Jean Chen Shih

Serotonin2A receptor gene polymorphism in mood disorders

Genes that regulate the serotonin (5-HT) system including 5-HT receptors may be involved in mood disorders. We studied 5-HT2A receptor exons and the adjacent intron regions in 102 patients with mood disorders (71 depressive disorders and 31 bipolar disorders). In 34 mood disorder cases, the gene encoding the 5-HT1A receptor had been sequenced, but no disease-specific polymorphism was found. The substitution of C for T at position 102 in exon 1, which had been reported by Warren et al., was confirmed. The corresponding amino acid, serine, did not change. The allele frequency of C [corrected] at position 102 was significantly higher in patients with depressive disorders than in those with bipolar disorders and healthy control subjects. Furthermore, the mean age of onset in the patients heterozyous for the T and C alleles was lower than that in those homozygous for the C allele. No other polymorphism in the gene was found.

Striatal dopamine metabolism in monoamine oxidase b-deficient mice : A brain dialysis study

Abstract : We have studied striatal dopamine (DA) metabolism in monoamine oxidase (MAO) B‐deficient mice using brain microdialysis. Baseline DA levels were similar in wild‐type and knock‐out (KO) mice. Administration of a selective MAO A inhibitor, clorgyline (2 mg/kg), increased DA levels and decreased levels of its metabolites in all mice, but a selective MAO B inhibitor, l‐deprenyl (1 mg/kg), had no effect. Administration of 10 and 50 mg/kg l‐DOPA, the precursor of DA, increased the levels of DA similarly in wild‐type and KO mice. The highest dose of l‐DOPA (100 mg/kg) produced a larger increase in DA in KO than wild‐type mice. This difference was abolished by pretreating wild‐type mice with l‐deprenyl. These results suggest that in mice, DA is only metabolized by MAO A under basal conditions and by both MAO A and B at high concentrations. This is in contrast to the rat, where DA is always metabolized by MAO A regardless of concentration.

Ketanserin and tetrabenazine abolish aggression in mice lacking monoamine oxidase A

Mice deficient in monoamine oxidase A (MAO A) have elevated brain levels of 5-HT and manifest enhanced aggression. We used these mice as a model to study the role of 5-HT in aggression. Our results show that ketanserin and tetrabenazine (TBZ) strikingly abolished the aggressive behavior of MAO A-deficient mice. The anti-aggressive effect of ketanserin may be primarily mediated by 5-HT(2A) receptors. Another specific 5-HT(2A) antagonist, [R-(+)-a-(2, 3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methan ol (MDL 100907), also blocks the aggression of mutant mice but was less dramatic. Ketanserin and TBZ are both antagonists of the vesicular monoamine transporter (VMAT2). The anti-aggressive effect of TBZ and part of the effect of ketanserin may be mediated by the VMAT2. Using radioligand binding and autoradiography, we also showed that the numbers of VMAT2, 5-HT(1A), 5-HT(2A) and 5-HT(2C) sites are decreased in brains of mutant mice, which may reflect down-regulation by excess 5-HT. This study suggests that ketanserin and TBZ may be developed as novel anti-aggressive agents.

Characterization of the human 5-HT2A receptor gene promoter

The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, it is important to characterize its promoter. In this report, the 5′ end of the human 5- HT2AR gene was cloned and characterized. Anchored PCR mapped multiple transcription initiation sites at nucleotides -1157, -1137, -1127, and - 496. Transfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT2AR-positive human cell lines (SHSY-5Y, neuroblastoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites between - 1157 and -1127 and 5′ of the downstream initiation site (at -496), exhibited significant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (the 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multiple cis and trans elements for this fragment, including Sp1, PEA3, cyclic AMP response element (CRE)-like sequence, and E- boxes. Two novel transcription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcription factors and promoter activities were low in the negative cell line NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.39 kb fragment, isolated from the 3′ end of the 0.74 kb fragment, exhibited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data are essential for further study of the regulation of 5-HT2AR gene expression.

Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling.

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.

Dual Effects of Ascorbate on Serotonin and Spiperone Binding in Rat Cortical Membranes

Abstract: Ascorbate‐induced lipid peroxidation, as measured by malonyldialdehyde (MDA) production, caused irreversible decreases in Bmax of both [3H]5‐HT and [3H]spiperone binding. CaCl2 (4 mM) inhibited ascorbateinduced MDA formation at ascorbate concentrations >0.57 mM, but not at ≤0.57 mM. Under the standard assay conditions (5.7 mM ascorbate and 4 mM CaCl2), CaCl2 inhibited the MDA production caused by ascorbate by 88%, and the loss in [3H]5‐HT binding by 57%. Ascorbate still decreased [3H]5‐HT binding when lipid peroxidation was completely inhibited by EDTA. This additional effect of ascorbate was reversible after washing the membranes. Other reducing agents (dithiothreitol, glutathione, and metabisulfite) also decreased the binding of [3H]serotonin. In contrast, [3H]spiperone binding was not affected by ascorbate in the absence of lipid peroxidation or by other reducing agents. These experiments demonstrate that ascorbate has a dual and differential effect on serotonin binding sites. First, ascorbate‐induced lipid peroxidation irreversibly inactivates both [3H]5‐HT and [3H]spiperone binding. Second, independent of lipid peroxidation, there is a direct, reversible effect of ascorbate on [3H]serotonin but not on [3H]spiperone binding, which is probably due to the difference in the biochemical nature of the two serotonin binding sites.

