RESUMO
Previous studies have consistently demonstrated that the amphetamine-related drug 3,4-methylenedioxymethamphetamine (MDMA) induces dopaminergic damage in the mouse brain, and that this effect is most marked in the nigrostriatal system. Moreover, it has been suggested that the overproduction of nitric oxide (NO) may participate in the dopaminergic damage induced by MDMA. To further elucidate this issue, we evaluated the levels of the enzyme nitric oxide synthase (nNOS), which catalyzes the production of NO, in mice treated with regimens of MDMA that induce progressive and persistent neurotoxicity in the dopaminergic nigrostriatal system. Mice received 14, 28, or 36 administrations of MDMA (10 mg/kg i.p.), twice a day/twice a week, and were sacrificed at different time-points after treatment discontinuation. Thereafter, the number of nNOS-positive neurons was quantified by immunohistochemistry in the caudate-putamen (CPu) and substantia nigra pars compacta (SNc). MDMA elevated the numbers of nNOS-positive neurons in the CPu of mice that received 28 or 36 drug administrations. This effect was still detectable at 3 months after treatment discontinuation. Moreover, MDMA elevated the numbers of nNOS-positive neurons in the SNc. However, this effect occurred only in mice that received 28 drug administrations and were sacrificed 3 days after treatment discontinuation. These results are in line with the hypothesis that activation of the NO cascade participates in the toxic effects induced by MDMA in the dopaminergic nigrostriatal system. Moreover, they suggest that activation of the NO cascade induces toxic effects that are more marked in striatal terminals, compared with nigral neurons.
Assuntos
Núcleo Caudado/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Óxido Nítrico Sintase Tipo I/metabolismo , Parte Compacta da Substância Negra/efeitos dos fármacos , Putamen/efeitos dos fármacos , Animais , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Parte Compacta da Substância Negra/enzimologia , Parte Compacta da Substância Negra/patologia , Putamen/enzimologia , Putamen/patologia , Distribuição AleatóriaRESUMO
We studied activity of lysosomal cysteine proteases, cathepsins B and L, in brain structures (frontal cortex, caudate nucleus, hippocampus, and hypothalamus) of C57Bl/6J mice with aggressive and depressive-like behavior formed under conditions of chronic social stress (repeated experience of victories and defeats within 20 days). Mice with depressive-like behavior showed increased activity of cathepsin Ð in the hypothalamus and nucleus caudatus and increased activity of cathepsin L in the hippocampus compared to control animals not subjected to agonistic confrontations. In mice with aggressive behavior, protease activity in the studied brain structures was not changed. In 4 h after immune system activation with LPS (250 µg/kg), cathepsin L activity in the hippocampus of control mice increased in comparison with mice receiving saline. In contrast to control animals, LPS caused a decrease in activity of the enzyme in the caudate nucleus and frontal cortex of aggressive mice and in the hippocampus of mice with depressive-like behavior.
Assuntos
Agressão/psicologia , Comportamento Agonístico , Catepsina B/metabolismo , Catepsina L/metabolismo , Depressão/enzimologia , Estresse Psicológico/enzimologia , Animais , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Núcleo Caudado/imunologia , Núcleo Caudado/fisiopatologia , Depressão/imunologia , Depressão/fisiopatologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/enzimologia , Lobo Frontal/imunologia , Lobo Frontal/fisiopatologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/imunologia , Hipocampo/fisiopatologia , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Hipotálamo/imunologia , Hipotálamo/fisiopatologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Estresse Psicológico/imunologia , Estresse Psicológico/fisiopatologiaRESUMO
Nicotine exerts its addictive influence through the meso-cortico-limbic reward system, where the striatum is essential. Nicotine addiction involves different neurotransmitters, nitric oxide (NO) being especially important, since it triggers the release of the others by positive feedback. In the nervous system, NO is mainly produced by nitric oxide synthase 1 (NOS1). However, other subtypes of synthases can also synthesize NO, and little is known about the specific role of each isoform in the process of addiction. In parallel, NOS activity and nicotine addiction are also affected by stress and sexual dimorphism. To determine the specific role of this enzyme, we analyzed both NOS expression and NO synthesis in the striatum of wild-type and NOS1-knocked out (KO) mice of both sexes in situations of nicotine sensitization and stress. Our results demonstrated differences between the caudate-putamen (CP) and nucleus accumbens (NA). With respect to NOS1 expression, the CP is a dimorphic region (27.5% lower cell density in males), but with a stable production of NO, exclusively due to this isoform. Thus, the nitrergic system of CP may not be involved in stress or nicotine addiction. Conversely, the NA is much more variable and strongly involved in both situations: its NO synthesis displays dimorphic variations at both basal (68.5% reduction in females) and stress levels (65.9% reduction in males), which disappear when nicotine is infused. Thus, the KO animals showed an increase in NO production (21.7%) in the NA, probably by NOS3, in an attempt to compensate the lack of NOS1.
