Alternate Promoters in the Rat Aromatic l-Amino Acid Decarboxylase Gene for Neuronal and Nonneuronal Expression: An In Situ Hybridization Study

Abstract: Aromatic l -amino acid decarboxylase (AADC) is found in both neuronal cells and nonneuronal cells, and a single gene encodes rat AADC in both neuronal and nonneuronal tissues. However, two cDNAs for this enzyme have been identified: one from the liver and the other from pheochromocytoma. Exons 1a and 1b are found in the liver cDNA and the pheochromocytoma cDNA, respectively. In the third exon (exon 2), there are two alternatively utilized splicing acceptors specific to these exons, 1a and 1b. Structural analysis of the rat AADC gene showed that both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. To demonstrate whether alternative promoter usage and splicing are tissue specific and whether the exons 1a and 1b are differentially and specifically transcribed in nonneuronal and neuronal cells, respectively, in situ hybridization histochemistry for the rat brain, adrenal gland, liver, and kidney was carried out using these two exon probes. The exon 1a probe specifically identified AADC mRNA only in nonneuronal cells, including the liver and kidney, and the exon 1b probe localized AADC mRNA to monoaminergic neurons in the CNS and the adrenal medulla. Thus, both alternative promoter usage and differential splicing are in fact operative for the tissue-specific expression of the rat AADC gene.     

Intrastriatal Grafting of Cos Cells Stably Expressing Human Aromatic l-Amino Acid Decarboxylase: Neurochemical Effects

Abstract: To study the possibility that increasing striatal activity of aromatic l -amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to l -3,4-dihydroxyphenylalanine ( l -DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos- haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of ¹⁴CO₂ from l -[¹⁴C]DOPA. The K _m value for l -DOPA in intact and disrupted cells was 0.60 and 0.56 m M , respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 m M . The pH optimum for enzyme activity was 6.8. Grafting Cos- haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of l -DOPA. When measured 2 h after l -DOPA administration, the mean dopamine level in the striata of Cos- haadc -grafted animals was 2 µg/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 µg/g. At 6–8 h after l -DOPA administration, striatal dopamine content in the Cos- haadc -grafted animals was 0.67 µg/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered l -DOPA.     

Regulation of Aromatic l-Amino Acid Decarboxylase by Dopamine Receptors in the Rat Brain

Decarboxylation of phenylalanine by aromatic L-amino acid decarboxylase (AADC) is the rate-limiting step in the synthesis of 2-phenylethylamine (PE), a putative modulator of dopamine transmission. Because neuroleptics increase the rate of accumulation of striatal PE, these studies were performed to determine whether this effect may be mediated by a change in AADC activity. Administration of the D1 antagonist SCH 23390 at doses of 0.01-1 mg/kg significantly increased rat striatal AADC activity in an in vitro assay (by 16-33%). Pimozide, a D2-receptor antagonist, when given at doses of 0.01-3 mg/kg, also increased AADC activity in the rat striatum (by 25-41%). In addition, pimozide at doses of 0.3 and 1 mg/kg increased AADC activity in the nucleus accumbens (by 33% and 45%) and at doses of 0.1, 0.3, and 1 mg/kg increased AADC activity in the olfactory tubercles (by 23%, 30%, and 28%, respectively). Analysis of the enzyme kinetics indicated that the Vmax increased with little change in the Km with L-3,4-dihydroxyphenylalanine as substrate. The AADC activity in the striatum showed a time-dependent response after the administration of SCH 23390 and pimozide: the activity was increased within 30 min and the increases lasted 2-4 h. Inhibition of protein synthesis by cycloheximide (10 mg/kg, 0.5 h) had no effect on the striatal AADC activity or on the increases in striatal AADC activity produced by pimozide or SCH 23390. The results indicate that the increases in AADC activity induced by dopamine-receptor blockers are not due to de novo synthesis of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Recombinant Human Aromatic l-Amino Acid Decarboxylase Expressed in COS Cells

The expression vector containing the full-length cDNA of human aromatic L-amino acid decarboxylase (EC 4.1.1.28) was transfected in COS cells by a modified calcium phosphate coprecipitation method. The cells transfected with plasmids that had a true direction of the cDNA gave a major immunoreactive band at 50 kDa. This expressed enzyme catalyzed the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA), L-5-hydroxytryptophan (L-5-HTP) and L-threo-3,4-dihydroxyphenylserine. The optimal pH of the enzyme activity with L-DOPA as a substrate was 6.5, whereas the enzyme had a broad pH optimum when L-5-HTP was used as a substrate. Addition of pyridoxal phosphate to the incubation mixture greatly enhanced the activity for both L-DOPA and L-5-HTP.     

