Role of Aromatic l-Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l -amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l -amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l -amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l -DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l -amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l -amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic l -amino acid decarboxylase produced less l -DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l -amino acid decarboxylase near striatal grafts producing l -DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

CHOLINE ACETYLTRANSFERASE, ACETYLCHOLINESTERASE AND AROMATIC l-AMINO ACID DECARBOXYLASE IN SINGLE IDENTIFIED NERVE CELL BODIES FROM SNAIL HELIX ASPERSA

—The distribution of choline acetyltransferase, aromatic l -amino acid decarboxylase and acetylcholinesterase in the nervous system of Helix aspersa has been studied using homogenates of whole ganglia, microdissection from freeze-dried sections and dissection of single neurons from fresh tissue. Choline acetyltransferase was found in both the cell body and neuropil layers of all the Helix ganglia. The enzyme was not specifically localized to any ganglion or region of ganglion. Between 10 and 30 per cent of the isolated single cell bodies contained the enzyme. The enzymic activity corresponded to 50–200 mmol ACh/1 cell bodies/h. Choline acetyltransferase is probably a specific marker for cholinergic cells in this species. Aromatic l -amino acid decarboxylase was more selectivity localized and its distribution corresponded well with that of monoamine containing cells as visualized by the fluorescence histochemical technique. A large proportion of cell bodies were localized in the boundary between the visceral and right parietal ganglia and in the pedal ganglion. The other ganglia contained few such cells. The activity of aromatic l -amino acid decarboxylase corresponded 10–50 mmol dopamine/1 cell bodies/h. A method was developed to measure the enzyme activity towards 5-hydroxytryptophan and DOPA in single cells simultaneously. The ratio between the activity towards both substrates did not vary significantly for the different cells. The enzyme is probably a specific marker for monoamine cells, but cannot be used to differentiate between the different monoamine cells. Acetylcholinesterase was uniformly distributed in the ganglia and was probably present in all nerve cells.     

DECARBOXYLATION OF EXOGENOUS l-5-HYDROXYTRYPTOPHAN AFTER DESTRUCTION OF THE CEREBRAL RAPHE SYSTEM

Abstract— l -5-Hydroxytryptophan ( l -5-HTP) was administered intravenously to rats (12 mg/kg) after inhibition of the peripheral aromatic l -amino acid decarboxylase with l -α-hydrazino-α-methyl dopa (MK 486). The accumulation of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid in the cerebral cortex was measured 1, 2 and 4 h after injection of 5-HTP with automated assay techniques. Besides controls two groups of rats were studied: rats after inhibition of tryptophan-5-hydroxylase with p -chlorophenylalanine (pcpa) and subjects with a chronic lesion in the area of the raphe nucleus. The net accumulation of both measured 5-hydroxyindoles was diminished in rat cerebral cortex after degeneration of 5-HT containing nerve endings, compared with control animals and pcpa-treated rats. These results indicate that the formation of 5-HT in the cerebral cortex from exogenous l -5-HTP, after inhibition of the peripheral aromatic amino acid decarboxylase, occurs predominantly in 5-HT containing nerve endings possibly by a specific 5-HTP-decarboxylating enzyme.     

OCCURRENCE AND DISTRIBUTION OF AROMATIC l-AMINO ACID (l-DOPA) DECARBOXYLASE IN THE HUMAN BRAIN

—The enzymatic decarboxylation of l -DOPA was measured in isotonic dextrose homogenates of different regions of the human brain by estimating ¹⁴CO₂ evolved from tracer amounts of d l -DOPA[carboxy1-¹⁴C]. Enzyme activity was linear with respect to tissue concentration and time of incubation. The reaction exhibited a pH maximum at 7·0, was completely dependent upon the presence of high concentrations of pyridoxal phosphate, proceeded at the same rate in an atmosphere of air and nitrogen, and produced dopamine in addition to CO₂ as a reaction product. The enzyme preparation behaved like an aromatic l -amino acid decarboxylase: it also decarboxylated o-tyrosine and when incubated with 5-hydroxytryptophan, serotonin was isolated as the reaction product; but it was devoid of activity towards d -DOPA[carboxy1-¹⁴C]. Within the human brain, l -DOPA decarboxylase was most active in the putamen and caudate nucleus; the pineal gland, hypothalamus, and the reticular formation and dorsal raphe areas of the mesencephalon exhibited considerable activity. Areas of cerebral cortex exhibited very low enzymatic activity and in regions composed predominantly of white matter, l -DOPA decarboxylase activity was not significantly above blank values. The activity of l -DOPA decarboxylase in the human putamen and caudate nucleus tended to decrease with the age of the patients; in comparatively young subjects (46 yr old) the enzyme activity compared favourably with that found, by means of the same assay technique, in the caudate nucleus of the cat.     

