John W. Haycock, Ph.D. - US grants

The long-term objectives of the research project are to describe the mechanisms by which catecholamine-containing neurons compensate for the diminution of endogenous catecholamine stores consequent to activity-related secretion of catecholamines. The compensatory replenishment of catecholamine stores results from an increase in catecholamine biosynthesis, the underlying basis of which is an increase in the catalytic activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Stimulation-dependent phosphorylation of tyrosine hydroxylase is one mechanism that catecholaminergic cells employ to activate tyrosine hydroxylase. The specific objective of the proposed research is to determine the phosphorylation sites and the protein kinase system(s) involved in the phosphorylation of tyrosine hydroxylase in intact bovine adrenal chromaffin cells as stimulated by acetylcholine (the splanchnic nerve neurotransmitter). Limit tryptic digestion will be used to produce tyrosine hydroxylase phosphopeptides. After separation of the peptides, site-specific phosphorylation of tyrosine hydroxylase produced by acetylcholine in situ will be compared with site-specific phosphorylation of tyrosine hydroxylase produced in situ by selective activators of specific protein kinases and in vitro by various purified protein kinases. In addition, the effects of injecting purified protein kinases and protein kinase inhibitors directly into chromaffin cells will be evaluated for effects upon tyrosine hydroxylase phosphorylation and catecholamine biosynthesis. These data should allow identification of the protein kinase system(s) involved in regulating tyrosine hydroxylase phosphorylation in situ. Such studies should provide important insight into the regulation of catecholamine biosynthesis as well as a firm biochemical basis for the further study of the relationship between the phosphorylation and catalytic activity of tyrosine hydroxylase with reference specifically to its regulation in situ.

The long-term objectives of the proposed research are to determine whether alterations in the levels and/or phosphorylation of tyrosine hydroxylase in the brain may be involved the etiology and/or pathogenesis of schizophrenia. In normal animals, activation of dopaminergic neurons results in the secretion of dopamine and a compensatory replenishment of dopamine stores. This replenishment of dopamine stores results from an increase in dopamine biosynthesis, the underlying basis of which is an increase in the catalytic activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis. One mechanism to increase the activity of tyrosine hydroxylase, described in other catecholamine-containing neurons, is the stimulation-dependent phosphorylation of tyrosine hydroxylase. Abberations in the dopaminergic systems in brain have been previously linked to schizophrenia; however, the exact relationship is as yet unknown. It is the premise of this grant application that alterations either in tyrosine hydroxylase or the protein kinase systems that attend tyrosine hydroxylase may be involved in schizophrenia. The specific objectives of the proposed research are 1.) to describe the regulation of tyrosine hydroxylase phosphorylation in dopaminergic nerve terminals in brain, 2.) to determine protein kinases systems that are involved in this regulation by using peptide mapping, and 3.) to assess the effects of acute and chronic drug treatments that have been shown to be effective in treating schizophrenia in a clinical setting. Specifically, the ability of treatments that cause the release of dopamine from rat, corpus striatum synaptosomes will be evaluated for their ability to increase the phosphorylation of tyrosine hydroxylase. The site-specificity of this phosphorylation will then be used to infer the protein kinase systems involved in this effect. Lastly, acute and chronic treatment of rats with haloperidol, clozapine, lithium chloride, or carbamazepine will be evaluated for their effects upon tyrosine hydroxylase levels, secretagogue- dependent phosphorylation of tyrosine hydroxylase, and the site- specificity of tyrosine hydroxylase phosphorylation.

biomedical equipment resource; biomedical equipment purchase;

