Reduced Tyrosine Hydroxylase Activity Research Articles

Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption.

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, serotonin, and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficacy of treatment. We applied active immunization against ADH to investigate its efficacy in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2% aluminum hydroxide-based adjuvant. The efficacy of immunization was demonstrated by the production of high titers of ADH-specific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats. Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum,while immunization neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum. The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and make available new prospects for developing innovative strategies for treatment of excessive alcohol intake.

Effects of ERK1/2 kinases inactivation on the nigrostriatal system of Krushinsky-Molodkina rats genetically prone to audiogenic seizures

Recently, we demonstrated that inhibition of ERK1/2 activity by SL-327 treatment blocks seizure behavior in Krushinsky-Molodkina (KM) rats, which was mediated by altering of GABA and glutamate release mechanism in the hippocampus. Basal ganglia representing various subcortical cell groups play a significant role in the regulation of motor activity, including epileptiform seizures.Objectives: To verify if nigrostriatal system could be also affected by SL-327 treatment we analyzed the expression of tyrosine hydroxylase, D1 and D2 dopamine receptors, NR2B subunit of NMDA receptor as well as vesicular glutamate transporter VGLUT2 and glutamic acid decarboxylases GAD65/67 in the striatum and substantia nigra of KM rats.Methods: Animals were injected i.p. with SL-327 (50 mg/kg) 60 min before audio stimulation. After audiogenic stimulation the brains of control and SL 327 treated rats were removed for further immunohistochemical and biochemical analysis.Results: Obtained results demonstrated a decrease activity in synapsin I, and accumulation of VGLUT2 in the striatum after blockade of audiogenic seizure (AGS) by SL 327 that could lead to inhibition of glutamate release. While in the striatum GAD65/67 level was diminished, in the substantia nigra GAD65/67 was increased showing enhanced inhibitory output to the compact part of the substantia nigra. Analysis of dopaminergic system showed a significant reduction of tyrosine hydroxylase activity and expression in the substantia nigra, and decreased D1 and D2 receptor expression in the striatum.In summary, we propose that changes in the nigrostriatal system could be mediated by inhibitory effect of SL 327 on AGS expression.

The N-Terminal Sequence of Tyrosine Hydroxylase Is a Conformationally Versatile Motif That Binds 14-3-3 Proteins and Membranes

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamine neurotransmitters, and a reduction in TH activity is associated with several neurological diseases. Human TH is regulated, among other mechanisms, by Ser19-phosphorylation-dependent interaction with 14-3-3 proteins. The N-terminal sequence (residues 1–43), which corresponds to an extension to the TH regulatory domain, also interacts with negatively charged membranes. By using X-ray crystallography together with molecular dynamics simulations and structural bioinformatics analysis, we have probed the conformations of the Ser19-phosphorylated N-terminal peptide [THp-(1-43)] bound to 14-3-3γ, free in solution and bound to a phospholipid bilayer, and of the unphosphorylated peptide TH-(1-43) both free and bilayer bound. As seen in the crystal structure of THp-(1-43) complexed with 14-3-3γ, the region surrounding pSer19 adopts an extended conformation in the bound state, whereas THp-(1-43) adopts a bent conformation when free in solution, with higher content of secondary structure and higher number of internal hydrogen bonds. TH-(1-43) in solution presents the highest mobility and least defined structure of all forms studied, and it shows an energetically more favorable interaction with membranes relative to THp-(1-43). Cationic residues, notably Arg15 and Arg16, which are the recognition sites of the kinases phosphorylating at Ser19, are also contributing to the interaction with the membrane. Our results reveal the structural flexibility of this region of TH, in accordance with the functional versatility and conformational adaptation to different partners. Furthermore, this structural information has potential relevance for the development of therapeutics for neurodegenerative disorders, through modulation of TH–partner interactions.

Protein Kinase C-Dependent Dephosphorylation of Tyrosine Hydroxylase Requires the B56δ Heterotrimeric Form of Protein Phosphatase 2A

Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKCδ) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKCδ negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKCδ–PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3–10 μM) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKCδ specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1–1 μM Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKCδ kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKCδ inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKCδ and PP2A activity.