Forebrain-specific Expression of Monoamine Oxidase A Reduces Neurotransmitter Levels, Restores the Brain Structure, and Rescues Aggressive Behavior in Monoamine Oxidase A-deficient Mice

Previous studies have established that abrogation of monoamine oxidase (MAO) A expression leads to a neurochemical, morphological, and behavioral specific phenotype with increased levels of serotonin (5-HT), norepinephrine, and dopamine, loss of barrel field structure in mouse somatosensory cortex, and an association with increased aggression in adults. Forebrain-specific MAO A transgenic mice were generated from MAO A knock-out (KO) mice by using the promoter of calcium-dependent kinase IIα (CaMKIIα). The presence of human MAO A transgene and its expression were verified by PCR of genomic DNA and reverse transcription-PCR of mRNA and Western blot, respectively. Significant MAO A catalytic activity, autoradiographic labeling of 5-HT, and immunocytochemistry of MAO A were found in the frontal cortex, striatum, and hippocampus but not in the cerebellum of the forebrain transgenic mice. Also, compared with MAO A KO mice, lower levels of 5-HT, norepinephrine, and DA and higher levels of MAO A metabolite 5-hydroxyindoleacetic acid were found in the forebrain regions but not in the cerebellum of the transgenic mice. These results suggest that MAO A is specifically expressed in the forebrain regions of transgenic mice. This forebrain-specific differential expression resulted in abrogation of the aggressive phenotype. Furthermore, the disorganization of the somatosensory cortex barrel field structure associated with MAO A KO mice was restored and became morphologically similar to wild type. Thus, the lack of MAO A in the forebrain of MAO A KO mice may underlie their phenotypes.

No association of the MAOA gene with alcoholism among Han Chinese males in Taiwan

A positive association of MAOA polymorphisms with alcoholism has been demonstrated in certain recent studies, however, this association is not universally supported. The haplotype status of the MAOA gene polymorphisms could provide more information than alleles at a single site alone tested for an association with a complex, heterogeous disorder. This study examines whether there is an association between alcoholism and either a variable number of tandem repeat located upstream of the MAOA gene or an EcoRV functional polymorphism of the MAOA gene. These are analyzed both individually and as haplotypes. The study consisted of 214 subjects meeting DSM-IV criteria for alcoholism from northern Taiwan and 77 control individuals without history of alcoholism from Taipei. All of the subjects were Chinese Han males. For the two polymorphic sites, significant linkage disequilibrium occurred. No significant intergroup difference was observed between the two subject groups with respect to the allele frequencies for the two polymorphisms at the MAO locus tested both individually and as haplotypes. This finding suggests that no association exists between genetic variation at the MAOA locus and alcoholism in Chinese Han males.

Phe208 and Ile199 in Human Monoamine Oxidase A and B Do Not Determine Substrate and Inhibitor Specificities as in Rat

Abstract: It has been reported previously that reciprocally switching Phe208 and Ile199 in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A‐F208I showed a sixfold decrease in the specificity constant kcat/Km for both the MAO A‐ and the MAO B‐preferring substrates 5‐hydroxytryptamine and β‐phenylethylamine, respectively. The reciprocal point mutant MAO B‐I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159‐214 in MAO A and 150‐205 in MAO B. Chimerics MAO AB159‐214A and MAO BA150‐205B had small changes in Km and IC50 values when compared with MAO A and B, respectively, but did not exhibit a preference switch. The results suggest that Phe208 in MAO A and amino acid segments 159‐214 and 150‐205 in MAO A and B, respectively, influence the enzyme active site. However, substrate and inhibitor preferences of human MAO A and B are not determined by the respective residues Phe108 and Ile199 as in rat MAO nor by their neighboring regions.

Determination of regions important for Monoamine Oxidase (MAO) A and B substrate and inhibitor selectivities

MAO-A and -B are defined by their substrate and inhibitor preferences. To determine which regions of the isoenzymes confer these preferences, we have constructed six chimeric MAO enzymes by reciprocally exchanging corresponding N-terminal, C-terminal, and internal segments of MAO-A and -B then determined the catalytic properties of these chimeric enzymes. N-terminal chimerics A45B and B36A were made by exchanging amino acid segments 1-45 and 1-36 of MAO-A and -B respectively. C-terminal chimerics A402B and B393A were made by exchanging amino acid segments 403-527 and 394-520 of MAO-A and -B respectively, and internal chimerics AB161-375A and BA152-366B were made by exchanging amino acid segments 161-375 and 152-366 of MAO-A and -B respectively. The enzymatic properties observed for the chimerics suggest that the exchanged internal regions but not the N- or C-terminal regions confer substrate and inhibitor preferences.