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1.
Eur J Med Chem ; 184: 111765, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629163

RESUMO

Synthesis and investigation of anti-Toxoplasma gondii activity of novel thiazoles containing benzo [b]thiophene moiety are presented. Among the derivatives, compound 3k with adamantyl group shows exceptionally high potency against Me49 strain with IC50 (8.74 µM) value which is significantly lower than the activity of trimethoprim (IC50 39.23 µM). In addition, compounds 3a, 3b and 3k showed significant activity against RH strain (IC50 51.88-83.49 µM). The results of the cytotoxicity evaluation showed that Toxoplasma gondii growth was inhibited at non-cytotoxic concentrations for the mammalian L929 fibroblast (CC30 ∼ 880 µM). The most active compound 3k showed tyrosinase inhibition effect, with IC50 value of 328.5 µM. The binding energies calculated for compounds 3a-3e, 3k are strongly correlated with the experimentally determined values of tyrosinase inhibition activity. Moreover, the binding energies corresponding to the same ligands and calculated for both tyrosinase and tyrosine hydroxylase are also correlated with each other, suggesting that tyrosinase inhibitors may also have an inhibitory effect on tyrosine hydroxylase. Compounds 3j and 3k have also very strong antioxidant activity (IC50 15.9 and 15.5 µM), respectively, which is ten times higher than well-known antioxidant BHT.


Assuntos
Antioxidantes/farmacologia , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Tiazóis/farmacologia , Tiofenos/farmacologia , Toxoplasma/efeitos dos fármacos , Animais , Antioxidantes/síntese química , Antioxidantes/química , Antiprotozoários/síntese química , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tiofenos/química , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
2.
Neurochem Int ; 125: 16-24, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30739038

RESUMO

Parkinson's disease (PD) is the second common neurodegenerative disorder. Deficit of the nigro-striatal dopaminergic neurons causes the motor symptoms of PD. While the oxidative stress is thought to be deeply involved in the etiology of PD, molecular targets for the oxidative insults has not been fully elucidated. 6R-5,6,7,8-Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase (TH), the rate-limiting enzyme for production of dopamine, and easily oxidized to its dihydro-form. In this study, we examined the alteration in the metabolism of BH4 caused by a parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP reduced the dopamine content and the in vivo activity of TH in the striatum prior to degeneration of the dopaminergic neurons. We found that administration of BH4 could restore the dopamine content and in vivo TH activity in the striatum of MPTP-treated mice. Unexpectedly, when BH4 was administered with MPTP, BH4 contents in the brain were far higher than those injected without MPTP even at 23 h after the last injection. Because MPTP has been shown to increase ROS production in the dopaminergic neurons, we assumed that the increased ROS oxidizes BH4 into its dihydro-form, excreted from the dopaminergic neurons, taken-up by the neighboring cells, reduced back to BH4, and then accumulated in the brain. We also investigated the action of MPTP in mice lacking quinonoid-dihydropteridine reductase (Qdpr), an enzyme catalyzing regeneration of BH4 from quinonoid dihydrobiopterin. The dopamine depletion induced by MPTP was severer in Qdpr-deficient mice than in wild-type mice. The present data suggest that perturbation of the BH4 metabolism would be the cause of early and persistent dopamine depletion in the striatum.


Assuntos
Biopterina/análogos & derivados , Corpo Estriado/metabolismo , Dopamina/metabolismo , Intoxicação por MPTP/metabolismo , Animais , Biopterina/administração & dosagem , Corpo Estriado/efeitos dos fármacos , Intoxicação por MPTP/tratamento farmacológico , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
3.
Pharmacol Biochem Behav ; 178: 42-50, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29289701

RESUMO

Efforts to replicate results from both basic and clinical models have highlighted problems with reproducibility in science. In psychiatry, reproducibility issues are compounded because the complex behavioral syndromes make many disorders challenging to model. We develop translatable tasks that quantitatively measure psychiatry-relevant behaviors across species. The behavioral pattern monitor (BPM) was designed to analyze exploratory behaviors, which are altered in patients with bipolar disorder (BD), especially during mania episodes. We have repeatedly assessed the behavioral effects of reduced dopamine transporter (DAT) expression in the BPM using a DAT knockdown (KD) mouse line (~10% normal expression). DAT KD mice exhibit a profile in the BPM consistent with acutely manic BD patients in the human version of the task-hyperactivity, increased exploratory behavior, and reduced spatial d (Perry et al., 2009). We collected data from multiple DAT KD BPM experiments in our laboratory to assess the reproducibility of behavioral outcomes across experiments. The four outcomes analyzed were: 1) transitions (amount of locomotor activity); 2) rearings (exploratory activity); 3) holepokes (exploratory activity); and 4) spatial d (geometrical pattern of locomotor activity). By comparing DAT KD mice to wildtype (WT) littermates in every experiment, we calculated effect sizes for each of the four outcomes and then calculated a mean effect size using a random effects model. DAT KD mice exhibited robust, reproducible changes in each of the four outcomes, including increased transitions, rearings, and holepokes, and reduced spatial d, vs. WT littermates. Our results demonstrate that the DAT KD mouse line in the BPM is a consistent, reproducible model of mania-relevant behaviors. More work must be done to assess reproducibility of behavioral outcomes across experiments in order to advance the field of psychiatry and develop more effective therapeutics for patients.


