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mTORC1 inhibition in the nucleus accumbens 'protects' against the expression of drug seeking and 'relapse' and is associated with reductions in GluA1 AMPAR and CAMKII? levels.
Neuropsychopharmacology
PUBLISHED: 01-14-2014
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The mechanistic target of rapamycin complex 1 (mTORC1) is necessary for synaptic plasticity, as it is critically involved in the translation of synaptic transmission-related proteins, such as Ca(2+)/Calmodulin-dependent kinase II alpha (CAMKII?) and AMPA receptor subunits (GluAs). Although recent studies have implicated mTORC1 signaling in drug-motivated behavior, the ineffectiveness of rapamycin, an mTORC1 inhibitor, in suppressing cocaine self-administration has raised questions regarding the specific role of mTORC1 in drug-related behaviors. Here, we examined mTORC1's role in three drug-related behaviors: cocaine taking, withdrawal, and reinstatement of cocaine seeking, by measuring indices of mTORC1 activity and assessing the effect of intra-cerebroventricular rapamycin on these behaviors in rats. We found that withdrawal from cocaine self-administration increased indices of mTORC1 activity in the nucleus accumbens (NAC). Intra-cerebroventricular rapamycin attenuated progressive ratio (PR) break points and reduced phospho-p70 ribosomal S6 kinase, GluA1 AMPAR, and CAMKII? levels in the NAC shell (NACsh) and core (NACc). In a subsequent study, we treated rats with intra-NACsh infusions of rapamycin (2.5??g/side/day for 5 days) during cocaine self-administration and then tracked the expression of addiction-relevant behaviors through to withdrawal and extinction. Rapamycin reduced drug seeking in signaled non-drug-available periods, PR responding, and cue-induced reinstatement, with these effects linked to reduced mTORC1 activity, total CAMKII?, and GluA1 AMPAR levels in the NACsh. Together, these data highlight a role for mTORC1 in the neural processes that control the expression and maintenance of drug reward, including protracted relapse vulnerability. These effects appear to involve a role for mTORC1 in the regulation of GluA1 AMPARs and CAMKII? in the NACsh.
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Dephosphorylation of CaMKII at T253 controls the metaphase-anaphase transition.
Cell. Signal.
PUBLISHED: 01-07-2014
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Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKII? in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKII? in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKII? significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase-anaphase transition.
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Catalytic Domain Surface Residues Mediating Catecholamine Inhibition in Tyrosine Hydroxylase.
J. Biochem.
PUBLISHED: 12-11-2013
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Tyrosine Hydroxylase (TH) performs the rate-limiting step in catecholamine synthesis and is a tetramer composed of regulatory, catalytic and tetramerisation domains. Catecholamines inhibit TH by binding two sites in the active site; one with high affinity and one with low affinity. Only high affinity catecholamine binding requires the regulatory domain, believed to interact with the catalytic domain in the presence of catecholamine. Without a crystal structure of the regulatory domain, the specific areas involved in this process are largely undefined. It is not clear whether the regulatory domain-catalytic domain interaction is asymmetrical across the tetramer to produce the high and low affinity sites. To investigate this, pure dimeric TH was generated through double substitution of residues at the tetramerisation interface and dimerisation salt bridge (K170E/L480A). This was shown to be the core regulatory unit of TH for catecholamine inhibition, possessing both high and low affinity catecholamine binding sites, indicating that there is symmetry between dimers of the tetramer. We also examined possible regulatory domain-interacting regions on the catalytic domain that mediate high affinity catecholamine binding. Using site-directed mutagenesis, A297, E362/E365 and S368 were shown to mediate high affinity dopamine inhibition through Vmax reduction and increasing the KM for the cofactor.
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Tyrosine hydroxylase: regulation by feedback inhibition and phosphorylation.
Adv. Pharmacol.
PUBLISHED: 09-24-2013
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Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short-term stimuli, TH activity is regulated by feedback inhibition by the catecholamines and relief of that inhibition by phosphorylation. This chapter examines the current understanding of these regulatory mechanisms and the roles that they play in different catecholaminergic cells. This chapter also examines hierarchical phosphorylation in TH and how it provides a mechanism for the differential regulation of the major human TH isoforms.
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Murine dopaminergic Müller cells restore motor function in a model of Parkinsons disease.
