Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Mutant PINK1 upregulates tyrosine hydroxylase and dopamine levels,leading to vulnerability of dopaminergic neurons

PINK1 mutations cause autosomal recessive forms of Parkinson disease (PD). Previous studies suggest that the neuroprotective function of wild-type (WT) PINK1 is related to mitochondrial homeostasis. PINK1 can also localize to the cytosol; however, the cytosolic function of PINK1 has not been fully elucidated. In this study we demonstrate that the extramitochondrial PINK1 can regulate tyrosine hydroxylase (TH) expression and dopamine (DA) content in dopaminergic neurons in a PINK1 kinase activity-dependent manner. We demonstrate that overexpression of full-length (FL) WT PINK1 can downregulate TH expression and DA content in dopaminergic neurons. In contrast, overexpression of PD-linked G309D, A339T, and E231G PINK1 mutations upregulates TH and DA levels in dopaminergic neurons and increases their vulnerability to oxidative stress. Furthermore transfection of FL WT PINK1 or PINK1 fragments with the PINK1 kinase domain can inhibit TH expression, whereas kinase-dead (KD) FL PINK1 or KD PINK1 fragments upregulate TH level. Our findings highlight a potential novel function of extramitochondrial PINK1 in dopaminergic neurons. Deregulation of these functions of PINK1 may contribute to PINK1 mutation-induced dopaminergic neuron degeneration. However, deleterious effects caused by PINK1 mutations may be alleviated by iron-chelating agents and antioxidant agents with DA quinone-conjugating capacity.     

Inactivation of tyrosine hydroxylase by reduced pterins

Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.     

Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro

Summary In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently of the action of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.     

Effect of TRH analog (DN-1417) on tyrosine hydroxylase activity in mesocortical, mesolimbic and nigrostriatal dopaminergic neurons of rat brain

Tyrosine hydroxylase (TH) was assayed in eight regions of rat brain following repeated treatment with a TRH analog, DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide). Repeated DN-1417 treatment (20 mg/kg/day, IP) for 7 days increased TH activity in the ventral tegmental area and decreased in the prefrontal cortex polar, medial and lateral fields and olfactory tubercles. No significant change in TH activity was found in the nucleus accumbens, striatum and substantia nigra. Kinetic analysis showed that the increased TH activity in the ventral tegmental area was due to an increase in Vmax, but not a change in the apparent Km of TH for a cofactor, 6-methyl-tetrahydropteridine. When TH was assayed at a suboptimal pH and in the presence of a subsaturating cofactor, the striatal TH was activated significantly after DN-1417. In the prefrontal cortex medial field, nucleus accumbens and olfactory tubercles, TH activity assayed under the suboptimal condition was not modified by DN-1417 treatment. These results suggest an intimate involvement of central dopaminergic systems in the actions of DN-1417.     

Substrate-mediated enhancement of phosphorylated tyrosine hydroxylase in nigrostriatal dopamine neurons: evidence for a role of alpha-synuclein

Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity. In wild-type animals, tyrosine administration potentiated the raclopride-induced increase in phosphorylated TH and enzyme activity. However, tyrosine administration did not enhance phosphorylated TH levels or enzyme catalytic activity in raclopride-stimulated NSDA neurons in alpha-synuclein knockout mice. These findings suggest that alpha-synuclein plays a role in the ability of tyrosine to either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity. The present study supports the hypothesis that alpha-synuclein functions as a molecular chaperone protein that regulates the phosphorylation state of TH in a substrate and activity-dependent manner.     

Distribution of tyrosine hydroxylase in human and animal brain

The activity of tyrosine hydroxylase (EC 1.10.3.1) when assayed under ideal conditions in young human brains, was comparable to that in brains of other species in level of activity and distribution. The highest levels of activity were in the putamen, caudate nucleus and substantia nigra, in keeping with data on other species. The caudate activity in human brain appeared to decrease substantially with increasing age. In both humans and baboons, the enzyme in the neostriatum was particle-bound and inhibited by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. In the substantia nigra it was soluble and stimulated by the 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine cofactor system. The data suggest that tyrosine hydroxylase may be produced in a soluble form in the cell bodies of the substantia nigra but become bound as it moves toward the nerve endings in the putamen and caudate nucleus. The bound form of the enzyme was unstable but the soluble form exhibited considerable stability.     

