Motor, memory, and anxiety-like behavioral impairments associated with brain-derived neurotrophic factor and dopaminergic imbalance after inhalational exposure to deltamethrin.

Believed to cause damage to the nervous system and possibly being associated with neurodegenerative diseases, deltamethrin (DM) is a type II pyrethroid used in pest control, public health, home environment, and vector control. The objective of this study was to evaluate the motor, cognitive and emotional changes associated with dopaminergic and BDNF imbalance after DM exposure in rats. Sixty Wistar rats (9-10 months-old) were used, under Ethics Committee on Animal Research license (ID 19/2017). The animals were randomly divided into four groups: control (CTL, 0.9% saline), DM2 (2 mg DM in 1.6 mL 0.9% saline), DM4 (4 mg of DM in 1.6 mL of 0.9% saline), and DM8 (8 mg of DM in 1.6 mL of 0.9% saline). DM groups were submitted to 9 or 15 inhalations, one every 48 h. Half of the animals from each group were randomly selected and perfused 24 h after the 9th or 15th inhalation. Throughout the experiment, the animal's behavior were evaluated using catalepsy test, open field, hole-board test, Modified Elevated Plus Maze, and social interaction. At the end of the experiments, the rats were perfused transcardially and their brains were processed for Tyrosine Hydroxylase (TH) and Brain derived neurotrophic factor (BDNF) immunohistochemistries. The animals submitted to 9 inhalations of DM showed a reduction in immunoreactivity for TH in the Substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), and dorsal striatum (DS) areas, and an increase in BDNF in the DS and CA1, CA3 and dentate gyrus (DG) hippocampal areas. Conversely, the animals submitted to 15 inhalations of DM showed immunoreactivity reduced for TH in the SNpc and VTA, and an increase in BDNF in the hippocampal areas (CA3 and DG). Our results indicate that the DM inhalation at different periods induce motor and cognitive impairments in rats. Such alterations were accompanied by dopaminergic system damage and a possible dysfunction on synaptic plasticity.

Exogenous glutathione exerts a therapeutic effect in ischemic stroke rats by interacting with intrastriatal dopamine.

We previously showed that oral administration of exogenous glutathione (GSH) exerted a direct and/or indirect therapeutic effect on ischemic stroke rats, but the underlying mechanisms remain elusive. In the current study, we conducted a quantitative proteomic analysis to explore the pathways mediating the therapeutic effect of GSH in cerebral ischemia/reperfusion (I/R) model rats. Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion. The rats were treated with GSH (250 mg/kg, ig) or levodopa (L-dopa, 100 mg/kg, ig) plus carbidopa (10 mg/kg, ig). Neurologic deficits were assessed, and the rats were sacrificed at 24 h after cerebral I/R surgery to measure brain infarct sizes. We conducted a proteomic analysis of the lesion side striatum samples and found that tyrosine metabolism and dopaminergic synapse were involved in the occurrence of cerebral stroke and the therapeutic effect of GSH. Western blot assay revealed that tyrosine hydroxylase (TH) mediated the occurrence of I/R-induced ischemic stroke and the therapeutic effect of GSH. We analyzed the regulation of GSH on endogenous small molecule metabolites and showed that exogenous GSH had the most significant effect on intrastriatal dopamine (DA) in I/R model rats by promoting its synthesis and inhibiting its degradation. To further explore whether DA-related alterations were potential targets of GSH, we investigated the therapeutic effect of DA accumulation on ischemic brain injury. The combined administration of the precursor drugs of DA (L-dopa and carbidopa) significantly ameliorated neurological deficits, reduced infarct size, and oxidative stress, and decreased pro-inflammatory cytokines levels in the striatum of I/R injury rats. More interestingly, exogenous L-dopa/carbidopa could also greatly enhance the exposure of intracerebral GSH by upregulating GSH synthetases and enhancing homocysteine (HCY) levels in the striatum. Thus, administration of exogenous GSH exerts a therapeutic effect on ischemic stroke by increasing intrastriatal DA, and the accumulated DA can, in turn, enhance the exposure of GSH and its related substances, thus promoting the therapeutic effect of GSH.