An immunohistochemical study of the catecholamine synthesizing enzymes and neuropeptides in the female guinea-pig uterus and vagina

Summary The uterus and vagina of the guinea pig have been examined, region by region, for acetylcholinesterase, tyrosine hydroxylase, dopamine -hydroxylase and aromatic amino acid decarboxylase activity, as well as for the neuropeptides, neuropeptide Y, vasoactive intestinal peptide, substance P, enkephalin and somatostatin. No acetylcholinesterase activity was localized in the uterus, though it was present in associated paracervical ganglion tissues. Of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine -hydroxylase activity was found virtually throughout the reproductive tract, whereas aromatic amino acid decarboxylase activity was restricted in its distribution. Neuropeptide distribution was quite varied. Neuropeptide Y was found throughout the endometrium/submucosa but only in the muscularis of the vagina and not in the myometrium. Substance P was localized in the vagina and uterine horn, though not the body of the uterus. Vasoactive intestinal peptide was present in all regions of the endometrium/submucosa, but not in the myometrium of the uterine horn. Enkephalin and somatostatin were not localized in any part of the reproductive tract examined, apart from paracervical ganglion tissues. The types and significance of the nerves supplying the reproductive tract are discussed.     

多巴脱羧酶及其与神经类疾病的关联性研究进展

多巴脱羧酶(dopa decarboxylase,DDC)又称作芳香族L-氨基酸脱羧酶,是儿茶酚胺生物合成途径中重要的酶之一,具有多种生物学功能。多巴脱羧酶可分别催化L-3,4-二羟基苯丙氨酸(L-多巴)和L-5-羟色氨酸合成两种神经递质多巴胺和五羟色胺。多巴胺和五羟色胺在脊椎动物和无脊椎动物的生殖、发育、行为和免疫应答过程中均具有重要作用。此外,它还与多种神经类疾病和社会行为有关。多巴脱羧酶一般以二聚体的形式存在于哺乳类和昆虫的多种神经和非神经组织中。本文从多巴脱羧酶的结构、催化机制、与神经类疾病及其攻击性社会行为的关联性研究进展等方面进行了综述。     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The V_max value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg⁻¹ protein h⁻¹. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.     

The presence of aromatic L-amino acid decarboxylase in certain intestinal nerve cells

Summary The presence of aromatic 1-amino acid decarboxylase (AADC) in nerve cell bodies of the intrinsic plexuses of the guinea-pig small intestine was demonstrated by incubating segments of intestine with 1-dopa in the presence of an inhibitor of monoamine oxidase, pargyline. After such incubation, some nerve cell bodies gave a fluorescence histochemical reaction indicative of the presence of a decarboxylated product of 1-dopa, probably dopamine. No fluorescence reaction occurred in the unincubated control or if the inhibitor of AADC, RO 4-4602, was included in the incubation mixture. The AADC-containing cell bodies apparently do not take up and store dopamine, because no fluorescence could be detected after incubation with dopamine and a monoamine oxidase inhibitor. The AADC-containing cells were found in about half of the ganglia of the submucous plexus of the guinea-pig small intestine, but were considerably less frequent in the myenteric plexus. They were also found in the other areas examined in this study, that is, in both enteric plexuses of the guinea-pig distal colon and of the small intestines of rabbits and rats.     

Restoration of Norepinephrine and Reversal of Phenotypes in Mice Lacking Dopamine β-Hydroxylase

Abstract: Mice with a targeted disruption of the dopamine β-hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, l -threo-3,4-dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25–30% of normal. NE levels typically peak ∼5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S (−)-Carbidopa, which does not cross the blood-brain barrier, inhibits aromatic l -amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind-limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine β-hydroxylase-deficient mice are all reversed by DOPS injection.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

3,4-Dihydroxyphenylalanine (dopa) decarboxylase activity in the arthropod nervous system