Aromatic l-Amino Acid Decarboxylase Activity of the Rat Retina Is Modulated In Vivo by Environmental Light Maria Hadjiconstantinou

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina is low in animals placed in the dark. When the room lights are turned on, activity rises for almost 3 h and reaches values that are about twice the values found in the dark. A study of the kinetics of the enzyme revealed that the apparent Km values for L-3,4-dihydroxyphenylalanine and pyridoxal-5'-phosphate were unchanged in light- and dark-exposed animals, whereas the Vmax increased in the light. Treating the animals with cycloheximide before exposure to light prevented the increase of enzyme activity. Immunotitration with antibodies to AAAD suggested that more enzyme molecules are present in the light than in the dark. When the room lights are turned off AAAD activity drops rapidly at first and then more slowly, suggesting that at least two processes are responsible for the fall of enzyme activity. Exposure to short periods of dark followed by light results in a rapid increase of AAAD activity. Mixing homogenates from light- and dark-exposed rats results in activity values that are less than expected, suggesting the presence of an endogenous inhibitor(s). These studies demonstrate that AAAD activity is modulated in vivo.     

Phorbol Ester Administration Transiently Increases Aromatic l-Amino Acid Decarboxylase Activity of the Mouse Striatum and Midbrain

Abstract: Aromatic l -amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12-myristate 13-acetate administered intracerebroventricularly transiently increased AAAD activity by 30–50% over control values within ∼30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of V _max with little change of K _m for either l -3,4-dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester-induced increase of AAAD. Moreover, okadaic acid, a serine/threonine-selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C-mediated pathways modulate AAAD activity in vivo.     

Modulation of Retinal Aromatic l-Amino Acid Decarboxylase via α2 Adrenoceptors

Aromatic L-amino acid decarboxylase (AAAD) activity of the rat retina increases when animals are placed in a lighted environment from the dark. The rise of activity can be inhibited by administering alpha 2 adrenoceptor agonists. In the dark, the enzyme activity can be made to increase by administering alpha 2 adrenoceptor antagonist drugs. Kinetic analysis indicates that the maximum velocity of the enzyme increases with little change of the Km for the substrate L-3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. The rise of activity in the light and in the dark after alpha 2 antagonists can be blocked by administering cycloheximide, suggesting that protein synthesis is needed for the response. We speculate that epinephrine released in the dark from a subpopulation of retinal amacrine cells onto alpha 2 receptors suppresses AAAD activity that is associated with dopaminergic amacrines.     

Structure of the Rat Aromatic L-Amino Acid Decarboxylase Gene: Evidence for an Alternative Promoter Usage

Abstract: Aromatic L-amino acid decarboxylase catalyzes the biosynthesis of the neurotransmitters dopamine and serotonin. This enzyme is also expressed in nonneuronal tissues. Two reported cDNA sequences show that the pheochromocytoma message differs from the liver message only at the 5'untranslated region. We present the complete exonal organization and promoter sequences of the rat gene encoding this enzyme. The rat aromatic L-amino acid decarboxylase gene is composed of two promoters and 16 exons spanning more than 80 kb in the genome. The first exon carries the majority of the 5'untranslated sequence of the liver cDNA, and the second exon carries that of the pheochromocytoma cDNA. In the third exon, there are two alternatively utilized splicing acceptors specific to the first and second exons. Therefore, both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. The sequence of each promoter region shows putative binding sites for octamer factors and AP-2.     