Progesterone decreases tyrosine hydroxylase phosphorylation state and increases protein phosphatase 2A activity in the stalk-median eminence on proestrous afternoon

The progesterone (P(4)) rise on proestrous afternoon is associated with dephosphorylation of tyrosine hydroxylase (TH) and reduced TH activity in the stalk-median eminence (SME), which contributes to the proestrous prolactin surge in rats. In the present study, we investigated the time course for P(4) effect on TH activity and phosphorylation state, as well as cAMP levels and protein phosphatase 2A (PP2A) activity and quantity, in the SME on proestrous morning and afternoon. P(4) (7.5 mg/kg, s.c.) treatment on proestrous afternoon decreased TH activity and TH phosphorylation state at Ser-31 and Ser-40 within 1 h, whereas morning administration of P(4) had no 1 h effect on TH. PP2A activity in the SME was enhanced after P(4) treatment for 1 h on proestrous afternoon without a change in PP2A catalytic subunit quantity, whereas P(4) treatment had no effect on PP2A activity or quantity on proestrous morning. cAMP levels in the SME were unchanged with 1 h P(4) treatment. At 5 h after P(4) treatment, TH activity and phosphorylation state declined coincident with an increase in plasma prolactin in both P(4)-treated morning and afternoon groups. PP2A activity in the SME was unchanged in 5 h P(4)-treated rat. Our data suggest that P(4) action on tuberoinfundibular dopaminergic (TIDA) neurons involves at least two components. A more rapid (1 h) P(4) effect engaged only on proestrous afternoon likely involves the activation of PP2A. The longer P(4) action on TIDA neurons is evident on both the morning and afternoon of proestrus and may involve a common, as yet unidentified, mechanism.

14-3-3ζ Contributes to Tyrosine Hydroxylase Activity in MN9D Cells: LOCALIZATION OF DOPAMINE REGULATORY PROTEINS TO MITOCHONDRIA

The 14-3-3 proteins stimulate the activation of tyrosine hydroxylase (TH), the rate-limiting catecholamine biosynthetic enzyme. To explore if particular endogenous 14-3-3 isoforms specifically affected TH activity and dopamine synthesis, we utilized rodent nigrostriatal tissues and midbrain-derived MN9D dopaminergic cells. Extracts from ventral midbrain and MN9D cells contained similar pools of 14-3-3 mRNAs, with 14-3-3zeta being relatively abundant in both. Protein levels of 14-3-3zeta were also abundant. [(32)P]Orthophosphate labeling of MN9D cells, followed by co-immunoprecipitation with pan-TH and pan-14-3-3 antibodies brought down similar amounts of phosphorylated TH in each, confirming that 14-3-3-bound phosphorylated TH in our cells. Co-immunoprecipitation of striatal tissues with a pan-TH antibody precipitated 14-3-3zeta but not another potential TH regulatory isoform, 14-3-3eta. In whole cell extracts from MN9D cells after 14-3-3 small interfering RNA treatments, we found that 14-3-3zeta knockdown significantly reduced TH activity and dopamine synthesis whereas knockdown of 14-3-3eta had no effect. 14-3-3zeta was found co-localized on mitochondria with TH, and its knockdown by small interfering RNA reduced TH phosphorylation and TH activity in the mitochondrial pool. Together the data support a role for 14-3-3zeta as an endogenous activator of TH in midbrain dopaminergic neurons and furthermore, identify mitochondria as a potential novel site for dopamine synthesis, with implications for Parkinson disease.

Reactive oxygen species‐dependent down regulation of protein phosphatase contributes to tyrosine hydroxylase activation by intermittent hypoxia

In a previous study we showed that intermittent hypoxia (IH) activates tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis via increased phosphorylation of Ser‐40 without altering total TH protein (Kumar et al., J. Appl. Physiol. 95: 536‐544, 2003). In the present study, we examined the role of protein phosphatase(s) in IH‐evoked TH activation in the brainstem of rats. IH increased TH activity in the medullary regions of the brainstem associated with regulation of cardio‐respiratory systems. This increase TH activity was associated with elevation of dopamine (DA) levels, and increase in TH phosphorylation at Ser‐31 and Ser‐40. IH also resulted in the down‐regulation of protein phosphatase 2A (PP2A) expression and activity. Reactive oxygen species (ROS) were elevated in the brainstem regions in IH exposed rats. Anti‐oxidants prevented IH‐induced down‐regulation of PP2A as well as increases in serine phosphorylation of TH with a concomitant reduction of TH activity and DA to normoxic control levels. These findings demonstrate that ROS‐dependent down regulation of protein phosphatase 2A plays a critical role in IH‐induced activation of TH and increase in DA synthesis which may in part contribute to altered catecholaminergic neurotransmission in the brainstem during IH (Supported by grants from NIH HL‐89616 and HL‐90554).