J. Neurochem.
PUBLISHED: 08-27-2013
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Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinsons disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting. Müller cells are the main glial cells in the retina. When these cells are cultured in the absence of neurons, they spontaneously express proteins of the dopaminergic phenotype, including the enzymes tyrosine hydroxylase (TH), L-DOPA-decarboxylase (DDC) and the dopamine transport system (DAT). In this study, we show this phenomenon is observed with Müller cells obtained from different species, including primates, and address the therapeutic potential of these cells, using a mouse model of Parkinsons disease (PD). Dopaminergic Müller cells synthesize dopamine and release most of this neurotransmitter to the extracellular space, constituting a natural dopaminergic pump. When transplanted to the striatum of PD mice, Müller cells decreased their apomorphine-induced rotational behavior and restored their overall motor functions, measured by rotarod and forelimb use asymmetry tests. Local restoration of dopaminergic signaling was also observed in grafted PD mice, by measuring striatum dopamine and its metabolite (DOPAC) levels (SB: 20µm).
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Neurobiological consequences of acute footshock stress; effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla.
J. Neurochem.
PUBLISHED: 07-19-2013
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Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40-min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area (VTA) and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone (ACTH), corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, VTA and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31phosphorylation than the mPFC, presumably due to their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma ACTH at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal-regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min.TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, presynaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the HPA axis. This article is protected by copyright. All rights reserved.
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Mechanism of action of salsolinol on tyrosine hydroxylase.
Neurochem. Int.
PUBLISHED: 07-05-2013
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Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in dopamine synthesis. Dopamine regulates TH as an end-product inhibitor through its binding to a high and low affinity site, the former being abolished by Ser40 phosphorylation only, and the latter able to bind and dissociate according to intracellular dopamine levels. Here, we have investigated TH inhibition by a dopamine metabolite found in dopaminergic brain regions, salsolinol (SAL). SAL is known to decrease dopamine in the nigrostriatal pathway and mediobasal hypothalamus, and to also decrease plasma catecholamines in rat stress models, however a target and mechanism for the effects of SAL have not been found. We found that SAL inhibits TH activity in the nanomolar range in vitro, by binding to both the high and low affinity dopamine binding sites. SAL produces the same level of inhibition as dopamine when TH is non-phosphorylated. However, it produces 3.7-fold greater inhibition of Ser40-phosphorylated TH compared to dopamine by competing more strongly with tetrahydrobiopterin, the cofactor of this enzymatic reaction. SALs potent inhibition of phosphorylated TH would prevent TH from being fully activated to synthesise dopamine.
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Functional programming of the autonomic nervous system by early life immune exposure: implications for anxiety.
PLoS ONE
PUBLISHED: 01-23-2013
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Neonatal exposure of rodents to an immune challenge alters a variety of behavioural and physiological parameters in adulthood. In particular, neonatal lipopolysaccharide (LPS; 0.05 mg/kg, i.p.) exposure produces robust increases in anxiety-like behaviour, accompanied by persistent changes in hypothalamic-pituitary-adrenal (HPA) axis functioning. Altered autonomic nervous system (ANS) activity is an important physiological contributor to the generation of anxiety. Here we examined the long term effects of neonatal LPS exposure on ANS function and the associated changes in neuroendocrine and behavioural indices. ANS function in Wistar rats, neonatally treated with LPS, was assessed via analysis of tyrosine hydroxylase (TH) in the adrenal glands on postnatal days (PNDs) 50 and 85, and via plethysmographic assessment of adult respiratory rate in response to mild stress (acoustic and light stimuli). Expression of genes implicated in regulation of autonomic and endocrine activity in the relevant brain areas was also examined. Neonatal LPS exposure produced an increase in TH phosphorylation and activity at both PNDs 50 and 85. In adulthood, LPS-treated rats responded with increased respiratory rates to the lower intensities of stimuli, indicative of increased autonomic arousal. These changes were associated with increases in anxiety-like behaviours and HPA axis activity, alongside altered expression of the GABA-A receptor ?2 subunit, CRH receptor type 1, CRH binding protein, and glucocorticoid receptor mRNA levels in the prefrontal cortex, hippocampus and hypothalamus. The current findings suggest that in addition to the commonly reported alterations in HPA axis functioning, neonatal LPS challenge is associated with a persistent change in ANS activity, associated with, and potentially contributing to, the anxiety-like phenotype. The findings of this study reflect the importance of changes in the perinatal microbial environment on the ontogeny of physiological processes.
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Mutational analysis of catecholamine binding in tyrosine hydroxylase.
