Mella Adlersberg

Attenuated 5‐HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase, phosphatidylinositol 3‐kinase, Akt and mitogen‐activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5‐HT)1A receptor antagonist‐binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5‐HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III‐R criteria. Measurements of [35S]GTPγS binding to Gαi/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5‐HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Gαi/o, Gαq/11 or Gαs proteins, or in the activity of cAMP‐dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5‐HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3‐kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5‐triphosphate. Finally, the activation of extracellular signal‐regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist‐stimulated 5‐HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5‐HT1A receptor transduction pathways that may have implications for cell survival.

Regulation of dopamine D1-receptor activation in vivo by protein phosphatase 2B (calcineurin)

Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist‐promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1‐agonist‐stimulated [35S]GTPγS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild‐type mice and D2‐receptor mutants. These studies revealed a decreased agonist‐stimulated G‐protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPγS binding in a concentration‐dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal andD1‐agonist‐stimulated phosphorylation of D1‐receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increasedkinase‐mediated phosphorylation.

Prenatal development of serotonin binding protein in relation to other transmitter-related characteristics of central serotonergic neurons

Serotonin binding protein (SBP) is a neuron-specific protein that binds serotonin (5-HT) with high affinity and is concentrated in synaptic vesicles. 5-HT has been shown to be stored in situ in a macromolecular complex with SBP. We have now investigated the ontogeny of SBP in the rat CNS. The time course of the appearance of SBP was related to the appearance of tryptophan hydroxylase, endogenous 5-HT and monoamine oxidase (MAO; types A and B). Binding of [3H]5-HT by SBP was assayed using molecular sieve chromatography. SBP had appeared by day E16; its activity then rose rapidly and reached adult levels (150 nmol [3H]5-HT/g protein) at days E18-E19. Tryptophan hydroxylase activity was measured by following the accumulation in vivo (30 min) of 5-hydroxytryptophan (5-HTP) in the brains of rat embryos whose mothers were treated with the aromatic L-amino acid decarboxylase inhibitor, NSD-1015, (100 mg/kg; i.p.). Tryptophan hydroxylase activity was first detectable at E15, remained present but at a low level through day E20 and then rapidly increased to reach 75% of the adult level at birth (747 pmol/g brain wet wt.). The development of stores of endogenous 5-HT paralleled the time course of development of tryptophan hydroxylase rather than that of SBP. 5-HT was first detected at E15, remained low until the end of intrauterine life and at birth was 50% of the adult level (2640 pmol/g brain wet wt.). MAO activity was determined in crude mitochondrial fractions by measuring 5-hydroxyindoleacetic acid produced from 5-HT as substrate. This activity was already present prior to day E15 (the activity of type B MAO was higher than that of type A) and reached adult levels at day E20 (55 pmol/mg protein/min; A, B). It is concluded that the potential of neurons to store 5-HT, as measured by the activity of SBP, develops more rapidly than their ability to produce 5-HT. Moreover, although the ratio of its two forms changes, MAO activity appears very early in development.

A Single Dose of Methamphetamine Rescues the Blunted Dopamine D1-Receptor Activity in the Neocortex of D2- and D3-Receptor Knockout Mice

Abstract: Knockout mice deficient for dopamine D2 and D3 receptors exhibit blunted c‐fos responses to D1‐agonist stimulation. A single dose of methamphetamine (METH), however, leads to a long‐term reversal of these blunted c‐fos responses in both mutants, and the same effect is obtained with a single administration of a full D1‐agonist. Consistent with the predominant c‐fos expression in the neocortex induced by METH itself, METH pretreatment leads to the largest D1‐agonist‐stimulated c‐fos responses in the neocortex of these mutants. For example, a pronounced blunting of neocortical c‐fos responses is detected in the prefrontal cortex, a region in which D1 receptors play a critical role in working memory. METH pretreated mutants, however, exhibit robust c‐fos responses in this region that are indistinguishable from wild type. Recent studies indicate that different mechanisms operate in brains of D2 and D3 mutants to lead to decreased D1‐receptor activity. For example, drug‐naive D2, but not D3, mutants show significantly decreased G protein activation in response to D1‐agonist stimulation, and METH pretreatment also rescues this abnormal molecular phenotype. Moreover, although the protein phosphatases (PP) 1/2A and 2B play a critical role in modulating G protein activation in wild type, their effect is either diminished (PP1/2A) or abolished (2B) in D2 mutants. Interestingly however, METH pretreatment does not rescue the activities of these phosphatases in the mutants, suggesting that the long‐term effects of a single dose of METH are mediated via effector systems that act downstream of G protein activation.

