receptors in noradrenergic nuclei of the brainstem (e.g. locus coeruleus), and in specific terminal fields of these nuclei. Utilization of the techniques of quantitative in situ hybridization and receptor autoradiography to measure mRNAs and receptors requires small amounts of tissue. Thus, multiple components of the noradrenergic system can be measured at the discrete neuroanatomical and cellular level in the same individual; and multivariate statistics can be used analyze data for systematic changes across individuals as well as across groups.

Converging evidence from years of intensive research has implicated that a disorder of central norepinephrine (NE) exists in depression. However, the basic neurobiology of depression has not yet been elucidated. The goal of this research proposal is to study the noradrenergic system in brain tissue from victims of suicide retrospectively identified as depressed. The components of the noradrenergic system studied here include that which responds to changes in noradrenergic neuronal activity (tyrosine hydroxylase mRNA), receptors which modulate NE release (alpha-2 adrenoceptors), receptors which are regulated by the concentrations of brain NE (alpha-2 adrenoceptors, beta adrenoceptors), neuropeptides which are colocalized with NE which modulate NE release (neuropeptide Y, galanin), and NE itself. The proposed studies are unique in that they will investigate noradrenergic chemistry in discrete brain regions, particularly the limbic system and the locus coeruleus, the principal source of NE in the brain. Furthermore, extensive psychological histories of the suicide completers will be obtained and used to determine if diagnoses of unipolar depression or bipolar affective disorder are correlated with alterations in noradrenergic chemistry. It is postulated here that an aberrant brain noradrenergic system in depression would manifest altered levels of mRNA encoding tyrosine hydroxylase and/or noradrenergic receptors in noradrenergic nuclei of the brainstem (e.g. locus coeruleus), and in specific terminal fields of these nuclei. Utilization of the techniques of quantitative in situ hybridization and receptor autoradiography to measure mRNAs and receptors requires small amounts of tissue. Thus, multiple components of the noradrenergic system can be measured at the discrete neuroanatomical and cellular level in the same individual; and multivariate statistics can be used analyze data for systematic changes across individuals as well as across groups.

Converging evidence from years of intensive research has implicated that a disorder of brain norepinephrine and/or serotonin occurs in major depression. However, the basic neurobiology of major depression has not yet been elucidated. The goal of this research is test the hypothesis that a distinct constellation of neurochemical/neuroanatomical deficits occurs in the noradrenergic system (in particular, the locus coeruleus) in major depression. Experiments are designed to address the issues of (1) the precise neurochemical and/or neuroanatomical alterations and (2) the specificity of alterations with respect to psychiatric illness. To address the first issue, concentrations of a number of proteins and substances will be measured throughout the locus coeruleus, and in a major limbic projection area (amygdala) in post-mortem brains from subjects with major depression and from psychiatrically normal control subjects. In particular, a number of noradrenergic and non-noradrenergic proteins which are sites of action of antidepressant drugs will be studied, along with proteins of which levels are modulated in the locus coeruleus by antidepressant treatment. Preliminary findings demonstrates that noradrenergic cell number is a major determinant of noradrenergic protein concentration in the locus coeruleus. Furthermore, cell loss in the locus coeruleus is associated with aging, Alzheimer's disease, and Parkinson's disease. Thus, if cell number in locus coeruleus is altered in psychiatric disease, such a change could complicate measurements of noradrenergic proteins in the locus coeruleus or lead to misinterpretation of neurochemical alterations in the locus coeruleus from major depressives. Thus, noradrenergic neuron density and number will be estimated stereologically in all subjects in parallel with neurochemical measurements. To address the issue of specificity, 4 groups of subjects will be studied: 1) no psychiatric diagnosis, age-matched control subjects dying of natural or accidental causes, 2) suicide victims with major depression, 3) non-suicide subjects with major depression, and 4) suicide victims with Axis I diagnoses other than major depression. This research will extend and clarify major findings of research in the first grant period. Elucidation of the biochemical and/or anatomical alterations of locus coeruleus neurons associated with major depression may yield important information for the development of more effective treatments for this devastating disorder.