Purification and Characterization of Aromatic L-Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme

Aromatic L-amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by polyacrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4-dihydroxyphenylalanine (dopa) decarboxylation of approximately 11,000 nmol/min/mg of protein at 37 degrees C. The purified enzyme also catalyzed the decarboxylation of 5-hydroxytryptophan, tyrosine, tryptophan, and phenylalanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60-5.85 in its absence. To examine the identity of aromatic L-amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody-affinity chromatography followed by SDS-polyacrylamide gel electrophoresis revealed that the enzymes from the striatum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross-reactivity and molecular size.     

Inhibition of Aromatic L-Amino Acid Decarboxylase and Tyrosine Aminotransferase by the Monoamine Oxidase Inhibitor Phenelzine

The concentration of p-tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect on p-tyramine levels in the first 1-2 h following injection. The high dose of phenelzine increased the p-tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatal p-tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine aminotransferase activity of rat liver homogenates; the IC50 was 50 microM. Phenelzine also inhibited the aromatic L-amino acid decarboxylase activity of rat brain homogenate with an IC50 of 25 microM. Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L-amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatal p-tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect on p-tyramine levels followed later by very large increases in p-tyramine levels.     

Evidence for Cyclic AMP-Mediated Increase of Aromatic L-Amino Acid Decarboxylase Activity in the Striatum and Midbrain

Abstract: Aromatic L-amino acid decarboxylase (AAAD) activity of mouse striatum and midbrain increased after an intracerebro-ventricular injection of either forskolin or 8-bromo-cyclic AMP. The increase was transient, peaking between 15 and 30 min and returning to baseline by ˜90 min. The increase of AAAD activity after forskolin was not affected by pretreatment with cycloheximide. Kinetic studies indicated an apparent increase of V _max with little change of the K _m for L-DOPA or pyridoxal 5'-phosphate. We conclude that AAAD activity of striatum and midbrain can be modulated by a cyclic AMP-dependent process.     

Specific Irreversible Monoamine Oxidase B Inhibitors Stimulate Gene Expression of Aromatic L-Amino Acid Decarboxylase in PC12 Cells

The effect of some selective monoamine oxidase (MAO) inhibitors on aromatic L-amino acid decarboxylase (AADC) gene expression in PC12 cells has been examined. Irreversible MAO B inhibitors [(-)-deprenyl, pargyline, and MDL 72,974A] stimulated AADC gene expression, whereas a selective irreversible MAO A inhibitor (clorgyline) and a reversible MAO B inhibitor (Ro 19-6327) had no effect. Because there is no apparent MAO B activity in PC12 cells, it is postulated that there is a novel site of action for these MAO B inhibitors and that the pharmacological profile of this site matches that of neuroprotective MAO B inhibitors. Finally, it is suggested that the stimulation of AADC gene expression may be relevant to the antiparkinsonian effects of MAO B inhibitors.     

Aromatic L-Amino Acid Decarboxylase Activity of Mouse Striatum Is Modulated via Dopamine Receptors

Abstract: Aromatic L-amino acid decarboxylase (AAAD) activity is enhanced in the striatum of control and MPTP-treated mice after administration of a single dose of the dopamine receptor antagonists haloperidol, sulpiride, and SCH 23390. MPTP-treated mice appear more sensitive to the antagonists, i.e., respond earlier and to lower doses of antagonists than control mice. The rise of AAAD activity induced by the antagonists is prevented by pretreatment with cycloheximide. The apparent K _m values for L-3,4-dihydroxyphenylalanine (L-DOPA) and pyridoxal 5-phosphate appear unchanged after treatment with the antagonists. Increased AAAD activity was observed also after subchronic administration of dopamine receptor antagonists or treatment with reserpine. A single dose of a selective dopamine receptor agonists had no effect on AAAD activity. In contrast, administration of L-DOPA, quinpirole, or SKF 23390 for 7 days lowers AAAD activity in the striatum. We conclude that AAAD is modulated in striatum via dopaminergic receptors.     