Assuntos
Comportamento Animal/fisiologia , Transtorno Bipolar/fisiopatologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Técnicas de Silenciamento de Genes , Animais , Antimaníacos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/psicologia , Estudos de Coortes , Agonistas de Dopamina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Quinolonas/uso terapêutico , Reprodutibilidade dos Testes , Tiofenos/uso terapêutico , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Ácido Valproico/uso terapêutico , alfa-Metiltirosina/farmacologia , alfa-Metiltirosina/uso terapêutico
4.
Mol Neurobiol ; 56(4): 2728-2740, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30056575

RESUMO

Reduced movement frequency or physical activity (bradykinesia) occurs with high prevalence in the elderly. However, loss of striatal tyrosine hydroxylase (TH) in aging humans, non-human primates, or rodents does not reach the ~ 80% loss threshold associated with bradykinesia onset in Parkinson's disease. Moderate striatal dopamine (DA) loss, either following TH inhibition or decreased TH expression, may not affect movement frequency. In contrast, moderate DA or TH loss in the substantia nigra (SN), as occurs in aging, is of similar magnitude (~ 40%) to nigral TH loss at bradykinesia onset in Parkinson's disease. In aged rats, increased TH expression and DA in SN alone increases movement frequency, suggesting aging-related TH and DA loss in the SN contributes to aging-related bradykinesia or decreased physical activity. To test this hypothesis, the SN was targeted with bilateral guide cannula in young (6 months old) rats, in a within-subjects design, to evaluate the impact of nigral TH inhibition on movement frequency and speed. The TH inhibitor, α-methyl-p-tyrosine (AMPT) reduced nigral DA (~ 40%) 45-150 min following infusion, without affecting DA in striatum, nucleus accumbens, or adjacent ventral tegmental area. Locomotor activity in the open-field was recorded up to 3 h following nigral saline or AMPT infusion in each test subject. During the period of nigra-specific DA reduction, movement frequency, but not movement speed, was significantly decreased. These results indicate that DA or TH loss in the SN, as observed in aging, contributes as a central mechanism of reduced movement frequency.


Assuntos
Movimento , Substância Negra/enzimologia , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Animais , Cateteres , Dopamina/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Movimento/efeitos dos fármacos , Ratos Endogâmicos BN , Reprodutibilidade dos Testes , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Metiltirosina/farmacologia
5.
Invest Ophthalmol Vis Sci ; 59(11): 4631-4638, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30372732

RESUMO

Purpose: To determine if myopia in albino guinea pigs is linked to altered ocular dopamine (DA) levels in both the retinal and uveal dopaminergic systems. Methods: Retina and retinal pigment epithelium (RPE)/choroid were dissected from eyes of 2-week-old albino myopic (AM) and pigmented hyperopic (PH) guinea pigs. The levels of DA, dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) were determined. Tyrosine hydroxylase (TH) and tyrosinase activities were also measured. PH animals received daily unilateral peribulbar injections of either kojic acid (tyrosinase inhibitor) or vehicle for 2 to 4 weeks. Refractive errors and ocular axial dimensions were measured by eccentric infrared photoretinoscopy and A-scan ultrasonography. Results: Retinal DA levels were similar between the two strains, but AM eyes had higher levels of DOPAC. RPE/choroid DA and tyrosinase activity in AM eyes were lower than in PH eyes (P < 0.01); however, the DA turnover was higher (P < 0.05). After 2 weeks of kojic acid treatment, PH eyes developed significant myopia, accompanied by elongated vitreous chambers and axial lengths. Inhibition of tyrosinase activity was linearly correlated with a myopic refraction shift (R = 0.79, P < 0.01). PH animals that received 62.5 ng/mL kojic acid treatment daily for 2 weeks followed by 625 ng/mL for 2 more weeks became more myopic and had deeper anterior chambers compared to those that received the 62.5 ng/mL dose over this period (P = 0.04). Conclusions: The uveal tyrosinase-dependent dopaminergic system is involved in the development of guinea pig refraction. Enhancing uveal tyrosinase activity might slow down the development of myopia.