Association of decreased triadin expression level with apoptosis of dopaminergic cells in Parkinson's disease mouse model.

BACKGROUND: Parkinson's disease (PD) represent a loss of dopaminergic neurons in the substantia nigra (SN) of the midbrain. However, its cause remains unknown and Triadin (TRDN) function in the brain is also unknown. To examine the relationship between TRDN and PD, the expression levels of protein related to PD in TRDN knockdown status were studied in the SH-SY5Y cells. Cell viability and apoptosis were assessed to examine the apoptosis effect on dopaminergic cells by decreased TRDN, and the levels of the proteins related to apoptosis were also confirmed. RESULTS: This study confirmed decreased TRDN expression level (P < 0.005) at the SN in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse model and identified the functional features of TRDN. Our results showed a relationship between TRDN expression and PD in that reduced TRDN level induced PD-like characteristics. Interestingly, there was TRDN expression in the regions where dopaminergic cells are in the SN, and the expression patterns of TRDN and tyrosine hydroxylase (TH) were similar. Decreased TRDN level also induced apoptotic characteristics and the Fluorescence-activated cell sorting analysis results showed that apoptosis increased (P < 0.05) as the TRDN small interfering RNA concentration increased. The cytotoxicity assay revealed that cell viability also decreased (P < 0.0005) in the same condition as that in the Fluorescence-activated cell sorting analysis. CONCLUSIONS: Decreased TRDN level could be related with the apoptotic death of dopaminergic cells at the SN in PD, and TRDN administration could give a positive effect on PD by reducing apoptotic cell death.

Bisphenol A exposure during the embryonic period: Insights into dopamine relationship and behavioral disorders in Drosophila melanogaster.

Environmental factors are involved in the pathogenesis of neurodevelopmental disorders in addition to genetic factors. In this sense, we demonstrated here that the embryonic exposure of Drosophila melanogaster to Bisphenol A (BPA) 1 mM resulted in changes in development, behavior, and biochemical markers punctuated below. BPA did not alter the oviposition and viability of the eggs, however, it was evidenced a decrease in the rate of pupal eclosion and life span of the hatched flies of the generation filial 1 (F1). F1 flies also developed behavioral changes such as incompatibility in the social interaction between them, and hyperactivity demonstrated by increased locomotion in open field tests, increased grooming, and aggression episodes. Furthermore, decreases in dopamine levels and tyrosine hydroxylase activity have also been observed in flies' heads, possibly related to oxidative damage. Through analyzes of oxidative stress biomarkers, carried out on samples of flies' heads, we observed an increase in malondialdehyde and reactive species, decrease in the activity of the superoxide dismutase and catalase, which possibly culminated in the reduction of cell viability. Thus, it is important to emphasize that BPA developed atypical behaviors in Drosophila melanogaster, reinforce the importance of the environmental factor in the development of neurobehavioral diseases.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via ß-adrenergic signaling. However, the potential of ß-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the ß-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

Latent alterations in swimming behavior by developmental methylmercury exposure are modulated by the homolog of tyrosine hydroxylase in Caenorhabditis elegans.

