Tissue-specific developmental requirements of Drosophila tyrosine hydroxylase isoforms

Drosophila tyrosine hydroxylase (DTH) is a key enzyme in dopamine (DA) biosynthesis, which is expressed in neural and hypodermal DA-synthesizing cells. We previously reported that two DTH isoforms are produced in flies through tissue-specific alternative splicing that show distinct regulatory properties. We have now selectively expressed each DTH isoform in vivo in a pale (ple, i.e., DTH-deficient) mutant background. We show that the embryonic lethality of ple can be rescued by expression of the hypodermal, but not the neural, DTH isoform in all DA cells, indicating that the hypoderm- isoform is absolutely required for cuticle biosynthesis and survival in Drosophila. In addition, we report new observations on the consequences of DTH overexpression in the CNS and hypoderm. Our results provide evidence that tissue-specific alternative splicing of the DTH gene is a vital process in Drosophila development.     

Maternal and embryonic sources of tyrosine hydroxylase during Drosophila embryogenesis

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is rate limiting in catecholamine biosynthesis, is biochemically expressed in late stage wild-type Drosophila oocytes as well as in early embryogenesis. Null mutant alleles of TH (pale) are embryonic lethals with death occurring in the late embryonic or early larval periods of development. Staging of embryos demonstrated that inhibition of the enzymatic activity of TH by alpha-methyl-p-tyrosine (alphaMT) retards the progression of embryos primarily during the organogenesis stages of embryonic development, with lesser effects on earlier and later stages. On the other hand, time of gene action studies with a conditional temperature sensitive pale mutant (ple(ts1)) at its restrictive temperature (29 degrees C) indicate an onset of tyrosine hydroxylase gene action beginning in the oocyte stage of development. Thus, maternal as well as embryonic effects on the secretion and/or functionality of this enzyme may play roles in the early developmental program of the organism.     

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain

Tyrosine hydroxylase (TH) is a rate‐limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4‐deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4‐deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post‐natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post‐natal period is critical for the developmental regulation of TH protein level.     

The Drosophila learning and memory gene linotte encodes a putative receptor tyrosine kinase homologous to the human RYK gene product

The linotte mutant was isolated on the basis of its learning and memory deficit. Interestingly, linotte individuals carrying a null mutation are viable, indicating that the linotte gene is not required for vital functions. We show here that the linotte gene encodes a putative receptor tyrosine kinase, homologous to the human protein RYK. These products are unique among receptor tyrosine kinases, since they possess a short extra cellular domain, and a modified intracellular catalytic domain. In particular, the subdomains directly involved in ATP binding and phosphotransfer reaction display remarkable variations. These results suggest that linotte is part of a novel signal transduction cascade involved in learning and memory.     

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.     

Nicotine induces tyrosine hydroxylase plasticity in the neurodegenerating striatum

It has been shown that nicotine prevents the loss of dopamine (DA) in the corpus striatum (CS) after 6-hydroxydopamine injection in the substantia nigra. To study the role of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) in this experimental paradigm, we have examined its activity by assessing the accumulation of l-3,4-dihydroxyphenylalanine after inhibiting the subsequent enzyme in the DA synthetic pathway, aromatic l-amino acid decarboxylase, with 3-hydroxybenzylhydrazine. In addition the amount of TH protein was assessed by western blotting and its distribution in the CS was examined using immunohistochemical methods. 6-hydroxydopamine injection produced a significant decrease in DA levels and l-3,4-dihydroxyphenylalanine accumulation, as well as decreases in TH protein and TH immunoreactive fibres in the CS. After nicotine treatment, the decrease in TH protein in the CS was significantly reduced, with a concomitant preservation of TH activity, but nicotine did not alter the number of TH immunoreactive fibres. The activity and amount of TH did not change in the contralateral (intact) CS. Thus, nicotine induces long lasting TH plasticity in the degenerating CS. A synergistic action of nicotine-activated and lesion-originated signals appears necessary for the expression of this neuronal molecular plasticity.     

Differential expression of tyrosine hydroxylase mRNA in the developing rat mesencephalon

1. With respect to the mesostriatal projection, the mesencephalon is composed of two dopaminergic (DA) cell populations, called dorsal tier and ventral tier. Strong evidence suggests differences in both the spatial and the temporal sequence of the innervation of the striatum between the two groups, with the ventral tier neurons innervating striatal patches prenatally and dorsal tier cells innervating striatal matrix postnatally. 2. Using in situ hybridization, we have examined the expression of the gene coding for tyrosine hydroxylase (TH) in mesencephalic DA neurons with respect to their postnatal development. Two ontogenic patterns of expression were observed: (a) dorsal tier neurons of the medial mesencephalon exhibited a sharp increase in expression beginning after birth, peaking on day 14, then decreasing and, finally, stabilizing; and (b) ventral tier neurons and dorsal tier cells from the lateral and the medial-dorsal mesencephalon showed only a slight increase in TH mRNA, reaching a plateau at P10. 3. The time course of the observed increase in TH gene expression in the first group, generally parallels the innervation of their target cells in the striatal matrix, suggesting that TH gene expression in these cells may be influenced by their postsynaptic cells or by the innervation process.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.     