Assuntos
Dopamina/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Erros de Refração/metabolismo , Retina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Antioxidantes/farmacologia , Biometria , Western Blotting , Cromatografia Líquida de Alta Pressão , Cobaias , Ácido Homovanílico/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Pironas/farmacologia , Retinoscopia , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Ultrassonografia
6.
Int J Mol Sci ; 19(8)2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042336

RESUMO

Caffeic acid derivatives represent promising lead compounds in the search for tyrosinase inhibitors to be used in the treatment of skin local hyperpigmentation associated to an overproduction or accumulation of melanin. We recently reported the marked inhibitory activity of a conjugate of caffeic acid with dihydrolipoic acid, 2-S-lipoylcaffeic acid (LCA), on the tyrosine hydroxylase (TH) and dopa oxidase (DO) activities of mushroom tyrosinase. In the present study, we evaluated a more lipophilic derivative, 2-S-lipoyl caffeic acid methyl ester (LCAME), as an inhibitor of tyrosinase from human melanoma cells. Preliminary analysis of the effects of LCAME on mushroom tyrosinase indicated more potent inhibitory effects on either enzyme activities (IC50 = 0.05 ± 0.01 µM for DO and 0.83 ± 0.09 µM for TH) compared with LCA and the reference compound kojic acid. The inhibition of DO of human tyrosinase was effective (Ki = 34.7 ± 1.1 µM) as well, while the action on TH was weaker. Lineweaver⁻Burk analyses indicated a competitive inhibitor mechanism. LCAME was not substrate of tyrosinase and proved nontoxic at concentrations up to 50 µM. No alteration of basal tyrosinase expression was observed after 24 h treatment of human melanoma cells with the inhibitor, but preliminary evidence suggested LCAME might impair the induction of tyrosinase expression in cells stimulated with α-melanocyte-stimulating hormone. All these data point to this compound as a valuable candidate for further trials toward its use as a skin depigmenting agent. They also highlight the differential effects of tyrosinase inhibitors on the human and mushroom enzymes.


Assuntos
Ácidos Cafeicos/química , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Preparações Clareadoras de Pele/farmacologia , Ácido Tióctico/análogos & derivados , Agaricales/enzimologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Melaninas/metabolismo , Melanoma/enzimologia , Pironas/farmacologia , Preparações Clareadoras de Pele/química , Ácido Tióctico/química , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores
7.
Biochem Biophys Res Commun ; 502(4): 435-441, 2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-29856999

RESUMO

Methylmercury (MeHg) is the causative substance of Minamata disease, which is associated with various neurological disorders such as sensory disturbance and ataxia. It has been suggested low-level dietary intake of MeHg from MeHg-containing fish during gestation adversely affects the fetus. In our study, we investigated the toxicological effects of MeHg exposure on neuronal differentiation focusing on epigenetics. We used human fetal brain-derived immortalized cells (LUHMES cells) as a human neuronal differentiation model. Cell viability, neuronal, and catecholamine markers in LUHMES cells were assessed after exposure to MeHg (0-1000 nM) for 6 days (from day 2 to day 8 of neuronal differentiation). Cell viability on day 8 was not affected by exposure to 1 nM MeHg for 6 days. mRNA levels of AADC, DBH, TUJ1, and SYN1 also were unaffected by MeHg exposure. In contrast, levels of TH, the rate-limiting enzyme for dopamine synthesis, were significantly decreased after MeHg exposure. Acetylated histone H3, acetylated histone H3 lysine 9, and tri-methyl histone H3 lysine 9 levels at the TH gene promoter were not altered by MeHg exposure. However, tri-methylation of histone H3 lysine 27 levels, related to transcriptional repression, were significantly increased at the TH gene promotor after MeHg exposure. In summary, MeHg exposure during neuronal differentiation led to epigenetic changes that repressed TH gene expression. This study provides useful insights into the toxicological mechanisms underlying the effects of developmental MeHg exposure during neuronal differentiation.


