Shu-chi Hsiung

Serotonin 1A Receptors, Serotonin Transporter Binding and Serotonin Transporter mRNA Expression in the Brainstem of Depressed Suicide Victims

Suicide and depression are associated with reduced serotonergic neurotransmission. In suicides, there is a reduction in serotonin transporter (SERT) sites and an increase in postsynaptic 5-HT1A receptors in localized regions of the prefrontal cortex. In depression, there is a diffuse decrease in SERT binding throughout the dorsoventral extent of the prefrontal cortex. Serotonergic innervation of the prefrontal cortex arises predominantly from neurons in the brainstem dorsal raphe nucleus (DRN). We, therefore, examined postmortem SERT binding and mRNA expression, as well as 5-HT1A autoreceptor binding in the DRN of 10 matched pairs of controls and depressed suicide victims. The concentration of SERT sites, SERT mRNA, and 5-HT1A binding was not different between controls and suicides (p > .05). In the DRN of suicides, the volume of tissue defined by 5-HT1A binding was 40% smaller than controls. An index of the total number of 5-HT1A receptors (receptor binding × volume of receptor distribution) was 43.3% lower in the DRN of suicides, compared with controls. The suicide group had 54% fewer DRN neurons expressing SERT mRNA compared with controls. In the serotonin neurons that expressed the SERT gene, expression per neuron was greater in suicides. Less total 5-HT1A and SERT binding is consistent with results of in vivo studies in depression. Less feedback inhibition of serotonin DRN firing via 5-HT1A autoreceptors and enhancement of serotonin action due to less uptake of serotonin, is consistent with compensatory changes in response to hypofunction in depressed suicides.

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.

Roles of extracellular signal‐regulated kinase and Akt signaling in coordinating nuclear transcription factor‐κB‐dependent cell survival after serotonin 1A receptor activation

To investigate the functional consequences of cross‐talk between multiple effectors of serotonin (5‐HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5‐HT1A receptor stimulated extracellular signal‐regulated kinase (ERK)1/2, Akt and nuclear transcription factor‐κB (NF‐κB). Stimulation of cells with 5‐HT1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5‐HT1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3‐kinase (PI3K). Stimulation of 5‐HT1A receptor also resulted in activation of NF‐κB through a decrease in inhibitor of nuclear transcription factor‐κB. In support of the importance of 5‐HT1A receptor signaling for cell survival, inhibition of NF‐κB facilitated caspase 3 activation and cleavage of poly (ADP‐ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist‐dependent NF‐κB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant‐negative Akt. These findings suggest a role for 5‐HT1A receptor signaling in the Ras/Raf‐dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF‐κB activation were required for 5‐HT1A receptor‐dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5‐HT1A receptor activation.

In vitro autoradiography of serotonin 5-HT2A/2C receptor-activated G protein: Guanosine-5?-(?-[35S]thio)triphosphate binding in rat brain

Agonist activation of G protein‐coupled receptors induces an increase in the binding of guanosine 5′‐(γ‐[35S]thio)triphosphate ([35S]GTPγS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide‐binding regulatory protein (G protein). The present study uses agonist‐stimulated [35S]GTPγS binding to characterize serotonin 5‐HT2A/2C receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide‐mounted sections. The stimulatory effect of the agonist [1‐(2,5‐dimethoxy‐4‐iodophenyl)]‐2 aminopropane (DOI) is compared to that of serotonin (5‐HT). Autoradiography revealed a similar localization of DOI‐ and 5‐HT‐stimulated binding of [35S]GTPγS in distinct areas of prefrontal and parietal cortex, consistent with previously reported 5‐HT2A receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbital‐insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5‐HT2A antagonist, and ketanserin, an antagonist of 5‐HT2A/2C receptors, blocked DOI stimulation in all labeled areas, whereas 5‐HT stimulation was only partially blocked (70–80%). A small but significant inhibition was observed with the specific antagonist of 5‐HT2C/2B, SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor–Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions. J. Neurosci. Res. 61:674–685, 2000.