Biochemistry
PUBLISHED: 01-31-2011
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Tyrosine hydroxylase (TH) performs the first and rate-limiting step in the synthesis of catecholamines, which feed back to regulate the enzyme by irreversibly binding to a high-affinity site and inhibiting TH activity. Phosphorylation of Ser40 relieves this inhibition by allowing dissociation of catecholamine. We have recently documented the existence of a low-affinity catecholamine binding which is dissociable, is not abolished by phosphorylation, and inhibits TH by competing with the essential cofactor, tetrahydrobiopterin. Here, we have substituted a number of active site residues to determine the structural nature of the low- and high-affinity sites. E332D and Y371F increased the IC(50) of dopamine for the low-affinity site 10-fold and 7 0-fold, respectively, in phosphorylated TH, indicating dramatic reductions in affinity. Only 2-4-fold increases in IC(50) were measured in the nonphosphorylated forms of E332D and Y371F and also in L294A and F300Y. This suggests that while the magnitude of low-affinity site inhibition in wild-type TH remains the same upon TH phosphorylation as previously shown, the active site structure changes to place greater importance on E332 and Y371. Changes to high affinity binding were also measured, including a loss of competition with tetrahydrobiopterin for E332D, A297L, and Y371F and a decreased ability to inhibit catalysis (V(max)) for A297L and Y371F. The common roles of E332 and Y371 indicate that the low- and high-affinity catecholamine binding sites are colocalized in the active site, but due to simultaneous binding, may exist in separate monomers of the TH tetramer.
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Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons.
J. Neurochem.
PUBLISHED: 01-19-2011
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Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinsons disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1??M tetrodotoxin decreased TH expression beginning around 20?h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300?nM apamin increased TH mRNA and protein between 12 and 24?h later in slices of adult midbrain. Two-week infusions of 300?nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ?20?h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.
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The effect of social defeat on tyrosine hydroxylase phosphorylation in the rat brain and adrenal gland.
Neurochem. Res.
PUBLISHED: 08-19-2010
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Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by protein phosphorylation and chronically by protein synthesis. No studies have systematically investigated the phosphorylation of these sites in vivo in response to stressors. We specifically investigated the phosphorylation of TH occurring within the first 24 h in response to the social defeat stress in the rat adrenal, the locus coeruleus, substantia nigra and ventral tegmental area. Five groups were investigated; home cage control (HCC), two groups that underwent social defeat (SD+) which were sacrificed either 10 min or 24 h after the end of the protocol and two groups that were put into the cage without the resident being present (SD-) which were sacrificed at time points identical to the SD+. We found at 10 min there were significant increases in serine 40 and 31 phosphorylation levels in the locus coeruleus in SD+ compared to HCC and increases in serine 40 phosphorylation levels in the substantia nigra in SD+ compared to SD-. We found at 24 h there were significant increases in serine 19 phosphorylation levels in the ventral tegmental area in SD+ compared to HCC and decreases in serine 40 phosphorylation levels in the adrenal in SD+ compared to SD-. These findings suggest that the regulation of TH phosphorylation in different catecholamine-producing cells varies considerably and is dependent on both the nature of the stressor and the time at which the response is analysed.
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Human neuroblastoma cells transfected with tyrosine hydroxylase gain increased resistance to methylmercury-induced cell death.
Toxicol In Vitro
PUBLISHED: 05-11-2010
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In a previous study we demonstrated that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH+TH cells) were substantially more resistant to cell death induced by pro-oxidants than wild type SH-SY5Y cells (SH cells). In the present communication we used methylmercury as a model of cell stress in order to test whether SH+TH cells would behave in a similar manner in response to this stressor. Incubation with methylmercury (0.1-3 microM) for 24h caused a significant reduction in cell viability and increased apoptotic markers in both cell types. However, the effects were significantly reduced in the SH+TH cells when compared to the SH cells. Activation of p38(MAPK) was also reduced in the SH+TH compared to the SH cells after methylmercury exposure. Since p38(MAPK) is known to participate in signal transduction pathways during cell stress, our data suggest that SH+TH cells develop an increased resistance to environmental stress caused by neurotoxins such as methylmercury. In conclusion our results show that insertion of the human TH gene in cells that originally do not express this protein leads to alterations in cell homeostasis and triggers defense mechanisms against pro-oxidative insults.
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Signal transduction pathways and tyrosine hydroxylase regulation in the adrenal medulla following glucoprivation: an in vivo analysis.
Neurochem. Int.