Assuntos
Epigênese Genética/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Neurônios/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genética , Animais , Diferenciação Celular , Linhagem Celular , Feminino , Peixes , Contaminação de Alimentos , Humanos , Intoxicação do Sistema Nervoso por Mercúrio/etiologia , Intoxicação do Sistema Nervoso por Mercúrio/genética , Intoxicação do Sistema Nervoso por Mercúrio/patologia , Metilação , Modelos Neurológicos , Neurônios/citologia , Neurônios/enzimologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Regiões Promotoras Genéticas , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores
8.
Mol Neurobiol ; 55(3): 2443-2453, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28365874

RESUMO

Developmental vitamin D (DVD) deficiency has been proposed as an important risk factor for schizophrenia. Our previous study using Sprague Dawley rats found that DVD deficiency disrupted the ontogeny of mesencephalic dopamine neurons by decreasing the mRNA level of a crucial differentiation factor of dopamine cells, the nuclear receptor related 1 protein (Nurr1). However, it remains unknown whether this reflects a reduction in dopamine cell number or in Nurr1 expression. It is also unclear if any particular subset of developing dopamine neurons in the mesencephalon is selectively affected. In this study, we employed state-of-the-art spinning disk confocal microscopy optimized for the imaging of tissue sections and 3D segmentation to assess post-mitotic dopamine cells on a single-cell basis in the rat mesencephalon at embryonic day 15. Our results showed that DVD deficiency did not alter the number, morphology, or positioning of post-mitotic dopamine cells. However, the ratio of Nurr1+TH+ cells in the substantia nigra pars compacta (SNc) compared with the ventral tegmental area (VTA) was increased in DVD-deficient embryos. In addition, the expression of Nurr1 in immature dopamine cells and mature dopamine neurons in the VTA was decreased in DVD-deficient group. Tyrosine hydroxylase was selectively reduced in SNc of DVD-deficient mesencephalon. We conclude that DVD deficiency induced early alterations in mesencephalic dopamine development may in part explain the abnormal dopamine-related behaviors found in this model. Our findings may have broader implications for how certain environmental risk factors for schizophrenia may shape the ontogeny of dopaminergic systems and by inference increase the risk of schizophrenia.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/metabolismo , Mitose/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Tirosina 3-Mono-Oxigenase/biossíntese , Deficiência de Vitamina D/metabolismo , Animais , Neurônios Dopaminérgicos/patologia , Expressão Gênica , Mesencéfalo/patologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/genética , Deficiência de Vitamina D/genética , Deficiência de Vitamina D/patologia
9.
Gen Comp Endocrinol ; 252: 236-238, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716505

RESUMO

In this article, we show that the tyrosine hydroxylase inhibitor α-Methyl-l-tyrosine (AMPT) decreased the responsiveness of the zebrafish stress axis to an acute stressful challenge. These effects were specific for responses to stimulation, since unstimulated (basal) cortisol levels were not altered by AMPT. Moreover, AMPT decreased the stress response 15min after stimulation, but not after that time period. To our knowledge, this is the first report about the effects of AMPT on the neuroendocrine axis of adult zebrafish in acute stress responses. Overall, these results suggest a mechanism of catecholamine-glucocorticoid interplay in neuroendocrine responses of fish, pointing an interesting avenue for physiological research, as well as an important endpoint that can be disrupted by environmental contamination. Further experiments will unravel the mechanisms by which AMPT blocked the cortisol response.


Assuntos
Inibidores Enzimáticos/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Peixe-Zebra/fisiologia , alfa-Metiltirosina/farmacologia , Animais , Feminino , Hidrocortisona/sangue , Masculino , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra/sangue
10.
ACS Chem Neurosci ; 8(9): 1880-1888, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28617576

RESUMO

Zebrafish (Danio rerio) have recently emerged as useful model organism for the study of neuronal function. Here, fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to measure locally evoked dopamine release and uptake in zebrafish whole brain preparations and results were compared with those obtained from brain slices. Evoked dopamine release ([DA]max) was similar in whole brain and sagittal brain slice preparations (0.49 ± 0.13 µM in whole brain and 0.59 ± 0.28 µM in brain slices). Treatment with α-methyl-p-tyrosine methyl ester (αMPT), an inhibitor of tyrosine hydroxylase, diminished release and the electrochemical signal reappeared after subsequent drug washout. No observed change in stimulated release current occurred after treatment with desipramine or fluoxetine in the whole brain. Treatment with the uptake inhibitors, nomifensine or GBR 12909 increased [DA]max, while treatment with sulpiride, a D2 dopamine autoreceptor antagonist, resulted in increased stimulated dopamine release in whole brain, but had no effect on release in slices. Dopamine release in whole brains increased progressively up to an electrical stimulation frequency of 25 Hz, while release in slices increased up to a frequency of only 10 Hz and then plateaued, highlighting another key difference between these preparations. We observed a lag in peak dopamine release following stimulation, which we address using diffusion models and pharmacological treatments. Collectively, these results demonstrate the electrochemical determination of dopamine release in the whole, intact brain of a vertebrate species ex vivo and are an important step for carrying out further experiments in zebrafish.


Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Estimulação Elétrica , Microeletrodos , Técnicas de Cultura de Tecidos , Animais , Autorreceptores/antagonistas & inibidores , Autorreceptores/metabolismo , Encéfalo/efeitos dos fármacos , Difusão , Antagonistas dos Receptores de Dopamina D2/farmacologia , Estimulação Elétrica/instrumentação , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Modelos Neurológicos , Inibidores da Captação de Neurotransmissores/farmacologia , Receptores de Dopamina D2/metabolismo , Técnicas de Cultura de Tecidos/métodos , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo , Peixe-Zebra
11.
Brain Res ; 1657: 253-261, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28041945

RESUMO

Leucine-rich repeat kinase 2 (LRRK2) has been linked to familial and sporadic Parkinson's disease. However, it is still unresolved whether LRRK2 in dopaminergic (DAergic) neurons may or may not aggravate the phenotype. We demonstrate that knocking down (KD) the Lrrk gene by RNAi in DAergic neurons untreated or treated with paraquat (PQ) neither affected the number of DAergic clusters, tyrosine hydroxylase (TH) protein levels, lifespan nor locomotor activity when compared to control (i.e. TH/+) flies. KD transgenic Lrrk flies dramatically increased locomotor activity in presence of TH enzyme inhibitor alpha-methyl-para-tyrosine (aMT), whereas no effect on lifespan was observed in both fly lines. Most importantly, KD Lrrk flies had reduced lipid peroxidation (LPO) index alone or in presence of PQ and the antioxidant minocycline (MC, 0.5mM). Taken together, these findings suggest that Lrrk appears unessential for the viability of DAergic neurons in D. melanogaster. Moreover, Lrrk might negatively regulate homeostatic levels of dopamine, thereby dramatically increasing locomotor activity, extending lifespan, and reducing oxidative stress (OS). Our data also indicate that reduced expression of Lrrk in the DAergic neurons of transgenic TH>Lrrk-RNAi/+ flies conferred PQ resistance and absence of neurodegeneration. The present findings support the notion that reduced/suppressed LRRK2 expression might delay or prevent motor symptoms and/or frank Parkinsonism in individuals at risk to suffer autosomal dominant Parkinsonism (AD-P) by blocking OS-induced neurodegenerative processes in the DAergic neurons.


Assuntos
Proteínas de Drosophila/antagonistas & inibidores , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Minociclina/farmacologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Paraquat , Transtornos Parkinsonianos/terapia , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Terapêutica com RNAi , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
12.
Expert Opin Ther Targets ; 21(2): 167-180, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27973928

RESUMO

INTRODUCTION: The ancient and ubiquitous monoamine signalling molecules serotonin, dopamine, norepinephrine, and epinephrine are involved in multiple physiological functions. The aromatic amino acid hydroxylases tyrosine hydroxylase (TH), tryptophan hydroxylase 1 (TPH1), and tryptophan hydroxylase 2 (TPH2) catalyse the rate-limiting steps in the biosynthesis of these monoamines. Genetic variants of TH, TPH1, and TPH2 genes are associated with neuropsychiatric disorders. The interest in these enzymes as therapeutic targets is increasing as new roles of these monoamines have been discovered, not only in brain function and disease, but also in development, cardiovascular function, energy and bone homeostasis, gastrointestinal motility, hemostasis, and liver function. Areas covered: Physiological roles of TH, TPH1, and TPH2. Enzyme structures, catalytic and regulatory mechanisms, animal models, and associated diseases. Interactions with inhibitors, pharmacological chaperones, and regulatory proteins relevant for drug development. Expert opinion: Established inhibitors of these enzymes mainly target their amino acid substrate binding site, while tetrahydrobiopterin analogues, iron chelators, and allosteric ligands are less studied. New insights into monoamine biology and 3D-structural information and new computational/experimental tools have triggered the development of a new generation of more selective inhibitors and pharmacological chaperones. The enzyme complexes with their regulatory 14-3-3 proteins are also emerging as therapeutic targets.


Assuntos
Desenho de Fármacos , Triptofano Hidroxilase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Proteínas 14-3-3/metabolismo , Animais , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Variação Genética , Humanos , Ligantes , Chaperonas Moleculares/farmacologia , Terapia de Alvo Molecular , Triptofano Hidroxilase/genética , Tirosina 3-Mono-Oxigenase/genética
13.
eNeuro ; 3(6)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27957531