The long-term objectives are to determine the mechanisms by which catecholamine (CA)-containing cells replenish endogenous CA stores lost via neuronal activity/secretion and, ultimately, the rules governing whether these mechanisms result in homeostasis vs a more or less lasting change in secretory output. Repletion of CA stores results from an increase in CA biosynthesis consequent to an increase in the catalytic activity of tyrosine hydroxylase (TH), the initial and rate-limiting enzyme. One mechanism by which CA cells increase TH activity is phosphorylation of the enzyme. In intact CA systems, TH is phosphorylated at four different residues, and the phosphorylation of three of these sites is regulated physiologically: Ser19 and Ser40 (phosphorylated by Ca/CaM- and cAMP-dependent protein kinase, respectively) and Ser31. This latter site and the protein kinase system responsible for its phosphorylation (ERKs, MAP kinases) represent a quite recently identified kinase/substrate classification, and little is known yet regarding their importance in regulating CA function. The specific objectives of the proposal are to (1.) characterize the regulation and relative involvement of different ERK species in Ser31 phosphorylation, (2.) establish the characteristics of site-specific TH phosphorylation in vitro using the appropriate, purified protein kinases, (3.) determine whether site-specific phosphorylation influences phosphorylation at other sites in vitro, (4.) evaluate and characterize the effects of site-specific phosphorylation and multiple-site phosphorylations upon the catalytic activity of TH in vitro, (5.) evaluate the interactions in vitro between CA-dependent and phosphorylation-dependent alterations in TH activity, and (6.) determine the effects of manipulating multiple-site TH phosphorylation in intact cells upon CA biosynthesis rates and upon the catalytic activity and CA binding characteristics of TH isolated from the cells. In terms of methods, (1.) Antisense oligonucleotides, to inhibit the expression of different ERK species in PC12 cells, will be tested for effects on Ser31 phosphorylation (relative to Ser8, Ser19 and Ser40) in intact PC12 cells. (2-3.) Kinetic parameters of site-specific TH phosphorylation (verified by HPLC of limit tryptic digests) will be studied in vitro using purified protein kinases (Ca/CaM-dependent, cAMP- dependent, ERK) with native PC12 TH and unphosphorylated recombinant wild type TH. Initial rates will be used to study interactions among sites. Mutant THs with A replacing Ser19, Ser31, or Ser40) will provide null/non-phosphorylatable controls while TH mutants with E as replacement will provide "phosphorylated" controls. (4-5.) The catalytic activity of TH (selected phosphorylation conditions/TH forms) will be measured in terms of cofactor/substrate kinetics and CA inhibition. A novel CA-TH interaction with recombinant TH will also be studied. (6.) Using previously established treatment profiles, specific patterns of multiple-site TH phosphorylation in situ will be tested for effects on CA biosynthesis rates and predicted effects upon catalytic activity and CA content of TH isolated from the cells.

The long-range goals of this project are to study the involvement of brain catecholamine systems in mental health. Dopaminergic systems have been implicated in schizophrenia, and noradrenergic systems have been implicated in depression. One common feature of both catecholaminergic systems is the initial, rate-limiting enzyme in their biosynthesis-tyrosine hydroxylase. The activity of tyrosine hydroxylase (TH) is regulated by a number processes which include, in the short-term, protein phosphorylation/dephosphorylation and, in the long-term, induction of enzyme synthesis. In humans, the possibility of an additional level of regulation also exists. Human TH mRNA (mRNAHTH) undergoes alternative splicing, and two different species of mRNAHTH have been identified in post-mortem human brain samples. From work on TH in the applicant's laboratory, the products of the two forms of mRNAHTH present in brain are predicted to possess different substrate specificities for protein kinases. Thus, in humans, changes in alternative splicing may also contribute to the regulation of TH activity. To date, studies published on multiple forms of human TH have analyzed only the nucleic acids and recombinant products therefrom. The present application proposes to determine the presence and regional distribution of the different forms of TH protein per se in post-mortem human brain samples. Antibodies will be raised to synthetic peptides representing the unique amino acid sequences created by the alternative splicing. Selective recognition of the different forms of TH will be tested against immunogens and against human TH holoenzymes produced from recombinant vectors. As necessary, preadsorption with the heteroimmunogens will be used to produce monospecific antibodies. Quantitative Western blot analyses will be developed to analyze the levels and distribution of the different HTH forms in post-mortem human brain. Immunocytochemistry will be used to analyze the regional, cellular and subcellular distribution of HTH forms in brain. Alternative splicing is predicted to change the phosphorylation of serine 31 from that of being nerve growth factor and phorbol ester sensitive to that of being a substrate for calcium/calmodulin-dependent protein kinase II. Site-specific phosphorylation of recombinant HTH in AtT-20 cells, of HTH in LA-N human neuroblastoma cells, of HTH in intact post-mortem human tissues (synaptosomes from corpus striatum and nucleus accumbens, chromaffin cells from adrenal medullae), and of HTH in cells isolated from human pheochromocytoma (as available) will be investigated.