DESCRIPTION (from applicant's abstract): The human norepinephrine transporter (hNET) is a high affinity binding site for many psychotherapeutic compounds, including those with antidepressant efficacy (e.g. tricyclic antidepressants). The uptake of norepinephrine (NE) by the NET is the principal mechanism by which the action of NE is terminated at the noradrenergic synapse. Regulation of NET activity in the plasma membrane, therefore, represents an important candidate mechanism through which modulation of noradrenergic ransmission can occur. Despite the fact that many psychoactive compounds bind to the NET, the regulation of NET function induced by these ligands is poorly understood. Our preliminary data demonstrate that certain inhibitors (those which are antidepressants) of NET down-regulate NET function. In fact, exposure to certain NET inhibitors reduces NET function in he absence of the inhibitor for a period of time greater than the initial exposure period. The implication of this data is that occupation-induced down-regulation of NET function may contribute to the therapeutic/ pharmacological action of drugs that bind to the NET. The goals of this proposal are to examine the ability of NET ligands to induce NET down-regulation in in vitro and in vivo preparations, and to elucidate the molecular mechanisms responsible for ligand-induced NET down-regulation. Following continuous exposure of intact NET-expressing cells to NET ligands, studies will examine: (1) NET uptake capacity (function) in in vitro uptake assays, (2) the possibility that there is a rapid redistribution of NET from the plasma membrane surface,(3) the role of protein kinases in ligand-induced NET regulation, and (4) the turnover of NET protein and levels of NET messenger RNA. The biological relevance of NET ligand-induced regulation of NET in vitro will be established by studying NET function in brain slices following treatment of rats with NET ligands. Emphasis in in vitro and in vivo studies will be placed on the temporal aspects of regulation and recovery, because slow recovery from inhibitorinduced down-regulation may imply that antidepressant compounds do not need to be present at the NET in order to inhibit uptake. This information may have a significant impact on treatment regimens of NET inhibitors in the management of psychiatric diseases. Overall, the proposed studies will reveal the basic principles of the relationship between drug exposure and NET regulation and will provide important clues relevant to the pharmacological actions of psychoactive agents that bind to the net.

tissue /cell culture

DESCRIPTION (provided by applicant): Psychiatric neuroscience is an exciting and burgeoning field that has contributed to many recent major discoveries regarding the relationship between neurobiology and behavior. An enhanced commitment to this research in the next decade holds the promise of major advances in the diagnosis and treatment of psychiatric illnesses such as depression, schizophrenia, and substance abuse disorders. The University of Mississippi Medical Center (UMMC) has made a major commitment to this field of medicine over the past 8 years by the recruitment of a number of prominent scientists and clinicians engaged in psychiatric neuroscience research. UMMC proposes to build on this existing strength by establishing the Center for Research Excellence in Psychiatric Neuroscience (CREPN). The CREPN will create and maintain a quality research environment highly conducive to productive and clinically-oriented basic research in the psychiatric neurosciences. A major goal of CREPN will be to nurture the transition of junior faculty neuroscientists to independent researchers through a multi-disciplinary program of cutting-edge psychiatric neuroscience research. The CREPN will foster a diverse and nationally competitive environment that will facilitate research careers of junior faculty and will foster collaborations between psychiatric neuroscientists at UMMC and throughout Mississippi. These goals will be achieved by establishing formal mentoring relationships between junior psychiatric neuroscientists and senior faculty with established records in NIH-funded psychiatric neuroscience research. The CREPN will enhance the methodological sophistication of junior investigators by providing access to state-of-the-art core facilities including a core facility for behavioral studies, a cellular neuroimaging facility, an interactive web-based psychiatric neuroscience resource, and a collection of psychiatrically-characterized post-mortem brains. In addition, a neuroscientist that utilizes predominantly molecular biological approaches to psychiatric neuroscience research will be recruited to the Center. Overall, the establishment of the CREPN will enrich and diversify the academic environment in Mississippi for junior investigators in psychiatric neuroscience and thereby enhance their ability to compete nationally through traditional granting mechanisms.