RESUMO

Normally, rapid eye movement sleep (REMS) does not appear during waking or non-REMS. Isolated, independent studies showed that elevated noradrenaline (NA) levels inhibit REMS and induce REMS loss-associated cytomolecular, cytomorphological, psychosomatic changes and associated symptoms. However, the source of NA and its target in the brain for REMS regulation and function in health and diseases remained to be confirmed in vivo. Using tyrosine hydroxylase (TH)-siRNA and virus-coated TH-shRNA in normal freely moving rats, we downregulated NA synthesis in locus coeruleus (LC) REM-OFF neurons in vivo. These TH-downregulated rats showed increased REMS, which was prevented by infusing NA into the pedunculo-pontine tegmentum (PPT), the site of REM-ON neurons, normal REMS returned after recovery. Moreover, unlike normal or control-siRNA- or shRNA-injected rats, upon REMS deprivation (REMSD) TH-downregulated rat brains did not show elevated Na-K ATPase (molecular changes) expression and activity. To the best of our knowledge, these are the first in vivo findings in an animal model confirming that NA from the LC REM-OFF neurons (1) acts on the PPT REM-ON neurons to prevent appearance of REMS, and (2) are responsible for inducing REMSD-associated molecular changes and symptoms. These observations clearly show neuro-physio-chemical mechanism of why normally REMS does not appear during waking. Also, that LC neurons are the primary source of NA, which in turn causes some, if not many, REMSD-associated symptoms and behavioral changes. The findings are proof-of-principle for the first time and hold potential to be exploited for confirmation toward treating REMS disorder and amelioration of REMS loss-associated symptoms in patients.


Assuntos
Locus Cerúleo/metabolismo , Neurônios/metabolismo , Norepinefrina/metabolismo , Núcleo Tegmental Pedunculopontino/metabolismo , Tegmento Pontino/metabolismo , Sono REM/fisiologia , Animais , Masculino , Neurônios/patologia , Núcleo Tegmental Pedunculopontino/patologia , Tegmento Pontino/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos Wistar , Privação do Sono/metabolismo , Privação do Sono/patologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
14.
Drug Des Devel Ther ; 10: 3947-3957, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27980392

RESUMO

Hyperpigmentation caused by melanin overproduction is a major skin disorder in humans. Inhibition of tyrosinase, a key regulator of melanin production, has been used as an effective strategy to treat hyperpigmentation. In this study, we investigated the use of solid lipid nanoparticles (SLNs) as a highly effective and nontoxic means to deliver a newly synthesized potent tyrosinase inhibitor, MHY498, and to target melanocytes through the skin. MHY498-loaded SLNs (MHY-SLNs) were prepared by an oil-in-water emulsion solvent-evaporation method, and their morphological and physicochemical properties were characterized. MHY-SLNs showed a prolonged drug-release profile and higher skin permeation than that of MHY solution. In an in vivo evaluation of antimelanogenic activity, MHY-SLNs showed a prominent inhibitory effect against ultraviolet B-induced melanogenesis, resulting in no change in the skin color of C57BL/6 mouse, compared with that observed in an MHY solution-treated group and an untreated control group. The antimelanogenic effect of MHY-SLNs was further confirmed through Fontana-Masson staining. Importantly, MHY-SLNs did not induce any toxic effects in the L929 cell line. Overall, these data indicate that MHY-SLNs show promise in the topical treatment of hyperpigmentation.


Assuntos
Portadores de Fármacos , Inibidores Enzimáticos/farmacologia , Lipídeos/química , Melaninas/metabolismo , Nanopartículas , Pigmentação da Pele/efeitos dos fármacos , Pele/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Administração Cutânea , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Composição de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Cinética , Camundongos Endogâmicos C57BL , Nanotecnologia , Permeabilidade , Pele/enzimologia , Pele/efeitos da radiação , Absorção Cutânea , Solubilidade , Tecnologia Farmacêutica/métodos , Tiazolidinedionas/administração & dosagem , Tiazolidinedionas/química , Tiazolidinedionas/toxicidade , Tirosina 3-Mono-Oxigenase/metabolismo
15.
Int J Mol Sci ; 17(11)2016 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-27879654

RESUMO

Prescription of antipsychotic drugs (APDs) to children has substantially increased in recent years. Whilst current investigations into potential long-term effects have uncovered some alterations to adult behaviours, further investigations into potential changes to neurotransmitter systems are required. The current study investigated potential long-term changes to the adult dopamine (DA) system following aripiprazole, olanzapine and risperidone treatment in female and male juvenile rats. Levels of tyrosine hydroxylase (TH), phosphorylated-TH (p-TH), dopamine active transporter (DAT), and D1 and D2 receptors were measured via Western blot and/or receptor autoradiography. Aripiprazole decreased TH and D1 receptor levels in the ventral tegmental area (VTA) and p-TH levels in the prefrontal cortex (PFC) of females, whilst TH levels decreased in the PFC of males. Olanzapine decreased PFC p-TH levels and increased D2 receptor expression in the PFC and nucleus accumbens (NAc) in females only. Additionally, risperidone treatment increased D1 receptor levels in the hippocampus of females, whilst, in males, p-TH levels increased in the PFC and hippocampus, D1 receptor expression decreased in the NAc, and DAT levels decreased in the caudate putamen (CPu), and elevated in the VTA. These results suggest that early treatment with various APDs can cause different long-term alterations in the adult brain, across both treatment groups and genders.