The Harman and Norharman Reduced Dopamine Content and Induced Cytotoxicity in PC12 Cells

The effects of harman and norharman on dopamine content and L-DOPA-induced cytotoxicity were investigated in PC12 cells. Harman and norharman decreased the intracellular dopamine content for 24 h. The <TEX>$IC_{50}$</TEX> values of harman and norharman were 20.4 <TEX>${\mu}M$</TEX> and 95.8 <TEX>${\mu}M$</TEX>, respectively. Tyrosine hydroxylase (TH) activity and TH mRNA levels were also decreased by 20 <TEX>${\mu}M$</TEX> harman and 100 <TEX>${\mu}M$</TEX> norharman. Under the same conditions, the intracellular cyclic AMP levels were decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 <TEX>${\mu}M$</TEX> and 150 <TEX>${\mu}M$</TEX> caused cytotoxicity at 24 h in PC12 cells. Non-cytotoxic ranges of 10-30 <TEX>${\mu}M$</TEX> harman and 50-150 <TEX>${\mu}M$</TEX> norharman inhibited L-DOPA (20-50 <TEX>${\mu}M$</TEX>)-induced increases of dopamine content at 24 h. Harman at 20-150 <TEX>${\mu}M$</TEX> and norharman at 100-300 <TEX>${\mu}M$</TEX> also enhanced LDOPA (20-100 <TEX>${\mu}M$</TEX>)-induced cytotoxicity at 24 h. These results suggest that harman and norharman decrease dopamine content by reducing TH activity and aggravate L-DOPA-induced cytotoxicity in PC12 cells.

Effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of harman and norharman on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Harman and norharman at a concentration of 20 μM and 100 μM showed 49.4% and 49.5% inhibition of dopamine content for 48 h, respectively. The IC 50 values of harman and norharman were 21.2 μM and 103.3 μM. Dopamine content, tyrosine hydroxylase (TH) activity and TH mRNA levels were decreased during the first 6 h, maintained for up to 48 h and then gradually recovered at 72 h after exposure to 20 μM harman and 100 μM norharman. Under the same conditions, the intracellular cyclic AMP levels and Ca 2+ concentrations were also decreased by harman and norharman. In addition, harman and norharman at concentrations higher than 80 μM and 150 μM caused cytotoxicity at 48 h in PC12 cells. Non-cytotoxic ranges of 10–30 μM harman and 50–150 μM norharman inhibited L-DOPA (20–50 μM)-induced increases in dopamine content at 48 h. Harman at 20–150 μM and norharman at 100–300 μM also enhanced L-DOPA (20–100 μM)-induced cytotoxicity at 48 h with an apoptotic process. These results suggest that harman and norharman inhibit dopamine biosynthesis by reducing TH activity and enhance L-DOPA-induced cytotoxicity in PC12 cells.

Developmental plasticity of the carotid chemoafferent pathway in rats that are hypoxic during the prenatal period

The chemoreflex pathway undergoes postnatal maturation, and the perinatal environment plays a critical role in shaping respiratory control system. We investigated the role of prenatal hypoxia on the maturation of the chemoreflex neural circuits regulating ventilation in rat. Effects of hypoxia (10% O2) from the 5th to the 20th day of gestation were studied on male offspring at birth and on postnatal days 3, 7, 21 and 68. Maturation of the respiratory control system was assessed by in vivo tyrosine hydroxylase (TH) activity measurement in peripheral chemoreceptors (carotid bodies, petrosal ganglia), and in brainstem catecholaminergic cell groups (A2C2c and A1C1 areas in the medulla, A5 and A6 areas in the pons). Resting ventilation and ventilatory response to hypoxia were evaluated as functional sequelae. In peripheral structures, prenatal hypoxia reduced TH activity within the first postnatal week and enhanced it later. In contrast, in central areas, prenatal hypoxia upregulated TH activity within the first postnatal week and downregulated it later. The in vivo TH activity impairment is therefore tissue specific, with an opposite effect on the peripheral and central neural circuits. A shift of the effect of prenatal hypoxia occurred between 1 and 3 weeks, indicating a postnatal temporal effect of prenatal hypoxia. An important period in the development of the chemoafferent pathway occurred between the first and the third postnatal week. Functionally, prenatal hypoxia impaired resting ventilation and ventilatory response to hypoxia. The alterations of the catecholaminergic components of the chemoafferent pathway resulting from prenatal hypoxia might contribute to impair postnatal respiratory behaviour.

Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells

The inhibitory effects of liriodenine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced dopamine content increases in PC12 cells were investigated. Treatment of PC12 cells with 5-10 microM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 microM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 microM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 microM liriodenine to 20-70% and 10-14% of control levels at 3-12 h, respectively; TH activity was more influenced than AADC activity. The levels of TH mRNA, intracellular cyclic AMP and basal Ca2+ concentration were also decreased by 10 microM liriodenine. In addition, 10 microM liriodenine reduced L-DOPA (20-100 microM)-induced increases in dopamine content. However, 10 microM liriodenine resulted in a protective effect against L-DOPA (50-100 microM)-induced cytotoxicity. These results suggest that liriodenine regulates dopamine biosynthesis by partially reducing TH activity and TH gene expression and has protective effects against L-DOPA-induced cytotoxicity in PC12 cells.

Acute and chronic exposure to hypoxia alters ventilatory pattern but not minute ventilation of mice overexpressing erythropoietin

Apart from enhancing red blood cell production, erythropoietin (Epo) has been shown to modulate the ventilatory response to reduced oxygen supply. Both functions are crucial for the organism to cope with increased oxygen demand. In the present work, we analyzed the impact of Epo and the resulting excessive erythrocytosis in the neural control of normoxic and hypoxic ventilation. To this end, we used our transgenic mouse line (Tg6) that shows high levels of human Epo in brain and plasma, the latter leading to a hematocrit of approximately 80%. Interestingly, while normoxic and hypoxic ventilation in Tg6 mice was similar to WT mice, Tg6 mice showed an increased respiratory frequency but a decreased tidal volume. Knowing that Epo modulates catecholaminergic activity, the altered catecholaminergic metabolism measured in brain stem suggested that the increased respiratory frequency in Tg6 mice was related to the overexpression of Epo in brain. In the periphery, higher response to hyperoxia (Dejours test), as well as reduced tyrosine hydroxylase activity in carotid bodies, revealed a higher chemosensitivity to oxygen in transgenic mice. Moreover, in line with the decreased activity of the rate-limiting enzyme for dopamine synthesis, the intraperitoneal injection of a highly specific peripheral ventilatory stimulant, domperidone, did not stimulate hypoxic ventilatory response in Tg6 mice. These results suggest that high Epo plasma levels modulate the carotid body's chemotransduction. All together, these findings are relevant for understanding the cross-talk between the ventilatory and erythropoietic systems exposed to hypoxia.

Short-term regulation of tyrosine hydroxylase activity and expression by endothelin-1 and endothelin-3 in the rat posterior hypothalamus

Brain catecholamines are involved in several biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and -3 (ET-1 and ET-3) modulate norepinephrine release in the anterior and posterior hypothalamus. As tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, the aim of the present work was to investigate the effects of ET-1 and ET-3 on TH activity, total enzyme level and the phosphorylated forms of TH in the rat posterior hypothalamus. Results showed that ET-1 and ET-3 diminished TH activity but the response was abolished by both selective ET A and ET B antagonists (BQ-610 and BQ-788, respectively). In addition ET A and ET B selective agonists (sarafotoxin S6b and IRL-1620, respectively) failed to affect TH activity. In order to investigate the intracellular signaling coupled to endothelins (ETs) response, nitric oxide (NO), phosphoinositide, cAMP/PKA and CaMK-II pathways were studied. Results showed that N ω-nitro- l-arginine methyl ester and 7-nitroindazole (NO synthase and neuronal NO synthase inhibitors, respectively), 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinozalin-1-one and KT-5823 (soluble guanylyl cyclase, and PKG inhibitors, respectively) inhibited ETs effect on TH activity. Further, sodium nitroprusside and 8-bromoguanosine-3′,5′-cyclic monophosphate (NO donor and cGMP analog, respectively) mimicked ETs response. ETs-induced reduction of TH activity was not affected by a PKA inhibitor but it was abolished by PLC, PKC and CaMK-II inhibitors as well as by an IP 3 receptor antagonist. On the other hand, both ETs did not modify TH total level but reduced the phosphorylation of serine residues of the enzyme at positions 19, 31 and 40. Present results suggest that ET-1 and ET-3 diminished TH activity through an atypical ET or ET C receptor coupled to the NO/cGMP/PKG, phosphoinositide and CaMK-II pathways. Furthermore, TH diminished activity may result from the reduction of the phosphorylated sites of the enzyme without changes in its total level. Taken jointly present and previous results support that ET-1 and ET-3 may play a relevant role in the modulation of catecholaminergic neurotransmission in the posterior hypothalamus of the rat.