Methylmercury (MeHg) is a persistent environmental neurotoxicant that may cause adverse neurodevelopmental effects. Previous studies showed that developmental MeHg exposure caused damage to brain functions that were unmasked after a silent period of years or decades. However, the underlying mechanisms of the latent neurotoxicity associated with MeHg exposure from earlier developmental stages have yet to be fully understood. Herein, we established a Caenorhabditis elegans (C. elegans) model of developmental MeHg latent toxicity. Synchronized L1 stage worms were exposed to MeHg (0, 0.05, 0.5 and 5 µM) for 48 h. Swimming moving speeds at adulthood were analyzed in worms exposed to MeHg exposure at early larvae stages. Worms developmentally exposed to MeHg had a significant decline in swimming moving speed on day 10 adult stage, but not on day 1 or 5 adult stage, even though the mercury level in the worms exposed to 0.05 or 0.5 µM MeHg were below the quantification limit on day 10 adult. Day 10 adult worms treated with MeHg showed a significant decrease in bending angle and bending frequency during swimming. Furthermore, their reduced moving speeds tended to increase during the 300-s swimming experiment. Dopamine signaling is known to be involved in the regulation of worms' moving speed. Accordingly, the moving speed of worms with cat-2 (mammalian tyrosine hydroxylase homolog) mutation or dat-1 deletion were assayed on day 10 adult. The cat-2 mutant worms did not show a decline in moving speeds, body bends or bending angles during swimming on day 10 adult stage. Analyses of moving speeds of worms with dat-1 deletion showed that the moving speeds were further reduced after MeHg exposure. However, the effects of MeHg and dat-1 deletion were not synergistic, as the interaction between these parameters did not attain statistical significance. Altogether, our results suggest that developmental MeHg exposure reduced moving speed, and this latent toxicity was less pronounced in the context of deficient production of dopamine synthesis. Tyrosine hydroxylase plays an important role in regulating dopamine-mediated modulation of neurobehavioral functions. These findings uncovered a pivotal role of dopamine and its metabolism in the latent neurotoxic effects of MeHg.

Involvement of the transcription factor E75 in adult cuticular formation in the red flour beetle Tribolium castaneum.

Insect adult metamorphosis generally proceeds with undetectable levels of juvenile hormone (JH). In adult development of the red flour beetle Tribolium castaneum, biosynthesis of adult cuticle followed by its pigmentation and sclerotization occurs, and dark coloration of the cuticle becomes visible in pharate adults. Here, we examined the molecular mechanism of adult cuticular formation in more detail. We noticed that an exogenous JH mimic (JHM) treatment of Day 0 pupae did not inhibit pigmentation or sclerotization, but instead, induced precocious pigmentation of adult cuticle two days in advance. Quantitative RT-PCR analyses revealed that ecdysone-induced protein 75B (E75) is downregulated in JHM-treated pupae. Meanwhile, tyrosine hydroxylase (Th), an enzyme involved in cuticular pigmentation and sclerotization, was precociously induced, whereas a structural cuticular protein CPR27 was downregulated, by exogenous JHM treatment. RNA interference-mediated knockdown of E75 resulted in precocious adult cuticular pigmentation, which resembled the phenotype caused by JHM treatment. Notably, upregulation of Th as well as suppression of CPR27 were observed with E75 knockdown. Meanwhile, JHM treatment suppressed the expression of genes involved in melanin synthesis, such as Yellow-y and Laccase 2, but E75 knockdown did not result in marked reduction in their expression. Taken together, these results provided insights into the regulatory mechanisms of adult cuticular formation; the transcription of genes involved in adult cuticular formation proceeds in a proper timing with undetectable JH, and exogenous JHM treatment disturbs their transcription. For some of these genes such as Th and CPR27, E75 is involved in transcriptional regulation. This study shed light on the molecular mode of action of JHM as insecticides; exogenous JHM treatment disturbed the expression of genes involved in the adult cuticular formation, which resulted in lethality as pharate adults.

Perillyl Alcohol Mitigates Behavioural Changes and Limits Cell Death and Mitochondrial Changes in Unilateral 6-OHDA Lesion Model of Parkinson's Disease Through Alleviation of Oxidative Stress.

In this study, we aim to assess the phytomedicinal potential of perillyl alcohol (PA), a dietary monoterpenoid, in a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease (PD). We observed that PA supplementation alleviated behavioural abnormalities such as loss of coordination, reduced rearing and motor asymmetry in lesioned animals. We also observed that PA-treated animals exhibited reduced oxidative stress, DNA fragmentation and caspase 3 activity indicating alleviation of apoptotic cell death. We found reduced mRNA levels of pro-apoptotic regulator BAX and pro-inflammatory mediators IL18 and TNFα in PA-treated animals. Further, PA treatment successfully increased mRNA and protein levels of Bcl2, mitochondrial biogenesis regulator PGC1α and tyrosine hydroxylase (TH) in lesioned animals. We observed that PA treatment blocked BAX and Drp1 translocation to mitochondria, an event often associated with the inception of apoptosis. Further, 6-OHDA exposure reduced expression of electron transport chain complexes I and IV, thereby disturbing energy metabolism. Conversely, expression levels of both complexes were upregulated with PA treatment in lesioned rats. Finally, we found that protein levels of Nrf2, the transcription factor responsible for antioxidant gene expression, were markedly reduced in cytosolic and nuclear fraction on 6-OHDA exposure, and PA increased expression of Nrf2 in both fractions. We believe that our data hints towards PA having the ability to provide cytoprotection in a hemiparkinsonian rat model through alleviation of motor deficits, oxidative stress, mitochondrial dysfunction and apoptosis.