Assuntos
Núcleo Caudado/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Núcleo Accumbens/enzimologia , Putamen/enzimologia , Estresse Psicológico/enzimologia , Tabagismo/enzimologia , Animais , Núcleo Caudado/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Isoenzimas/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I/genética , Núcleo Accumbens/efeitos dos fármacos , Putamen/efeitos dos fármacos , Caracteres SexuaisRESUMO
Acetylcholinesterase activity was quantitatively evaluated by cytochemical method in brain structures (layers III and V of the sensorimotor cortex, caudate nucleus, nucleus accumbens, hippocampus CA3 field) of August and Wistar rats demonstrating high and low motor activity in the open field test. In August rats, acetylcholinesterase activity in the analyzed brain structures prevailed in animals with high motor activity in comparison with rats with low motor activity. In Wistar rats, the differences between the animals demonstrating high and low motor activity were less pronounced, but varied depending on the experimental series of studies. Comparisons of August rats with low motor activity and Wistar rats with high motor activity (maximum difference of motor function in these animals) revealed significant excess of acetylcholinesterase activity in layer III of the sensorimotor cortex in August rats and no differences in other brain structures of the examined animals.
Assuntos
Acetilcolinesterase/metabolismo , Núcleo Caudado/enzimologia , Hipocampo/enzimologia , Atividade Motora/fisiologia , Núcleo Accumbens/enzimologia , Córtex Sensório-Motor/enzimologia , Animais , Química Encefálica , Núcleo Caudado/química , Núcleo Caudado/fisiologia , Hipocampo/química , Hipocampo/fisiologia , Masculino , Núcleo Accumbens/química , Núcleo Accumbens/fisiologia , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Ratos Wistar , Córtex Sensório-Motor/química , Córtex Sensório-Motor/fisiologia , Especificidade da EspécieRESUMO
Adenosine to inosine (A-to-I) RNA editing is a base recoding process within precursor messenger RNA, catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family. A notable example occurs at the Q/R site of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor subunit GluA2. Abnormally, low editing at this site leads to excessive calcium influx and cell death. We studied hippocampus and caudate samples from Alzheimer's disease (AD) patients and age-matched healthy controls, using direct sequencing and a high accuracy primer-extension technique to assess RNA editing at the Q/R GluA2 site. Both techniques revealed lower, more variable RNA editing in AD, specific to the hippocampus and the GluA2 site. Deficient editing also characterized the hippocampus of apolipoprotein ε4 allele carriers, regardless of clinical diagnosis. In AD, messenger RNA expression of neuronal markers was decreased in the hippocampus, and expression of the Q/R-site editing enzyme ADAR2 was decreased in caudate. These findings provide a link between neurodegenerative processes and deficient RNA editing of the GluA2 Q/R site, and may contribute to both diagnosis and treatment of AD.
Assuntos
Doença de Alzheimer/genética , Hipocampo/metabolismo , Hipocampo/patologia , Edição de RNA/genética , RNA Mensageiro/genética , Receptores de AMPA/genética , Adenosina Desaminase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Apolipoproteína E4/genética , Cálcio/metabolismo , Núcleo Caudado/enzimologia , Morte Celular , Feminino , Humanos , Masculino , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.