Involvement of nitric oxide pathways in short term modulation of tyrosine hydroxylase activity by endothelins 1 and 3 in the rat anterior hypothalamus

The ability of endothelins 1 and 3 (ET-1 and ET-3) to reduce neuronal norepinephrine release through ETB receptor activation involving nitric oxide (NO) pathways in the rat anterior hypothalamus region (AHR) was previously reported. In the present work, we studied the effects of ET-1 and -3 on tyrosine hydroxylase (TH) activity and the possible involvement of NO pathways. Results showed that ET-1 and -3 (10 nM) diminished TH activity in AHR and this effect was blocked by a selective ETB receptor antagonist (100 nM BQ-788), but not by a ET A receptor antagonist (BQ-610). To confirm these results, 1 μM IRL-1620 (ET B agonist) reduced TH activity whereas 300 nM sarafotoxin S6b falled to modify it. N ω-Nitro- l-arginine methyl ester (10 μM), 7-nitroindazole (10 μM), 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-ona (10 μM), KT5823 (2 μM), inhibitors of nitric oxide synthase, neuronal nitric oxide synthase, NO-sensitive-guanylyl cyclase, and protein kinase G, respectively, did not modify the reduction of TH activity produced by ETs. In addition, both 100 μM sodium nitroprusside and 50 μM 8-bromoguanosine-3′,5′-cyclic monophosphate (NO donor and guanosine-3′,5′-cyclic monophosphate analog, respectively) diminished TH activity. Present results showed that ET-1 and ET-3 diminished TH activity through the activation of ET B receptors involving the NO/guanosine-3′,5′-cyclic monophosphate/protein kinase G pathway. Taken jointly present and previous results it can be concluded that both ETs play an important role as modulators of norepinephrine neurotransmission in the rat AHR.

Cannabinoids provide neuroprotection against 6-hydroxydopamine toxicity in vivo and in vitro: Relevance to Parkinson's disease

Cannabinoids have been reported to provide neuroprotection in acute and chronic neurodegeneration. In this study, we examined whether they are also effective against the toxicity caused by 6-hydroxydopamine, both in vivo and in vitro, which may be relevant to Parkinson's disease (PD). First, we evaluated whether the administration of cannabinoids in vivo reduces the neurodegeneration produced by a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. As expected, 2 weeks after the application of this toxin, a significant depletion of dopamine contents and a reduction of tyrosine hydroxylase activity in the lesioned striatum were noted, and were accompanied by a reduction in tyrosine hydroxylase-mRNA levels in the substantia nigra. None of these events occurred in the contralateral structures. Daily administration of delta9-tetrahydrocannabinol (delta9-THC) during these 2 weeks produced a significant waning in the magnitude of these reductions, whereas it failed to affect dopaminergic parameters in the contralateral structures. This effect of delta9-THC appeared to be irreversible since interruption of the daily administration of this cannabinoid after the 2-week period did not lead to the re-initiation of the 6-hydroxydopamine-induced neurodegeneration. In addition, the fact that the same neuroprotective effect was also produced by cannabidiol (CBD), another plant-derived cannabinoid with negligible affinity for cannabinoid CB1 receptors, suggests that the antioxidant properties of both compounds, which are cannabinoid receptor-independent, might be involved in these in vivo effects, although an alternative might be that the neuroprotection exerted by both compounds might be due to their anti-inflammatory potential. As a second objective, we examined whether cannabinoids also provide neuroprotection against the in vitro toxicity of 6-hydroxydopamine. We found that the non-selective cannabinoid agonist HU-210 increased cell survival in cultures of mouse cerebellar granule cells exposed to this toxin. However, this effect was significantly lesser when the cannabinoid was directly added to neuronal cultures than when these cultures were exposed to conditioned medium obtained from mixed glial cell cultures treated with HU-210, suggesting that the cannabinoid exerted its major protective effect by regulating glial influence to neurons. In summary, our results support the view of a potential neuroprotective action of cannabinoids against the in vivo and in vitro toxicity of 6-hydroxydopamine, which might be relevant for PD. Our data indicated that these neuroprotective effects might be due, among others, to the antioxidant properties of certain plant-derived cannabinoids, or exerted through the capability of cannabinoid agonists to modulate glial function, or produced by a combination of both mechanisms.