This is a revised application for competitive renewal of K02 support for Dr. John W. Haycock. This award will enable Dr. Haycock to continue his scientific development and to pursue his recently funded research program (MH55208) on brain monoaminergic systems in mental illnesses, which was initiated and developed over the course of the current award. The ability to study clinical issues related to catecholamine function concurrently with his previously established fundamental neuroscience research program (NS25134) has realized one of the candidate's major long-term goals. As a result, the candidate's short-term goals are focused upon development of his laboratory by recruiting younger scientists to participate in effecting the scientific goals of both the clinical and fundamental research projects. In addition to developing his role as a laboratory director, the candidate will continue his scientific development by pursuing a novel, multidisciplinary approach which he has recently developed, using antibodies to labile epitopes for studying the regulation of signaling molecules in vivo. The proposed research plan focuses upon tyrosine hydroxylase (TyrOH) and tryptophan hydroxylase (TrpOH), which catalyze the initial and rate- limiting steps in catecholamine (dopamine and norepinephrine) and serotonin biosynthesis, respectively. Alterations in each of these systems have been implicated in mental disorders and, in particular, schizophrenia. TyrOH is highly regulated--by protein phosphorylation in the short-term and by transcriptional control in the long-term; and, alternative splicing (which occurs exclusively in monkeys and humans) produces multiple TyrOH isoforms and perhaps, an additional level of regulation. By contrast, comparatively little is known about TrpOH, despite its evolutionary and functional proximity to TyrOH. Postmortem human brain tissue will be analyzed using quantitative blot immunolabeling techniques and a bank of antibodies developed for this purpose by the applicant. TyrOH and TrpOH protein levels, as well as the relative abundances of TyrOH isoforms, will be measured in neurochemically appropriate brain regions dissected from cryostatic sections. DOPA decarboxylase (immediately downstream of both TyrOH and TrpOH) and dopamine beta-hydroxylase (which converts dopamine to norepinephrine) protein levels will also be quantitated, and similar assays have been developed for another class of presynaptic monoaminergic markers--the vesicular and plasmalemmal monoamine transporters. The primary study groups will consist of (a.) suicide/sudden death victims having confirmed diagnoses of schizophrenia and (b.) age-matched, sudden-death control subjects having no Axis 1 mental disorder. Parallel, collaborative studies of major depressives will provide comparison groups and allow identification of potential disease-specific differences.

DESCRIPTION: (Adapted from applicant's abstract) Tyrosine hydroxylase (TyrOH) and tryptophan hydroxylase (TrpOH) catalyze the rate-limiting steps in catecholamine (dopamine and norepinephrine) and serotonin biosynthesis, respectively. Alterations in each of these systems have been implicated in mental disorders and, in particular schizophrenia. TyrOH is highly regulated--by protein phosphorylation in the short-term and by transcriptional control in the long-term. Alternative splicing (which occurs exclusively in monkeys and humans) produces multiple TyrOH isoforms having different phosphorylation sites, such that changes in RNA splicing or in relative mRNA/protein turnover rates among isoforms could represent an additional level of regulation. By contrast, despite the close evolutionary and functional similarities between TrpOH and TyrOH, very little is known about the regulation of TrpOH. Moreover, even less is known regarding the status of these enzymes in mental disorders. Postmortem human brain tissue will be analyzed using quantitative blot immunolabeling techniques. TyrOH and TrpOH protein levels, as well as the relative abundances of TyrOH isoforms, will be determined in several brain regions. Depending upon the brain region/monoamine system being studied, DOPA decarboxylase (which catalyzes the second step in catecholamine and serotonin biosynthesis) and dopamine beta-hydroxylase (which converts dopamine to norepinephrine) protein levels will also be determined. The primary study groups will be (a) suicide/sudden death victims having diagnoses of schizophrenia from psychiatric autopsy and (b) age-matched, sudden-death control subjects having no axis 1 mental disorder. Additional toxicological and neuropathological screening criteria will be applied to all cases, and samples will be processed in a blind, matched-pairs design for the neurochemical analyses. Coordination of experimental procedures with ongoing studies of major depressives from the same collection of brains will enable direct comparisons between the disorders. An independent cohort of schizophrenics and age-matched controls (and bipolar affective disorder comparison group) will be used for replicating effects in selected brain regions.