Assuntos
Corpo Estriado/enzimologia , Dependovirus/genética , Dopa Descarboxilase/genética , Técnicas de Transferência de Genes , Animais , Cateterismo , Núcleo Caudado/enzimologia , Dopa Descarboxilase/metabolismo , Feminino , Humanos , Macaca mulatta , Imageamento por Ressonância Magnética , Neurônios/enzimologia , Neurônios/patologia , Putamen/enzimologia , Putamen/patologia , Técnicas Estereotáxicas , TransgenesRESUMO
The Leucine Rich Repeat Kinase-2 (LRRK2) gene is a common mutation target in Parkinson's disease (PD), but the cellular mechanisms by which such mutations underlie the pathophysiology of PD remain poorly understood. Thus, to better characterize the neuronal target sites of LRRK2 mutations in the primate brain, we studied the cellular and ultrastructural localization of Lrrk2 immunoreactivity in the monkey basal ganglia. As previously described, the monkey striatum was the most enriched basal ganglia structure in Lrrk2 labeling. Both projection neurons and parvalbumin-containing GABAergic interneurons displayed Lrrk2 immunoreactivity. At the electron microscopic level, striatal Lrrk2 labeling was associated predominantly with dendritic shafts and subsets of putative glutamatergic axon terminals. At the pallidal level, moderate cellular Lrrk2 immunostaining was found in the external globus pallidus (GPe), while neurons in the internal globus pallidus (GPi) were devoid of Lrrk2 immunoreactivity. Strong labeling was associated with cholinergic neurons in the nucleus basalis of Meynert. Midbrain dopaminergic neurons in the primate substantia nigra pars compacta (SNc) and ventral tegmental area harbored a significant level of Lrrk2 labeling, while neurons in the subthalamic nucleus were lightly immunostained. Most thalamic nuclei were enriched in Lrrk2 immunoreactivity, except for the centromedian nucleus that was completely devoid of labeling. Thus, Lrrk2 protein is widely distributed in the monkey basal ganglia, suggesting that gene mutations in PD may result in multifarious pathophysiological effects that could impact various target sites in the functional circuitry of the primate basal ganglia.
Assuntos
Gânglios da Base/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Tálamo/enzimologia , Animais , Gânglios da Base/ultraestrutura , Núcleo Caudado/enzimologia , Núcleo Caudado/ultraestrutura , Macaca mulatta , Putamen/enzimologia , Putamen/ultraestrutura , Tálamo/ultraestruturaRESUMO
We investigated hyposensitivity after amphetamine in early (postnatal Day 30; P30) and late (P45) adolescent rats compared to adults (P70) in experiment 1. Locomotor activity was measured for 1 hr after the first (acute) and second (24 hr later) injection of amphetamine (0.5 or 1.5 mg/kg). P30 and P45 rats were transiently hypoactive compared to adults, as indicated by reduced locomotor activity after acute amphetamine and enhanced activity after the second injection in adolescents only. In experiment 2, ovariectomy did not alter locomotor activity during habituation at any age compared to intact rats, and, as for intact adolescents, ovariectomized adolescents continued to be less active after amphetamine than adults, suggesting gonadal immaturity alone cannot account for age differences in experiment 1. However, ovariectomy attenuated the increase in activity after the second treatment. In experiment 3 involving untreated rats, tyrosine hydroxylase immunoreactivity was reduced in P30, P40, and P50 compared to P90 rats in the nucleus accumbens core and the medial prefrontal cortex. Thus, adolescents may have an increased threshold of behavioral activation that can be overcome with either a higher dose or with repeated amphetamine treatment, and may be related to changes in the dopamine system over development.