Prevention of MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) dopaminergic neurotoxicity in mice by chronic lithium: involvements of Bcl-2 and Bax

Lithium has been reported to exert neuroprotective activity in several neuronal cell cultures and in vivo models against glutamate toxicity. Since this action was reported to be associated with alterations in the antiapoptotic Bcl-2 family proteins, the effect of chronic lithium diet on the ability of the parkinsonism neurotoxin, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to deplete striatal dopamine in mice was determined. Mice were fed for with a diet containing 1.1, 2.2, 3.3, and 4.4 g/kg lithium chloride (LiCl) for 4 weeks, during which time serum levels of lithium were monitored. The 3.3 g/kg lithium diet gave serum level value very similar to what is observed in lithium therapy in man and the 4.4 g/kg well above this. At the end of this period the mice received 24 mg/kg MPTP i.p. once daily for 3 days. A direct relation was established with the increase in serum lithium and its ability to prevent MPTP induced depletion of striatal dopamine (DA) and its metabolites DPOAC and HVA. With the diet containing the highest lithium concentration there was an almost complete prevention of striatal dopamine depletion and the reduction in tyrosine hydroxylase activity and protein and it prevented the increase in dopamine turnover (DOPAC+HVA/DA) normally observed in MPTP treatment. Lithium did not interfere with the metabolism of MPTP, or with its brain uptake, since, the level of its monoamine oxidase (MAO) B derived metabolite, MPP+, in the striata of lithium and non-lithium treated mice were almost identical. Striatal Bcl-2 was significantly decreased, while Bax was increased in MPTP treated mice. Lithium treatment not only increased striatal Bcl-2 in control mice, but also prevented its reduction as induced by MPTP, and an opposing effect was seen with Bax. The neuroprotective action of lithium in this model of Parkinson’s disease has been attributed to its antiapoptotic activity which among other factors includes induction of Bcl-2 and reduction of Bax.

Short-term effects of 3,4-methylenedioximethamphetamine on noradrenergic activity in locus coeruleus and hippocampus of the rat

This study was undertaken to investigate the effects of the administration of 3,4-methylenedioxymethamphetamine (MDMA) on the locus coeruleus firing rate, on the sensitivity of the α 2-adrenoceptors which regulate neuronal activity and on the in vivo tyrosine hydroxylase activity in hippocampus. The basal firing rate was not modified by either a single dose or repeated doses of MDMA, although the latter produced a shift to the right in the dose-response curve for clonidine-induced inhibition of the firing rate (ED 50 increased by 59%) and a reduction in tyrosine hydroxylase activity (20%) in the hippocampus. However, 8 days after the final dose α 2-adrenoceptor sensitivity and tyrosine hydroxylase activity had returned to control values. Our results show a desensitization of α 2-adrenoceptors in locus coeruleus and the existence of short-term changes in the noradrenergic system.

A role for alpha-synuclein in the regulation of dopamine biosynthesis.

The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

Desensitization of α2-Adrenoceptors which Regulate Noradrenaline Synthesis and Release after Chronic Treatment with Clorgyline in the Rat Brain

The sensitivity of α₂-adrenoceptors which regulate synthesis and release of noradrenaline was investigated in hippocampus, parietal cortex, and hypothalamus of rats treated with clorgyline. After administering a DOPA decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the noradrenaline content were evaluated. Acute and chronic treatment with clorgyline led to both increases of noradrenaline levels and decreases of tyrosine hydroxylase activity, determined as the accumulation of DOPA. Whereas the α₂-adrenoceptor agonist clonidine induced a similar reduction in tyrosine hydroxylase activity in the group subjected to the acute treatment and in the control group, it failed to do so after chronic clorgyline treatment. In hippocampal and cortical synaptosomes, a reduction in the sensitivity of α₂-adrenoceptors which regulate [³H]noradrenaline release, reflected by the shift to the right of the concentration-effect curves for oxymetazoline, was also found after the repeated treatment. These results indicate a desensitization of α₂-adrenoceptors after chronic treatment with clorgyline.