Targeted mutagenesis and genetic analysis of a Drosophila receptor-linked protein tyrosine phosphatase gene

Several Drosophila receptor-linked protein tyrosine phosphatases (R-PTPs) are selectively expressed on axons of the developing embryonic central nervous system. The extracellular domains of these axonal R-PTPs are homologous to neural adhesion molecules. Thus, R-PTPs may directly couple cell recognition to signal transduction via control of tyrosine phosphorylation. To examine the function of these molecules during nervous system development, we wished to generate mutations in R-PTP genes. It was unclear whether a mutation in a single R-PTP gene would confer lethality, however, because the similarities in sequence and expression pattern between the axonal R-PTPs suggest that they may have partially redundant functions. To circumvent this problem, we developed a directed mutagenesis strategy based on local transposition of P elements, and used this approach to isolate a null mutation in the DPTP99A gene. This strategy, which we describe in detail here, should be applicable to any Drosophila gene within a lettered division of an appropriately marked P element. Flies lacking DPTP99A expression are viable and fertile, and we have been unable to detect any alterations in the embryonic nervous system of DPTP99A embryos using a variety of antibody markers.     

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Development of neuropeptide Y and tyrosine hydroxylase immunoreactive innervation in postnatal rat heart

Neuropeptide Y (NPY), immunoreactive (IR), and tyrosine hydroxylase (TH)-IR nerve fibers were scarce at birth in rat heart, but increased rapidly during the first 2 postnatal weeks, reaching approximately adult levels by the third week. The sequence of development was: interatrial septum and atrial wall, free ventricular wall starting from the epicardium, and finally the atrial appendages and interventricular septum. In ventricles and atrial appendages both fiber types developed similarly. In interatrial septum and atrial walls more NPY-IR than TH-IR fibers were evident, and NPY-IR, but not TH-IR, neurons were detected in intrinsic ganglia. Doublelabel immunohistochemistry provided further evidence that NPY is located in ventricular and atrial noradrenergic nerves, but is also located in nonnoradrenergic nerves in atria.     

Dopaminergic mediation of Y-aminobutyric acid in the control of prolactin release: Plasma prolactin and brain tyrosine hydroxylase levels in ovariectomized conscious rats

The present experiments were carried out to further elucidate the mechanism by which dopamine mediates the actions of Y-aminobutyric acid on prolactin release from anterior pituitary following its intraventricular injection in overiectomized conscious rats, Y-Aminobutyric acid significantly suppressed the prolactin levels at 0.1 Μmol concentration while at 4 Μmol dose, the level was elevated. The activity of tyrosine hydroxylase was increased significantly in the anterior pituitary at the lower dose while the higher concentration of Y-aminobutyric acid did not bring about any change in the activity both in the hypothalamus and the anterior pituitary. The results presented suggest that intracellular dopamine in the anterior pituitary may directly inhibit prolactin release; the plasma prolactin level is elevated by Y-aminobutyric acid, by way of either inhibiting dopaminergic tone or possible stimulation of a physiological prolactin releasin g hormone.     

Effects of temperature stress on NO-synthase and tyrosine hydroxylase activities in the central nervous system of bivalve molluscs

Effects of temperature stress on activities of NO-synthase (NOS) and tyrosine hydroxylase (TH) in the CNS of two species of bivalve molluscs, Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and immunocytochemistry. General and specific peculiarities in distribution and relative proportion of TH- and NO-containing neurons in the CNS nerve ganglia were revealed in norm and under stress at 30°C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been found to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In intact Chlamys farreri nipponensis, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.     

Ciliary neurotrophic factor stimulates tyrosine hydroxylase activity

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.     

Diencephalo-telencephalic changes of tyrosine hydroxylase in rats and grass frogs after sleep deprivation

Based on sleep deprivation-produced changes of electrographic parameters of the wakefulness-sleep cycle (WSC) in rats and frogs (Rana temporaria), dynamics of activity of tyrosine hydroxylase, the key enzyme of dopamine synthesis, was studied immunohistochemically in substantia nigra and nigrostriatal pathway in rats and in striatum, paraventricular organ, and extrahypothalamic pathways in frogs. Changes in dynamics of tyrosine hydroxylase in rats and in frogs are revealed after the 6-h sleep deprivation and after 2 h of postdeprivation sleep. This allows determining the degree of participation of corticostriatal neuroregulatory and hypothalamo-pituitary neurosecretory systems and their role in regulation of WSC. Possible evolutionary peculiarities of morphofunctional differences in homoiothermal and poikilothermal animals are discussed.     

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.