低温菌启动子分析及异源蛋白高效表达

在已构建的能在低温菌和E. coli中正常复制的启动子探针质粒的基础上, 筛选到了强启动子, 通过RT-PCR评估了启动子活性, 并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白表达质粒, 使一个热不稳定a-淀粉酶在低温下(7℃)得到了高效表达, 表达量达胞外总蛋白的35%。显示出该表达系统具有高效表达热不稳定蛋白质的潜在应用价值。     

低温菌启动子分析及异源蛋白高效表达

在已构建的能在低温菌和E. coli中正常复制的启动子探针质粒的基础上, 筛选到了强启动子, 通过RT-PCR评估了启动子活性, 并通过引物延伸实验确定了转录起始位点和启动子核心序列。利用其中最强的启动子构建了低温蛋白表达质粒, 使一个热不稳定a-淀粉酶在低温下(7℃)得到了高效表达, 表达量达胞外总蛋白的35%。显示出该表达系统具有高效表达热不稳定蛋白质的潜在应用价值。     

Inhibition of Monoamine Oxidase Selectively in Brain Monoamine Nerves Using the Bioprecursor (E-β-Fluoromethylene-m-Tyrosine (MDL 72394), a Substrate for Aromatic L-Amino Acid Decarboxylase

(E)-beta-Fluoromethylene-m-tyrosine (FMMT) is a dual-enzyme-activated inhibitor of monoamine oxidase (MAO). The compound is not an inhibitor per se but is decarboxylated by aromatic L-amino acid decarboxylase (AADC) to yield a potent enzyme-activated irreversible inhibitor of MAO, (E)-beta-fluoromethylene-m-tyramine, which shows some selectivity for inhibition of MAO type A. Decarboxylation of FMMT was demonstrated in vitro using hog kidney AADC and in vivo in rats by the ability of alpha-monofluoromethyldopa (MFMD), a potent inhibitor of AADC, to prevent MAO inhibition produced by FMMT. In isolated synaptosomes, FMMT was decarboxylated by AADC, and, furthermore, the compound was actively transported into these isolated nerve endings. An active transport into the CNS has also been demonstrated in vivo by performing competition experiments with leucine. To demonstrate that FMMT is preferentially decarboxylated within monoamine nerves of the CNS, the nigrostriatal 3,4-dihydroxyphenylethylamine (dopamine) pathway of rats was unilaterally lesioned with 6-hydroxydopamine or infused with MFMD. Under these conditions, MAO inhibition produced by orally administered FMMT in the striatum ipsilateral to the lesion or infusion was markedly attenuated. Combination of FMMT with an inhibitor of extracerebral AADC, such as carbidopa, protected peripheral organs against the MAO inhibitory effects and concomitantly enhanced MAO inhibition in the CNS. Such combinations had a greatly reduced propensity to augment the cardiovascular effects of intraduodenally administered tyramine, when compared with FMMT given alone or with clorgyline, a selective inhibitor of MAO type A. The results obtained with FMMT suggest the possibility of achieving selective inhibition of MAO within monoamine nerves of the CNS and, further, suggest that combination of FMMT with an inhibitor of extracerebral AADC will reduce the propensity of this inhibitor to produce adverse interactions with tyramine.     

Nuclear Magnetic Resonance Spectroscopy Study on Energy Metabolism, Intracellular pH, and Free Mg2+ Concentration in the Brain of Transgenic Mice Overexpressing Human Ornithine Decarboxylase Gene

We have generated a transgenic mouse line strikingly overexpressing the human ornithine decarboxylase (ODC) gene in their brain. Brain ODC activity was increased in the transgenic animals by a factor of 70 in comparison with their nontransgenic littermates. The content of brain putrescine, the product of ODC, was greater than 60 mumol/g of tissue in the transgenic mice, whereas in the normal animals it was below the level that could be detected by an HPLC method. The concentrations of the higher polyamines (spermidine and spermine) were not significantly different from control values. 31P nuclear magnetic resonance (31P NMR) spectroscopy analyses revealed a significantly reduced (40%) free Mg2+ concentration as calculated from the chemical shift differences of the nucleoside triphosphate alpha and beta peaks in the brains of the transgenic animals. The lower free Mg2+ concentration in the brains of ODC transgenic mice was not a consequence of altered intracellular pH or changes in cellular high-energy metabolites. 1H NMR showed no differences in brain choline/N-acetylaspartate and total creatine/N-acetylaspartate ratios between the two animal groups. These ODC transgenic animals may serve as models in vivo for studies on cerebral postischemic events and on epilepsy, as polyamines are supposed to be involved in these processes.     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.