Identification of Serotonin Receptors Recognized by Anti‐Idiotypic Antibodies

Anti‐idiotypic antibodies were generated by immunizing rabbits with affinity‐purified antibodies to serotonin (5‐hydroxytryptamine; 5‐HT). Anti‐5‐HT activity was removed from the resulting antisera by chromatography through a 5‐HT affinity column. The anti‐idiotypic antibodies were demonstrated by enzyme‐linked immunosorbent assay to bind to affinity‐purified whole anti‐5‐HT antibodies and their Fab fragments. Anti‐idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5‐HT were coupled, competed with 5‐HT (covalently bound to protein) for the binding sites on anti‐5‐HT antibodies and serotonin binding protein. The anti‐idiotypic antibodies antagonized the binding of [3H]5‐HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti‐ idiotypic antibodies also blocked the binding of the 5‐HT1B‐ selective ligand (‐)‐[125I]iodocyanopindolol (in the presence of 30 μM isoproterenol) to cortical membranes. In contrast, anti‐idiotypic antibodies failed to inhibit binding of the 5‐ HT1A‐selective ligand 8‐hydroxy‐2‐(di‐n)‐[3H]propylamino)‐ tetralin ([3H]8‐OH‐DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti‐idiotypic antibodies may recognize some 5‐HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti‐idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5‐ HT1C or 5‐HT2 receptor or with a genomic clone encoding the 5‐HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5‐ HT1C and 5‐HT2 receptors, but not on the surfaces of HeLa cells that expressed 5‐HT1A receptors. Immunostaining of cells by the anti‐idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5‐HT1C receptors was blocked by mesulergine (but not ketanserin, 8‐OH‐DPAT. or spiperone), whereas that of cells expressing 5‐HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8‐OH‐DPAT). The anti‐ idiotypic antibodies failed to inhibit the uptake of [3H]5‐HT by serotonergic neurons. It is concluded that the anti‐idiotypic antibodies generated with anti‐5‐HT serum recognize the 5‐ HT1B, 5‐HTlC, and 5‐HT2 receptor subtypes; however, neither 5‐HT1A receptors nor 5‐HT uptake sites appear to react with these antibodies.

Inhibition of 5-HT1A receptor-dependent cell survival by cAMP/protein kinase A: Role of protein phosphatase 2A and Bax

Serotonergic 5‐HT1A receptor signaling leading to nuclear factor‐κB (NF‐κB) activation appears to be critical for cell survival. Adenylyl cyclase and protein kinase A (AC/PKA) are effectors of the 5‐HT1A receptor that are inhibited by Gαi subunits. Conversely, Gβγi subunits downstream from the 5‐HT1A receptor participate in the activation of extracellular signal‐regulated kinases (ERK1/2), phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB. To model the contribution of pro‐ and antiapoptotic signaling cascades downstream of activated 5‐HT1A receptor in cell survival, Chinese hamster ovarian (CHO) cells were employed that exogenously overexpress 5‐HT1A receptors. Stimulation with the 5‐HT1A receptor agonist 8‐OH‐DPAT and pharmacological agonists of AC induced PKA and protein phosphatase 2A (PP2A) activity, which in turn inhibited: Akt activity, IκBα degradation, nuclear translocation of NF‐κB, and expression of X‐linked inhibitor of apoptosis protein (XIAP/BIRC4). Pharmacological inhibition of PP2A with calyculin A potentiated Akt activity while attenuating ERK1/2 signaling via increased inhibitory phosphorylation of Raf (pSer259). In contrast, increased cAMP levels enhanced Bax translocation to the mitochondria, resulting in the release of cytochrome c, caspase‐3 activation, and apoptosis induction. Our data suggest a central role of cAMP/PKA‐dependent PP2A in shifting the homeostasis of intracellular signaling downstream of activated 5‐HT1A receptor toward cell death in biological systems linked to neuropsychiatric disorders.

A Ca2+‐Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

Abstract: The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5‐hydroxytryptamine (5‐HT, serotonin). A protein kinase activity that copurifies with SBP (SBP‐kinase) was partially characterized and compared with calcium/calmodulin‐dependent protein kinase II (CAM‐PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5‐HT. SBP‐kinase and CAM‐PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+‐dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule‐associated proteins well and did not phosphorylate myosin, phosphorylase b., and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP‐kinase and CAM‐PK II were observed: (1) CAM enhanced CAM‐PK II activity, but had no effect on SBP‐kinase; (2) synapsin I was an excellent substrate for CAM‐PK II, but not for SBP‐kinase; (3) 5‐HT inhibited both the autophosphorylation of SBP‐kinase and the phosphorylation of SBP, but had no effect on CAM‐PK II. These data indicate that SBP‐kinase is different from CAM‐PK II. Phosphopeptide maps of SBP and SBP‐kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5‐HT within neurons.

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.

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.