A vast amount of research reveals that central dopamine (DA) containing systems are neuronal substrates of a broad spectrum of behaviors related to reward-seeking, motivation, and environmental responsivity. Disruption of these behaviors leads to anhedonia, social isolation, and psychomotor retardation that form core symptoms of depression. While there is little debate for a critical role of limbic structures, e.g. amygdala, in the regulation of mood and affect, the role of limbic DA in the pathobiology of depressive illness is not known. Functional imaging studies report involvement of limbic structures in depression, but few have focused on dopaminergic indices. Furthermore, there is a paucity of neurochemical studies of depression that have utilized postmortem brain tissue. Studies using postmortem brain tissue offer much higher anatomical resolution than it is offered by functional imaging. In preliminary studies, we have found lower dopamine transporter (DAT) and up-regulation of D2 receptors in the basal and central nuclei of the amygdala, but not in the other nuclei of this complex region, in major depression as compared to psychiatrically normal controls. These and other findings compel us to examine the possibility that there is diminished DA neurotransmission in subjects diagnosed with major depression (who died of suicide or natural causes). The central hypothesis of this proposal is that subjects with major depression have diminished mesolimbic DA activity that can be revealed by neurochemical abnormalities in discrete regions of the mesolimbic dopaminergic system. These neurochemical measures will be performed in discrete regions of the amygdala, (Aim 1), in the nucleus accumbens (Aim 2), and in the ventral tegmental area (VTA, Aim 3), core limbic regions of the brain. We also hypothesize that a distinct constellation of neurochemical abnormalities within limbic structures is specific for major depression (Aim 4), and will differentiate the pathobiology of major depression from that of suicide or schizophrenia. Groups of subjects to be studied will be: a) subjects with major depression who committed suicide, b) subjects with major depression not dying by suicide, c) sudden death non-psychiatric controls, and d) schizophrenics not dying by suicide. The proposed research will be the first focused investigation of potential abnormalities of limbic DA in major depression utilizing psychiatrically characterized subjects. The research will establish the specificity of neurochemical findings with respect to major depression and with respect to regional brain anatomy. Uncovering the potential role of DA in the pathobiology of depression may lead to advancements in the pharmacological, and possibly genetic, intervention of major depression.

Project Summary Currently available antidepressants produce remission in approximately two-thirds of patients with major depressive disorder (MDD), a disorder affecting over 10 million people in the US each year. This leaves an enormous number of depressed patients without the opportunity for a mentally healthy life, and demonstrates that novel therapeutic strategies to treat depression are sorely needed. A better understanding of the brain pathology associated with MDD will yield critical insights for the development of novel therapeutic approaches. Pathology of white matter (WM) in MDD has been demonstrated in in vivo imaging and postmortem brain tissue studies, but the molecular and cellular basis of WM pathology in MDD are poorly understood. The myelinating oligodendrocyte (OL) is a cellular resident of WM; OLs provide insulation, and trophic and metabolic support to axons passing through WM. Hence, OLs support neurotransmission and disruption of OL function has deleterious consequences on axonal conduction and transport. Interestingly, OLs are normally highly susceptible to oxidative damage. We previously have shown that OLs from MDD brain donors demonstrate reduced gene expression of antioxidant enzymes that protect cells from oxidative stress. Here, strong preliminary data are provided showing that OLs in WM from MDD brain donors demonstrate elevated DNA oxidation and upregulation of DNA base excision repair. Based on these findings, we hypothesize that the consequences of the cellular vulnerability of OLs to reactive oxygen species in MDD is increased oxidation of DNA in WM, and an elevated activation of DNA base excision repair that results in facilitation of inflammatory pathways and increased consumption of cellular energy supplies. Because OLs are critical components of saltatory conduction along axons, compromised OLs could disrupt neural activity related to regulation of emotion in depression. The aims of this study will directly test our hypothesis by using postmortem brain tissues already collected from behaviorally characterized human brain donors (MDD and matched psychiatrically normal control) for whom psychiatric/behavioral status at the time of death was confirmed by psychiatric autopsy. The proposed research will 1) investigate oxidative damage to DNA in brain WM OLs by measuring DNA oxidation and gene expression of base excision repair and related enzymes 2) interrogate deleterious cellular events resulting from upregulation of the DNA base excision repair process, e.g. cellular accumulation of toxic metabolic products (poly [ADP-ribose] polymers), cellular depletion of an energy source (NAD+) used in DNA repair, and increased expression of inflammatory mediators (NF-?B and related cytokines) facilitated by key base excision repair enzymes. A rigorous consideration will be made of potentially contributing factors to changes in dependent variables in MDD, such as suicide/suicidal behavior, other behavioral dimensions, antidepressant exposure, and gender. The outcome of this research has the potential to reveal novel therapeutic targets to improve the treatment of depression.