Pb-induced alterations in tyrosine hydroxylase activity in rat brain

It was concluded that cytochrome oxidase was a strange enzyme for three reasons. (1) The thermodynamic flux-force relationship of this enzyme was inverse in some conditions: flux decreased when force increased. (2) The flux-force relationship was not unique and depended on the way in which the thermodynamic span of cytochrome oxidase was changed. (3) The regulation of cytochrome oxidase was different in the same conditions when different external parameters (energy demand, oxygen concentration) were changed.It was also shown that the flux control coefficient of cytochrome oxidase, small at saturating oxygen concentration, increases when oxygen pressure diminishes, approaching unity at very low oxygen concentrations. (Mol Cell Biochem 174: 137–141, 1997)     

Central dopaminergic and serotoninergic systems in the regulation of adrenal tyrosine hydroxylase.

Administration of the dopamine receptor agonists apomorphine, piribedil and bromocryptine caused an increase in adrenal tyrosine hydroxylase (TH; tyrosine-3-monooxygenase, EC 1.14.16.2) which could be partially abolished by prior injection of the dopamine blocker haloperidol. Injection of L-dihydroxyphenylalanine, along with the decarboxylase inhibitor carbidopa, also led to a highly significant increase in adrenal TH activity. Intraventricular injection of 5,7-dihydroxytryptamine (DHT), which destroys serotonin neurons, doubled adrenal TH activity in both normal and hypophysectomized rats. Splanchnicotomy abolished this effect of DHT. The increase in enzyme activity mediated by DHT could be partially prevented by peripheral administration of L-5-hydroxytryptophan together with carbidopa. Blockade of serotoninergic functions with the antagonist methiothepin also increased adrenal TH activity. The interrelationship between the dopamine and the presumed serotonin system was investigated. Intraventricular injection of 6-hydroxydopamine partially prevented the DHT-induced increase in adrenal TH activity. Administration of haloperidol to DHT-treated rats had the same effect. This suggests that an intact dopaminergic system is required. When DHT and either apomorphine or piribedil were adminstered simultancously the dopamine agonist-induced increase was potentiated. An intact serotoninergic system is therefore not required for dopamine function. Thus, the increase in adrenal TH activity is associated with either stimulation of central dopamine receptors or destruction of serotonin neurons. It is suggested that dopaminergic and serotoninergic systems are involved in the regulation of adrenal TH and that these systems have net excitatory and inhibitory roles, respectively. Furthermore, the present evidence favors the view that the interaction between the two systems is sequential, with the serotonin system preceding the dopamine one.     

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Purification and immunochemical properties of tyrosine hydroxylase in human brain

Tyrosine hydroxylase (TH) was isolated from human brain (caudate nucleus + putamen). The major form of the active enzyme in the cytoplasmic fraction was purified to apparent homogeneity. The molecular weight of the purified enzyme was estimated to be 280 kdalton by gel filtration. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) of the purified enzyme gave a single subunit with mol. wt 60 kdalton, which is similar to the subunit of human adrenal TH. Using a sandwich enzyme immunoassay (EIA), the presence of inactive form(s) of TH in human brain was demonstrated, and the total content of this immunoinactive form(s) was approx. 8 times higher than that of the active form. By the Western blot technique after two-dimensional (2-D) electrophoresis, TH in the crude fraction of the human brain was found to consist of multiple forms with different pI-values and with the same molecular weight. The pl of the major spots ranged from 5.3 to 5.8, and that of the minor spot was 6.0. Because the pl of the purified enzyme preparation was 6.0, this protein with pI at 6.0 may be the active form of TH.