1. When homogenates of brains from mature adult locusts (Locusta migratoria) were incubated with l-3-(3,4-dihydroxyphenyl)[3-(14)C]alanine the major radioactive metabolite was dopamine, suggesting the presence of a dopa (3,4-dihydroxyphenylalanine) decarboxylase. 2. Decarboxylation of l-dopa by this tissue, measured under optimum conditions by a radiochemical method, was 21mumol of CO(2)/h per g wet wt. Apparent decarboxylation of l-tyrosine proceeded at 0.34mumol of CO(2)/h per g wet wt. There was no detectable decarboxylation of l-tryptophan, l-histidine or l-phenylalanine. 3. Dopa decarboxylase activity was found in all major regions of the ventral nerve cord of the mature locust (range: 4-7mumol of CO(2)/h per g wet wt.) but was low or absent in thoracic peripheral nerve. 4. Marked decarboxylation of l-dopa was found in homogenates of brains of four other species of insects, and in brain and ventral nerve cord, but not in the claw nerve, of the crayfish. 5. The activity of the locust brain enzyme may be slightly lower at the time of imaginal ecdysis than during the mature period. By contrast, the dopa decarboxylase that produces dopamine as an intermediate in cuticle biosynthesis is known to be high in activity at the time of ecdysis and low in activity during the intermoult stages.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

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.     

A method for the direct detection of aromatic amino acid decarboxylase in electrophoretic media

Aromatic amino acid decarboxylase is an enzyme of broad specificity which decarboxylates a range of aromatic amino acids including 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, which yield dopamine and serotonin, respectively. The name aromatic amino acid decarboxylase was applied to the enzyme by workers (1–3) who found that, on purification, a single enzyme appeared to be responsible for activities previously ascribed to two separate enzymes. The separate enzymes had been named 5-hydroxy-l-tryptophan carboxylyase (EC 4.1.1.28) and 3,4-dihydroxy-l-phenylalanine carboxylyase (EC 4.1.1.26). More recent papers (4–7) still leave unresolved the question of whether one enzyme or two is responsible for the two activities.     

Effect of neurotropic agents on total pyridoxal phosphate and on the activity of the decarboxylose of aromatic amino acids as well as of other pyridoxal phosphate-dependent enzymes in rat brains

—(1) Rats received single intraperitoneal injections of various neuroactive chemicals in order to compare the changes of gross behaviour and the level of pyridoxal phosphate as well as the activity of the decarboxylase of aromatic amino acids, of glutamate decarboxylase and of tyrosine transaminase in the brain. (2) The majority of excitatory agents tested (i.e. convulsives like amino-oxyacetate, thiosemicarbazide, pentylenetetrazol and oxotremorine; stimulants such as amphetamine, theophylline and methylphenidate; the amphetamine-like monoamine oxidase inhibitor tranylcypromine as well as the classical monoamine oxidase inhibitor iproniazid when combined with the monoamine releaser Ro 4-1284) caused a decrease in aromatic decarboxylase activity which was coexistent with maximal changes of gross behaviour and partly preceded the latter. The level of pyridoxal phosphate was only partially parallel. As an exception, depression of aromatic decarboxylase was lacking after cocaine (which reduced pyridoxal phosphate only), atropine, the hallucinogens lysergic acid diethylamide and mescaline as well as the combination of the dopamine precursor l -DOPA and the aromatic decarboxylase inhibitor Ro 4-4602. (3) Depression of obvious central nervous functions was almost regularly accompanied and in part preceded by increase of DCA activity (i.e. with the anaesthetics pentobarbitone, diethyl ether and chloroform, the neuroleptics chlorpromazine, haloperidol, reserpine and the benzoquinolizine Ro 4-1284 as well as the tranquillizers diazepam and chlordiazepoxide). Pyridoxal phosphate was increased during or after maximal behavioural changes by pentobarbitone and chlorpromazine only. As an exception, activation of aromatic decarboxylase was absent after morphine. (4) The activity of glutamate decarboxylase was significantly reduced by thiosemicarbazide only, whereas a distinct increase in enzyme activity was exclusively observed after atropine. (5) Tyrosine transaminase activity was significantly diminished by amino-oxyacetate only and showed a late increase after tranylcypromine. (6) It is concluded that there is an inverse relationship, in the majority of neuroactive chemicals tested, between changes of gross behaviour and cerebral aromatic decarboxylase activity. Thereby, the latter is neither regularly related to corresponding variations of the total cerebral pyridoxal phosphate nor to hitherto described alterations of the monoamine turnover nor to effects on other vitamin B₆-dependent enzymes.     

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.     

Gene Expression of Tyrosine Hydroxylase in the Developing Fetal Brain

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.