Regulation of dopaminergic neuronal phenotypes by the estrogen-related receptor gamma ligand GSK4716 via the activation of CREB signaling.

Midbrain dopaminergic (DAergic) neurotransmission plays a crucial role in regulating motor, cognitive, and emotional functions. The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the adult brain and in the developing fetal brain. Our previous study showed the relevance of ERRγ in the regulation of the DAergic neuronal phenotype with the upregulation of dopamine synthesizing tyrosine hydroxylase (TH) and dopamine transporter (DAT) and the possibility that ERRγ could be a novel target for regulating DAergic neuronal differentiation. In this study, we examined whether ERRγ ligands could be small molecule regulators of DAergic phenotypes. The ERRγ agonist GSK4716 increased DAT and TH expression, and the ERRγ inverse agonist GSK5182 attenuated the retinoic acid-induced upregulation of DAT and TH in differentiated SH-SY5Y cells. We found that biphasic activation of the protein kinase A/cyclic AMP response element-binding (CREB) protein signaling pathway was involved in the GSK4716-induced increase in the DAergic phenotype in SH-SY5Y cells. CREB signaling activated as early as 3 h after GSK4716 treatment in an ERRγ-independent manner, but increased following ERRγ activation after 3 days. Protein kinase A inhibitor H-89 attenuated GSK4716-induced DAT and TH upregulation. In primary cultured DAergic neurons, GSK4716 increased neurite length and the number of DAT and TH-double-positive (DAT + TH+) neurons compared to that in control cells. These findings suggest that ERRγ ligands could serve as useful chemical tools for obtaining a better understanding of the regulation of DAergic phenotypes and might facilitate the development of small molecule therapeutics to treat DA-related neurological diseases.

The pesticide fipronil injected into the substantia nigra of male rats decreases striatal dopamine content: A neurochemical, immunohistochemical and behavioral study.

Experimental evidence shows that the phenylpyrazole pesticide fipronil exerts neurotoxic effects at central level in rodents, and in particular on nigrostriatal dopaminergic neurons, whose degeneration is well known to cause motor and non-motor deficits in animals and in humans. In order to characterize better the central neurotoxic effect of fipronil, we injected fipronil (15 and 25 µg) dissolved in dimethyl sulfoxide (DMSO) unilaterally into the substantia nigra of male rats. Male rats injected with DMSO unilaterally into the substantia nigra were used as controls. Control and fipronil-treated rats were then tested in different motor (i.e., open field arena, rotarod, tail flick) and non motor tests (novel object recognition, social interaction) 15 days after injection. A systemic challenge dose of the dopamine-agonist apomorphine was also used to study the presence of a rotational behavior. Sixteen days after fipronil or DMSO injection into the substantia nigra, rats were sacrificed, and either striatal dopamine content or substantia nigra tyrosine hydroxylase (TH) immunoreactivity were measured. The results confirm that the unilateral injection of fipronil into the substantia nigra caused the degeneration of nigrostriatal dopaminergic neurons, which leads to a decrease around 50 % in striatal dopamine content and substantia nigra TH imunoreactivity. This occurred together with changes in motor activity and coordination, and in nociception but not in recognition memory and in social interaction, as revealed by the results of the behavioral experiments performed in fipronil-treated rats compared to vehicle-treated rats 15 days after treatment, as found with other compounds that destroy nigrostriatal dopaminergic neurons.