Assuntos
Antipsicóticos/efeitos adversos , Aripiprazol/efeitos adversos , Benzodiazepinas/efeitos adversos , Dopamina/metabolismo , Risperidona/efeitos adversos , Transmissão Sináptica/efeitos dos fármacos , Animais , Antipsicóticos/administração & dosagem , Aripiprazol/administração & dosagem , Benzodiazepinas/administração & dosagem , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Regulação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Olanzapina , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Putamen/efeitos dos fármacos , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Risperidona/administração & dosagem , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo
16.
J Pharmacol Exp Ther ; 358(3): 528-36, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27405316

RESUMO

Our objective was to study hypertension induced by chronic administration of synthetic glucocorticoid, dexamethasone (DEX), under nonstressful conditions and examine the role of catecholamine biosynthesis. To achieve this, we did the following: 1) used radiotelemetry to record mean arterial pressure (MAP) and heart rate (HR) in freely moving rats, and 2) administered different doses of DEX in drinking water. To evaluate the involvement of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine biosynthesis, we treated rats with the TH inhibitor, α-methyl-para-tyrosine (α-MPT), for 3 days prior to administration of DEX and assessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenal medulla. We observed a dose-dependent elevation in blood pressure with a DEX dose of 0.3 mg/kg administered for 10 days, significantly increasing MAP by +15.0 ± 1.1 mm Hg, while concomitantly reducing HR. Although this DEX treatment also significantly decreased body weight, pair-fed animals that showed similar decreases in body weight due to lowered food intake were not hypertensive, suggesting that body weight changes may not account for DEX-induced hypertension. Chronic DEX treatment significantly increased the TH mRNA and protein levels in the adrenal medulla, and α-MPT administration not only reduced DEX pressor effects, but also inhibited TH (serine(40)) phosphorylation. Our study thus validates a novel model to study hypertension induced by chronic intake of DEX in freely moving rats not subject to the confounding factors of previous models and establishes its dependence on concomitant activation of peripheral catecholamine biosynthesis.


Assuntos
Dexametasona/farmacologia , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo , Medula Suprarrenal/efeitos dos fármacos , Medula Suprarrenal/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Serina/metabolismo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/genética , alfa-Metiltirosina/farmacologia
17.
Sci Rep ; 6: 30390, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27462005

RESUMO

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.


Assuntos
Tirosina 3-Mono-Oxigenase/química , Domínio Catalítico , Dopamina/farmacologia , Estabilidade Enzimática , Humanos , Ligação Proteica , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
18.
Toxicol Lett ; 258: 36-45, 2016 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-27313094

RESUMO

Inflammatory responses are involved in mechanisms of neuronal cell damage in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). We investigated the mechanisms whereby inflammatory responses contribute to loss of dopaminergic neurons in fipronil (FPN)-treated rats. After stereotaxic injection of FPN in the substantia nigra (SN), the number of tyrosine hydroxylase (TH)-positive neurons and the levels of TH expression in the SN decreased at 7days, and a significant decrease was observed at 14days with a subsequent reduction in striatal TH expression. Decreases in dopamine (DA) levels, however, began at 3days post-injection, preceding the changes in TH expression. In contrast, glial fibrillary acidic protein (GFAP) expression was significantly increased at 3days and persisted for up to 14days post-lesion; these changes in GFAP expression appeared to be inversely correlated with TH expression. Furthermore, we found that FPN administration induced an inflammatory response characterized by increased levels of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α), which was mediated by activated microglia following infusion of FPN unilaterally into the SN. Intranigral injection of FPN underwent an inflammatory response with a resultant ongoing loss of dopaminergic neurons, indicating that pesticides may have important implication for the study of PD.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Encefalite/etiologia , Síndromes Neurotóxicas/patologia , Praguicidas/toxicidade , Pirazóis/toxicidade , Degeneração Estriatonigral/etiologia , Substância Negra/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Progressão da Doença , Dopamina/química , Dopamina/metabolismo , Neurônios Dopaminérgicos/imunologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Proteína Glial Fibrilar Ácida/agonistas , Proteína Glial Fibrilar Ácida/metabolismo , Injeções Intraventriculares , Masculino , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Pirazóis/administração & dosagem , Distribuição Aleatória , Ratos Sprague-Dawley , Técnicas Estereotáxicas , Substância Negra/imunologia , Substância Negra/metabolismo , Substância Negra/patologia , Fator de Necrose Tumoral alfa/agonistas , Fator de Necrose Tumoral alfa/metabolismo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/metabolismo
19.
Pain ; 156(12): 2595-2606, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26447701