PUBLISHED: 04-06-2010
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The regulation of tyrosine hydroxylase (TH, the rate limiting enzyme involved in catecholamine synthesis) is critical for the acute and sustained release of catecholamines from adrenal medullary chromaffin cells, however the mechanisms involved have only ever been investigated under in vitro/in situ conditions. Here we explored the effects on, TH phosphorylation and synthesis, and upstream signalling pathways, in the adrenal medulla evoked by the glucoprivic stimulus, 2-deoxy-d-glucose (2DG) administered intraperitoneally to conscious rats. Our results show that 2DG evoked expected increases in plasma adrenaline and glucose at 20 and 60min. We demonstrated that protein kinase A (PKA) and cyclin dependent kinases (CDK) were activated 20min following 2DG, whereas mitogen activated protein kinase (MAPK) was activated later and PKC was not significantly activated. We demonstrated that phosphorylation of Ser40TH peaked after 20min whereas phosphorylation of Ser31TH was still increasing at 60min. Serine 19 was not phosphorylated in this time frame. TH phosphorylation also occurred on newly synthesized protein 24h after 2DG. Thus 2DG increases secretion of adrenaline into the plasma and the consequent rise in glucose levels. In the adrenal medulla 2DG activates PKA, CDK and MAPK, and evokes phosphorylation of Ser40 and Ser31 in the short term and induces TH synthesis in the longer term all of which most likely contribute to increased capacity for the synthesis of adrenaline.
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Regulation of CaMKII by phospho-Thr253 or phospho-Thr286 sensitive targeting alters cellular function.
Cell. Signal.
PUBLISHED: 01-11-2010
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Calcium/calmodulin-stimulated protein kinase II (CaMKII) is an important mediator of synaptic function that is regulated by multi-site phosphorylation and targeting through interactions with proteins. A new phosphorylation site at Thr253 has been identified in vivo, that does not alter CaMKII activity, but does alter CaMKII function through interactions with binding proteins. To identify these proteins, as well as to examine the specific effects following Thr253 or Thr286 phosphorylation on these interactions, we developed an in vitro overlay binding assay. We demonstrated that the interaction between CaMKII and its binding proteins was altered by the phosphorylation state of both the CaMKII and the partner, and identified a CaMKII-specific sequence that was responsible for the interaction between CaMKII and two interacting proteins. By comparing CaMKII binding profiles in tissue and cell extracts, we demonstrated that the CaMKII binding profiles varied with cell type, and also showed that overexpression of a CaMKII Thr253 phospho-mimic mutant in human neuroblastoma and breast cancer cells dramatically altered the morphology and growth rates when compared to overexpression of non-phosphorylated CaMKII. This data highlights the importance of the microenvironment in regulating CaMKII function, and describes a potentially new mechanism by which the functions of CaMKII can be regulated.
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Expression of tyrosine hydroxylase increases the resistance of human neuroblastoma cells to oxidative insults.
Toxicol. Sci.
PUBLISHED: 10-08-2009
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In this study, we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine when compared to wild-type SH-SY5Y cells (SH cells). SH + TH cells exhibit increased levels of dopamine (DA) compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10-100microM) for 24 h caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH + TH cells when compared to the SH cells. The SH + TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activities, while catalase activity was unchanged. Our data suggest that a preconditioning-like mechanism linked to higher DA levels increased the resistance of SH + TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.
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Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells.
J. Neurochem.
PUBLISHED: 06-23-2009
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Manganese (Mn2+) is an essential metal involved in normal functioning of a range of physiological processes. However,occupational overexposure to Mn2+ causes neurotoxicity. The dopaminergic system is a particular target for Mn2+ neurotoxicity.Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn2+ exposure on the phosphorylation and activity of TH. Mn2+ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 lM, without altering the level of TH protein orPC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli didnot block the effects of Mn2+ on Ser40 phosphorylation.A substantial increase in H2O2 production occurred in response to 100 lM Mn2+. The antioxidant Trolox completely inhibited H2O2 production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn2+ and this provides a potential new mechanism for Mn2+-induced neuronal action that does not involve H2O2-mediated cell death.
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Differential regulation of human tyrosine hydroxylase isoforms 1 and 2 in situ: Isoform 2 is not phosphorylated at Ser35.