Gender differences in the effects of cannabidiol on ethanol binge drinking in mice.

The purpose of this study was to explore the effects of cannabidiol (CBD) on binge drinking and evaluate potential gender-related differences. To this aim, male and female C57BL/6J mice (n = 60 per sex) were exposed to the drinking in the dark (DID) model for 4 weeks (DID-1 to DID-4). Dose-response effects of CBD on the ethanol intake were tested by acute (day-4 of DID-3) or repeated administration (day-1 to 4 of DID-4) (experiment 1: CBD 15, 30, and 60 mg/kg, i.p.; experiment 2: CBD 90 mg/kg, i.p.). Finally, we analyzed the relative gene expression of tyrosine hydroxylase (TH) and µ-opioid receptor (OPRM1) and cannabinoid CB1 receptor (CB1 r) in the ventral tegmental area (VTA) and in the nucleus accumbens (NAc), respectively, by real-time quantitative PCR. Females exhibited higher ethanol intake during each DID session. Interestingly, females also showed higher expression of TH and OPRM1, without any difference in CB1 r. Only the acute administration of CBD at the highest dose (90 mg/kg) reduced significantly ethanol consumption in both sexes. Chronic CBD administration (30, 60 and 90 mg/kg) reduced ethanol intake in males, whereas in females a significant reduction was only achieved with the highest dose (90 mg/kg). Repeated administration with CBD (60 mg/kg) significantly reduced TH and OPRM1 in males. In addition, CBD (30 and 60 mg/kg) significantly reduced CB1 r in males. No effect was observed in females. Taken together, these findings suggest that CBD may be of interest for treating binge-drinking patterns and that gender-related difference may affect the treatment outcome.

The psychoactive cathinone derivative pyrovalerone alters locomotor activity and decreases dopamine receptor expression in zebrafish (Danio rerio).

INTRODUCTION: Pyrovalerone (4-methyl-ß-keto-prolintane) is a synthetic cathinone (beta-keto-amphetamine) derivative. Cathinones are a concern as drugs of abuse, as related street drugs such as methylenedioxypyrovalerone have garnered significant attention. The primary mechanism of action of cathinones is to inhibit reuptake transporters (dopamine and norepinephrine) in reward centers of the central nervous system. METHODS: We measured bioenergetic, behavioral, and molecular responses to pyrovalerone (nM-µM) in zebrafish to evaluate its potential for neurotoxicity and neurological impairment. RESULTS: Pyrovalerone did not induce any mortality in zebrafish larvae over a 3- and 24-hr period; however, seizures were prevalent at the highest dose tested (100 µM). Oxidative phosphorylation was not affected in the embryos, and there was no change in superoxide dismutase 1 expression. Following a 3-hr treatment to pyrovalerone (1-100 µM), larval zebrafish (6d) showed a dose-dependent decrease (70%-90%) in total distance moved in a visual motor response (VMR) test. We interrogated potential mechanisms related to the hypoactivity, focusing on the expression of dopamine-related transcripts as cathinones can modulate the dopamine system. Pyrovalerone decreased the expression levels of dopamine receptor D1 (~60%) in larval zebrafish but did not affect the expression of tyrosine hydroxylase, dopamine active transporter, or any other dopamine receptor subunit examined, suggesting that pyrovalerone may regulate the expression of dopamine receptors in a specific manner. DISCUSSION: Further studies using zebrafish are expected to reveal new insight into molecular mechanisms and behavioral responses to cathinone derivates, and zebrafish may be a useful model for understanding the relationship between the dopamine system and bath salts.

Impairment of Nrf2- and Nitrergic-Mediated Gastrointestinal Motility in an MPTP Mouse Model of Parkinson's Disease.