RESUMO

Physical exercise is a low-cost, safe, and efficient intervention for the reduction of neuropathic chronic pain in humans. However, the underlying mechanisms for how exercise reduces neuropathic pain are not yet well understood. Central monoaminergic systems play a critical role in endogenous analgesia leading us to hypothesize that the analgesic effect of low-intensity exercise occurs through activation of monoaminergic neurotransmission in descending inhibitory systems. To test this hypothesis, we induced peripheral nerve injury (PNI) by crushing the sciatic nerve. The exercise intervention consisted of low-intensity treadmill running for 2 weeks immediately after injury. Animals with PNI showed an increase in pain-like behaviors that were reduced by treadmill running. Reduction of serotonin (5-hydroxytryptamine) synthesis using the tryptophan hydroxylase inhibitor para-chlorophenylalanine methyl ester prevented the analgesic effect of exercise. However, blockade catecholamine synthesis with the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine had no effect. In parallel, 2 weeks of exercise increased brainstem levels of the 5-HT and its metabolites (5-hydroxyindoleacetic acid), decreased expression of the serotonin transporter, and increased expression of 5-HT receptors (5HT-1B, 2A, 2C). Finally, PNI-induced increase in inflammatory cytokines, tumor necrosis factor-alpha, and interleukin-1 beta, in the brainstem, was reversed by 2 weeks of exercise. These findings provide new evidence indicating that low-intensity aerobic treadmill exercise suppresses pain-like behaviors in animals with neuropathic pain by enhancing brainstem 5-HT neurotransmission. These data provide a rationale for the analgesia produced by exercise to provide an alternative approach to the treatment of chronic neuropathic pain.


Assuntos
Comportamento Animal/fisiologia , Tronco Encefálico/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Condicionamento Físico Animal , Receptores de Serotonina/metabolismo , Nervo Isquiático/lesões , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fenclonina/análogos & derivados , Fenclonina/farmacologia , Interleucina-1beta/metabolismo , Camundongos , Triptofano Hidroxilase/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , alfa-Metiltirosina/farmacologia
20.
Can J Anaesth ; 62(12): 1303-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26362800

RESUMO

PURPOSE: Pheochromocytomas (PHEOS) are rare catecholamine-secreting adrenal tumours requiring surgical resection. Preoperative alpha-adrenergic receptor blockade to prevent intraoperative hypertension has traditionally been achieved with phenoxybenzamine. Due to changes in the availability of phenoxybenzamine in Canada, alternate therapies are needed for patients. We report our first experience using metyrosine, a tyrosine hydroxylase inhibitor, for preoperative management in a symptomatic patient with a unilateral PHEO. CLINICAL FEATURES: A 50-yr-old male was referred to our centre with a history of symptoms suggestive of a catecholamine-secreting PHEO, including tachycardia, diaphoresis, nervousness, and tremor. Computerized tomography revealed a right adrenal mass, and additional positive imaging and elevated urine epinephrine levels supported a diagnosis of PHEO. The patient was admitted to hospital five days prior to surgery, and metyrosine therapy was initiated and titrated to 4 g daily over four days. Despite adequate blood pressure (BP) control leading up to the resection, the initial BP reading in the operating room was 191/106 mmHg, but it subsequently declined and was well controlled during induction (100-110 mmHg systolic BP). Significant hypertension (up to 201/110 mmHg) developed upon tumour manipulation and resolved with phentolamine administration and surgical isolation of the tumour. The patient's BP remained stable throughout the residual part of the procedure and in the recovery room and step-down unit. CONCLUSION: In the case of this patient's PHEO, the use of metyrosine was unsatisfactory in achieving sufficient inhibition of catecholamine synthesis as evidenced by significant intraoperative hypertension. Metyrosine could have a role in preoperative management of these patients, but it may not be optimal as monotherapy for some patients with actively secreting tumours.


Assuntos
Inibidores Enzimáticos/uso terapêutico , Feocromocitoma/cirurgia , alfa-Metiltirosina/uso terapêutico , Catecolaminas/metabolismo , Humanos , Hipertensão/etiologia , Complicações Intraoperatórias/prevenção & controle , Masculino , Pessoa de Meia-Idade , Fentolamina/uso terapêutico , Feocromocitoma/fisiopatologia , Cuidados Pré-Operatórios/métodos , Falha de Tratamento , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores
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