Biochim. Biophys. Acta
PUBLISHED: 05-28-2009
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The major human tyrosine hydroxylase isoforms (hTH1 and 2) differ in their ability to be phosphorylated in vitro. hTH1 is phosphorylated at Ser31 by extracellular signal-regulated kinase (ERK). This kinase is not capable of phosphorylating hTH2 at Ser35 (the residue that corresponds to Ser31 in hTH1). We have stably transfected SH-SY5Y cells with hTH1 or hTH2 to determine if hTH2 can be phosphorylated at Ser35 in situ. Forskolin increased the phosphorylation of Ser40 in hTH1 and Ser44 in hTH2. Muscarine increased the phosphorylation of both Ser19 and Ser40/44 in both hTH1 and hTH2. EGF increased the phosphorylation of Ser31 in hTH1. Phosphorylation of Ser35 in hTH2 was not detected under any of the conditions tested. Inhibition of ERK by UO126 decreased the phosphorylation of Ser31 and this lead to a 50% decrease in the basal level of phosphorylation of Ser40 in hTH1. The basal level of Ser44 phosphorylation in hTH2 was not altered by treatment with UO126. Therefore, phosphorylation of Ser31 contributes to the phosphorylation of Ser40 in hTH1 in situ; however, this effect is absent in hTH2. This represents a major difference between the two human TH isoforms, and has implications for the regulation of catecholamine synthesis in vivo.
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Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase.
Free Radic. Biol. Med.
PUBLISHED: 05-13-2009
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In this study, we investigated the involvement of glutathione peroxidase-GPx in methylmercury (MeHg)-induced toxicity using three models: (a) in mouse brain after treatment with MeHg (40 mg/L in drinking water), (b) in mouse brain mitochondrial-enriched fractions isolated from MeHg-treated animals, and (c) in cultured human neuroblastoma SH-SY5Y cells. First, adult male Swiss mice exposed to MeHg for 21 days showed a significant decrease in GPx activity in the brain and an increase in poly(ADP-ribose) polymerase cleavage, an index of apoptosis. Second, in mitochondrial-enriched fractions isolated from MeHg-treated mice, there was a significant reduction in GPx activity and a concomitant decrease in mitochondrial activity and increases in ROS formation and lipid peroxidation. Incubation of mitochondrial-enriched fractions with mercaptosuccinic acid, a GPx inhibitor, significantly augmented the toxic effects of MeHg administered in vivo. Incubation of mitochondrial-enriched fractions with exogenous GPx completely blocked MeHg-induced mitochondrial lipid peroxidation. Third, SH-SY5Y cells treated for 24 h with MeHg showed a significant reduction in GPx activity. There was a concomitant significant decrease in cell viability and increase in apoptosis. Inhibition of GPx substantially enhanced MeHg toxicity in the SH-SY5Y cells. These results suggest that GPx is an important target for MeHg-induced neurotoxicity, presumably because this enzyme is essential for counteracting the pro-oxidative effects of MeHg both in vitro and in vivo.
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The low affinity dopamine binding site on tyrosine hydroxylase: the role of the N-terminus and in situ regulation of enzyme activity.
Neurochem. Res.
PUBLISHED: 05-04-2009
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Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.
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Tyrosine hydroxylase phosphorylation in catecholaminergic brain regions: a marker of activation following acute hypotension and glucoprivation.
PLoS ONE
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The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased (prefrontal cortex) or in conjunction with pSer40 (caudal C1). Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the short term, in response to hypotension and glucoprivation.
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?CaMKII is differentially regulated in brain regions that exhibit differing sensitivities to ischemia and excitotoxicity.
J. Cereb. Blood Flow Metab.
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Different brain regions exhibit differing sensitivities to ischemia/excitotoxicity. Whether these differences are due to perfusion or intrinsic factors has not been established. Herein, we found no apparent association between sensitivity to ischemia/excitotoxicity and the level of expression or basal phosphorylation of calcium/calmodulin-stimulated protein kinase II (?CaMKII) or glutamate receptors. However, we demonstrated significant differences in CaMKII-mediated responses after ischemia/excitotoxic stimulation in striatum and cortex. In vivo ischemia and in vitro excitotoxic stimulation produced more rapid phosphorylation of Thr253-?CaMKII in striatum compared with cortex, but equal rates of Thr286-?CaMKII phosphorylation. Phosphorylation by CaMKII of Ser831-GluA1 and Ser1303-GluN2B occurred more rapidly in striatum than in cortex after either stimulus. The differences between brain regions in CaMKII activation and its effects were not accounted for by differences in the expression of ?CaMKII, glutamate receptors, or density of synapses. These results implicate intrinsic tissue differences in Thr253-?CaMKII phosphorylation in the differential sensitivities of brain regions to ischemia/excitotoxicity.
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The sustained phase of tyrosine hydroxylase activation in vivo.
Neurochem. Res.
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Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05 mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48 h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24 h, TH phosphorylation of Ser31 at 24 h, TH activity at 4 and 24 h and TH protein at 48 h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24 h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.