BACKGROUND: Gastrointestinal (GI) motility dysfunction is the most common non-motor symptom of Parkinson's disease (PD). Studies have indicated that GI motility functions are impaired before the onset of PD. AIMS: To investigate the underlying mechanism of PD-induced GI dysmotility in MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine)-induced animal model. METHODS: C57BL/6 mice were administered with or without a selective dopamine neurotoxin, MPTP, to induce parkinsonian symptoms. In addition to in vivo studies, in vitro experiments were also conducted in colon specimens using l-methyl-4-phenylpyridinium (MPP+), a metabolic product of MPTP. Gastric emptying, colon motility, nitrergic relaxation, and western blot experiments were performed as reported. RESULTS: MPTP-induced PD mice showed decreased expression of nuclear factor erythroid 2-related factor (Nrf2) and its target phase II genes in gastric and colon neuromuscular tissues. Decreased levels of tetrahydrobiopterin (BH4, a critical cofactor for nNOS dimerization) associated with uncoupling of nNOS in gastric and colon tissues exposed to MPTP. Impaired enteric nitrergic system led to delayed gastric emptying and slower colonic motility compared to the control mice. In vitro results in colon specimens confirm that activation of Nrf2 restored MPP+-induced suppression of alpha-synuclein, tyrosine hydroxylase (TH), Nrf2, and heme oxygenase-1. In vitro exposure to L-NAME [N(w)-nitro-L-arginine methyl ester], a NOS synthase inhibitor, reduced protein expression of TH in colon tissue homogenates. CONCLUSIONS: Loss of Nrf2/BH4/nNOS expression in PD impairs antioxidant gene expression, which deregulates NO synthesis, thereby contributing to the development of GI dysmotility and constipation. Nitric oxide appears to be important to maintain dopamine synthesis in the colon.

L-Tyr-Induced Phosphorylation of Tyrosine Hydroxylase at Ser40: An Alternative Route for Dopamine Synthesis and Modulation of Na+/K+-ATPase in Kidney Cells.

BACKGROUND/AIMS: Dopamine (DA) is a natriuretic hormone that inhibits renal sodium reabsorption, being Angiotensin II (Ang II) its powerful counterpart. These two systems work together to maintain sodium homeostasis and consequently, the blood pressure (BP) within normal limits. We hypothesized that L-tyrosine (L-tyr) or L-dihydroxyphenylalanine (L-dopa) could inhibit the Na+/K+-ATPase activity. We also evaluated whether L-tyr treatment modulates Tyrosine Hydroxylase (TH). METHODS: Experiments involved cultured LLCPK1 cells treated with L-tyr or L-dopa for 30 minutes a 37°C. In experiments on the effect of Dopa Descarboxylase (DDC) inhibition, cells were pre incubated for 15 minutes with 3-Hydroxybenzylhydrazine dihydrochloride (HBH), and them L-dopa was added for 30 minutes. Na+/K+-ATPase activity was quantified colorimetrically. We used immunoblotting and immunocytochemistry to identify the enzymes TH, DDC and the dopamine receptor D1R in LLCPK1 cells. TH activity was accessed by immunoblotting (increase in the phosphorylation). TH and DDC activities were also evaluated by the modulation of the Na+/K+-ATPase activity, which can be ascribed to the synthesis of dopamine. RESULTS: LLCPK1 cells express the required machinery for DA synthesis: the enzymes TH, and (DDC) as well as its receptor D1R, were detected in control steady state cells. Cells treated with L-tyr or L-dopa showed an inhibition of the basolateral Na+/K+-ATPase activity. We can assume that DA formed in the cytoplasm from L-tyr or L-dopa led to inhibition of the Na+/K+-ATPase activity compared to control. L-tyr treatment increases TH phosphorylation at Ser40 by 100%. HBH, a specific DDC inhibitor; BCH, a LAT2 inhibitor; and Sch 23397, a specific D1R antagonist, totally suppressed the inhibition of Na+/K+-ATPase activity due to L-dopa or L-tyr administration, as indicated in the figures. CONCLUSION: The results indicate that DA formed mainly from luminal L-tyr or L-dopa uptake by LAT2, can inhibit the Na+/K+-ATPase. In addition, our results showed for the very first time that TH activity is also significantly increased when the cells were exposed to L-tyr.

The effects of rotenone on TH, BDNF and BDNF-related proteins in the brain and periphery: Relevance to early Parkinson's disease.