Serotonin Binding Protein: Synthesis, Secretion, and Recycling

Abstract: Serotonin binding protein (SBP) is present in all neurectodermally derived cells that store serotonin (5‐HT). Three forms of SBP have been detected (68, 56, and 45 kDa), and antibodies to SBP that interfere with the binding of 5‐HT react with each of these proteins. The current experiments test two hypotheses: (a) that the 56‐ and 45‐kDa forms of SBP are produced by posttranslational cleavage of a 68‐kDa precursor molecule; and (b) that 45‐kDa SBP is a constituent of serotonergic secretory vesicles. Pulse‐chase experiments were carried out using medullary thyroid carcinoma cells as a model. These neurectodermally derived cells produce 5‐HT and all three forms of SBP. Following pulse labeling for 20 min with l‐[35S]methionine, the cells were incubated in the presence of an excess of unlabeled l‐methionine for 0, 30, 60, or 90 min at 37°C. Alternatively, the chase was performed under conditions (20°C, inhibition of ATP generation) that delay or stop transport of newly synthesized proteins from the rough endoplasmic reticulum through the Golgi apparatus. Following incubation, the cells were washed and solubilized, and SBP was immunoprecipitated. Radioactive proteins in the immunoprecipitate were electrophoretically resolved and quantified. Immediately after the pulse, each of the three forms of SBP was found to be labeled with 35S. The relative proportions of 35S‐labeled 68‐, 56‐, and 45‐kDa SBP remained the same at each interval of chase. These proportions were not changed when the chase was carried out at 20°C or under conditions that blocked the biosynthesis of ATP. These observations suggest that each form of SBP is a primary product of translation, that the smaller forms of SBP are not produced by cleavage from a larger molecule, and that the size of the primary products of translation is not altered by passage to the Golgi apparatus or a post‐Golgi compartment. When secretion was induced, 45‐kDa SBP, but not 56‐ or 68‐kDa SBP, was released to the medium. When antibodies to 45‐kDa SBP were added to the medium at the time secretion was induced, antibody binding sites appeared as patches on the cell surfaces. Because of these sites, cells were lysed when they were stimulated to secrete in the presence of antibodies to 45‐kDa SBP and guinea pig complement. Antibody binding sites disappeared from cell surfaces after 20 min, at which time antibodies to SBP were found inside the cells. It is suggested that 45‐kDa SBP is packaged with 5‐HT in secretory vesicles. Some 45‐kDa SBP is lost during secretion as a result of exocytosis; however, a fraction of the 45‐kDa SBP remains bound to the luminal surface of the membrane of secretory vesicles. This protein is exposed to the ambient medium as a consequence of exocytosis, but is reinternalized when the vesicular membrane is recaptured during vesicle recycling.

Synthesis of 3-deoxy-d-arabino-2-heptulosonic acid 7-phosphate by phosphorylation of methyl (methyl 3-deoxy-d-arabino-heptulopyranosid)onate

Abstract 3-Deoxy- d - arabino -2-heptulosonic acid 7-phosphate ( 5 ), the first committed intermediate in aromatic amino acid biosynthesis, has been synthesized in good yield by treatment of methyl (methyl 3-deoxy- d - arabino -2-heptulopyranosid)onate with diphenylphosphoric chloride under mild conditions to give the 7-diphenyl phosphate. Catalytic removal of the phenyl residues, followed by base-catalyzed hydrolysis resulted in formation of (methyl 3-deoxy- d - arabino -2-heptulopyranosid)onic acid dihydrogen 7-phosphate ( 4 ), which yielded a crystalline tris-(cyclohexylammonium) salt. Acid-catalyzed hydrolysis of 4 afforded 5 , which was used to purify 3-dehydroquinate synthase.