Loss of dopaminergic neurons in the substantia nigra (SN) is one of the pathological hallmarks in Parkinson's disease (PD). This neuron loss is accompanied by reduced protein and activity levels of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Reduced nigral brain-derived neurotrophic factor (BDNF) has been postulated to contribute to the loss of nigral dopaminergic neurons in PD by causing a lack of trophic support. Prior to this nigral cell loss many patients develop non-motor symptoms such as hyposmia, constipation and orthostatic hypotension. We investigated how TH, BDNF and BDNF related receptors are altered in the SN, olfactory bulb, adrenal glands and colon (which are known to be affected in PD) using rotenone-treated rats. Rotenone was administered to Sprague-Dawley rats at a dose of 2.75 mg/kg, 5 days/week for 4 weeks, via intraperitoneal injections. Rats underwent behavioural testing, and tissues were collected for western blot and ELISA analysis. This rotenone treatment induced reduced rears and distance travelled in the rearing and open field test, respectively but caused no impairments in forced movement (rotarod test). The SN had changes consistent with a pro-apoptotic state, such as increased proBDNF but no change in TH; whereas, the colon had significantly reduced TH and increased sortilin. Thus, our results indicate further investigation is warranted for this rotenone-dosing paradigm's capacity for reproducing the early stage of PD, as we observed impairments in voluntary movement and pathology in the colon without overt motor symptoms or nigral dopaminergic loss.

Antidepressant mechanisms of venlafaxine involving increasing histone acetylation and modulating tyrosine hydroxylase and tryptophan hydroxylase expression in hippocampus of depressive rats.

Venlafaxine (VEN) is a widely used antidepressant as a serotonin-reuptake and norepinephrine-reuptake inhibitor. It is used primarily in depression, especially with generalized anxiety disorder or chronic pain. This medicine is of interest because its mechanisms involved multiple aspects. In the current study, the antidepressant action of VEN was investigated by studying the histone acetylation and expression of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) in rats exposed to chronic unpredicted stress (CUS) for 28 days. Male Sprague-Dawley rats were divided into a control group, VEN-treated control group, CUS group, and VEN-treated CUS group. VEN (23.4 mg/kg once daily) was administered to rats by intragastric gavage, whereas the same volume of vehicle was given to rats in the control and model groups. Rat behaviors, acetylated H3 at lysine 9 (acH3K9), acetylated H3 at lysine 14 (acH3K14), acetylated H4 at lysine 12 (acH4K12), histone deacetylase 5, and TH and TPH expression in the hippocampus were determined. Chronic VEN treatment significantly relieved the anxiety- and depression-like behaviors, prevented the increase of histone deacetylase 5 expression and decrease of acH3K9 level, and promoted TH and TPH protein expression in the hippocampus of CUS rats. The results suggest that the preventive antidepressant mechanism of VEN is partly involved in the blocking effects on histone de-acetylated modification and then increasing TH, TPH expression.

Withaferin-A Protects the Nigral Dopamine Neuron and Recovers Motor Activity in Aged Rats.

Withaferin-A (WA) was evaluated for its neuroprotective efficacy on the dopamine (DA) neurons of the substantia nigra (SN) and striatum (ST) in aged rats. Wistar albino rats were divided into group I, young (3 months old); group II, aged (24 months old); group III, aged rats supplemented with WA (50 mg/kg bodyweight once per day for 30 days), and group IV, young rats supplemented with WA (50 mg/kg bodyweight). At the end of the experiment period, the animals were subjected to various motor behavior analyses, and were sacrificed by transcardial perfusion. The brains were dissected out and subjected to various analyses, including histological, histomorphometrical, and immunolocalization of the tyrosine hydroxylase (TH) enzyme. The data of rotarod analysis (p < 0.001) showed a significant motor impairment in aged rats (number of falls 10.2 ± 0.86) and reduction in retention time (31.23 ± 2.56 s) compared to young controls (2.41 ± 0.35 and 84.05 ± 5.15 s). The stride length was significantly reduced (p < 0.001) in aged rats (4.21 ± 0.57 and 4.38 ± 0.61 cm) when compared to young control rats (6.98 ± 0.25 and 7.13 ± 0.70 cm). The histomorphometric data of the aged animals showed a significant reduction in the neuronal diameter (p < 0.001), density (p < 0.001), and volume (p < 0.001) in the SN of aged rats when compared to young rats. Immunohistology demonstrated a marked reduction in the levels of TH enzyme in both the SN and ST of aged animals when compared to young rats. Both structural and functional impairments were reversed in the aged animals after the supplementation of WA (p < 0.001). The present study clearly indicates that WA attenuates the ageing-mediated motor degenerative changes in the SN and ST of aged rats and ascertains its neuroprotective potential.

FoxO1 regulates leptin-induced mood behavior by targeting tyrosine hydroxylase.

PURPOSE: While leptin has been associated with various psycho-physiological functions, the molecular network in leptin-mediated mood regulation remains elusive. METHODS: Anxiolytic behaviors and tyrosine hydroxylase (TH) levels were examined after leptin administration. Functional roles of STAT3 and FoxO1 in regulation of TH expression were investigated using in vivo and in vitro systems. A series of animal behavioral tests using dopaminergic neuron-specific FoxO1 KO (FoxO1 KODAT) were performed and investigated the roles of FoxO1 in regulation of mood behaviors. RESULTS: Here, we show that administration of leptin induces anxiolytic-like phenotype through the activation of signal transducer and activator of transcription 3 (STAT3) and the inhibition of forkhead box protein O1 (FoxO1) in dopaminergic (DA) neurons of the midbrain. Specifically, STAT3 and FoxO1 directly bind to and exert opposing effects on tyrosine hydroxylase (TH) expression, where STAT3 acts as an enhancer and FoxO1 acts as a prominent repressor. Accordingly, suppression of the prominent suppressor FoxO1 by leptin strongly increased TH expression. Furthermore, our previous results showed that specific deletion of FoxO1 in DA neurons (FoxO1 KODAT) led to a profound elevation of TH activity and dopamine contents. Finally, FoxO1 KODAT mice exhibited enhanced leptin sensitivity as well as displayed reduced anxiety- and depression-like behaviors. CONCLUSIONS: This work establishes a novel molecular mechanism of mood behavior regulation by leptin and suggests FoxO1 suppression by leptin might be a key for leptin-induced behavioral manifestation in DA neurons.

Role of the HIF-1α/Nur77 axis in the regulation of the tyrosine hydroxylase expression by insulin in PC12 cells.

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l-DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.

Pedunculopontine tegmentum cholinergic loss leads to a progressive decline in motor abilities and neuropathological changes resembling progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is the most common atypical Parkinsonism. Although PSP shares some symptomology with Parkinson's disease (PD), PSP has a different underlying pathology characterized by tau aggregation. Furthermore, PSP sufferers respond poorly to PD medications and there are no effective alternative therapeutics. The development of both palliative and disease altering therapeutics has been hampered by the lack of an animal model that displays relevant PSP-like pathology and behavioral deficits. Previously, our lab found that in rats the selective removal of cholinergic pedunculopontine neurons (whose axonal projections overlap with areas of PSP pathology), mimics the extensive loss of cholinergic pedunculopontine neurons seen in PSP, and produces a unique PSP-like combination of deficits in: startle reflex, attention, and motor function. The present study extends those findings by allowing the lesion to incubate for over a year and compares behavioral and post-mortem pathology of pedunculopontine-cholinergic-lesioned and sham-lesioned rats. There was an early startle reflex deficit which did not improve over time. Progressive declines in motor function developed over the course of the year, including an increase in the number of "slips" while navigating various beams and poorly coordinated transitions from an elevated platform into homecages. Histological analysis discovered that the loss off cholinergic pedunculopontine neurons precipitated a significant loss of substantia nigra tyrosine hydroxylase-positive neurons and a significant enlargement of the lateral ventricles. The latter is a distinguishing feature between PSP and PD. This preclinical animal model of PSP has the potential to further our understanding of PSP and aid in the testing of potential therapeutic agents.