Assuntos
Anfetamina/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo , Estimulantes do Sistema Nervoso Central/farmacologia , Locomoção/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/imunologia , Tirosina 3-Mono-Oxigenase/metabolismo , Fatores Etários , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/imunologia , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Núcleo Caudado/imunologia , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Hormônios Gonadais/metabolismo , Habituação Psicofisiológica , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/enzimologia , Núcleo Accumbens/imunologia , Ovariectomia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/enzimologia , Córtex Pré-Frontal/imunologia , Ratos , Ratos Long-EvansRESUMO
Glycogen synthase kinase 3 (GSK3) is a critical mediator of many intracellular signaling systems. The activity of GSK3 is regulated by several kinases, with inactivation occurring via phosphorylation of the inhibitory serine-21 (alpha-isoform) and serine-9 (beta-isoform) residues. Here, we investigated whether acute cocaine administration regulates GSK3 activity and if inhibition of GSK3 by valproate or the selective GSK3 inhibitor SB 216763 would attenuate cocaine-induced behaviors in mice. Mice injected with cocaine (20 mg/kg, i.p.) showed a reduction in the phosphorylation of GSK3beta in the caudate putamen, reflecting an increase in the activity of the kinase. To assess the role of GSK3 in cocaine-induced hyperactivity, mice were pretreated with valproate (50-300 mg/kg, i.p.), SB 216763 (0.25-7.5 mg/kg, i.p.), or the appropriate vehicle prior to saline or cocaine (20 mg/kg, i.p.). Valproate or SB 216763 produced significant dose-dependent reductions in cocaine-induced ambulatory and stereotypic activity. Repeated administration of cocaine can result in an augmentation of the locomotor-stimulatory effects of the drug, a phenomenon referred to as sensitization. Mice pretreated with SB 216763 (2.5 mg/kg, i.p.) prior to daily cocaine (20 mg/kg, i.p.) for 5 days showed a significant attenuation of the development of cocaine-induced behavioral sensitization following a cocaine challenge on day 13. These results indicate that cocaine activated GSK3beta in the caudate putamen and that pharmacological inhibition of GSK3 reduced both the acute behavioral responses to cocaine and the long-term neuroadaptations produced by repeated cocaine, therefore suggesting a role for GSK3 in the behavioral and neurochemical manifestations associated with cocaine exposure.
Assuntos
Núcleo Caudado/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/enzimologia , Cocaína/toxicidade , Quinase 3 da Glicogênio Sintase/fisiologia , Hipercinese/induzido quimicamente , Putamen/efeitos dos fármacos , Animais , Núcleo Caudado/enzimologia , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Hipercinese/enzimologia , Indóis/farmacologia , Locomoção/efeitos dos fármacos , Masculino , Maleimidas/farmacologia , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Putamen/enzimologia , Comportamento Estereotipado/efeitos dos fármacos , Comportamento Estereotipado/fisiologia , Ácido Valproico/farmacologiaRESUMO
We examined the relationship between COMT Val158Met genotype and temporal lobe volumes, including the caudate as a control region. Thirty-one healthy subjects completed 1.5T brain MRI and genotyping. After controlling for demographics, Val158 allele homozygotes exhibited significantly smaller temporal lobe and hippocampal volumes, with a trend for smaller amygdala volumes.
Assuntos
Catecol O-Metiltransferase/genética , Imageamento por Ressonância Magnética/estatística & dados numéricos , Polimorfismo de Nucleotídeo Único/genética , Lobo Temporal/anatomia & histologia , Adulto , Tonsila do Cerebelo/anatomia & histologia , Tonsila do Cerebelo/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/enzimologia , Encéfalo/metabolismo , Catecol O-Metiltransferase/metabolismo , Núcleo Caudado/anatomia & histologia , Núcleo Caudado/enzimologia , Núcleo Caudado/metabolismo , Feminino , Lateralidade Funcional/genética , Genótipo , Homozigoto , Humanos , Masculino , Metionina/genética , Metionina/metabolismo , Pessoa de Meia-Idade , Lobo Temporal/enzimologia , Lobo Temporal/metabolismo , Valina/genética , Valina/metabolismoRESUMO
Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinson's disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.
Assuntos
Encéfalo/enzimologia , Transtornos Parkinsonianos/enzimologia , Proteínas Serina-Treonina Quinases/genética , Encéfalo/patologia , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Humanos , Hibridização In Situ , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Corpos de Lewy/enzimologia , Corpos de Lewy/patologia , Mutação , Neurônios/enzimologia , Neurônios/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , Putamen/enzimologia , Putamen/patologia , Valores de ReferênciaRESUMO
Haloperidol (HAL) is a typical antipsychotic drug and known to cause extrapyramidal symptoms (EPS) that may be associated with the blockade of dopamine D2-receptors in nigrostriatal pathway by the drug. In contrast, quetiapine (QTP) is an atypical antipsychotic drug that has the lowest incidence of producing EPS in patients with schizophrenia, while improving psychosis symptoms. In the present study, we investigated the possibility of reversing the HAL-induced changes in locomotor activity and in striatal tyrosine hydroxylase (TH) of rats. Rats were administered HAL (2mg/kg/day, p.o.) for 3 months, followed by vehicle (VEH), QTP (10mg/kg/day), HAL, or HAL+QTP for another 5 weeks. The locomotor activity and TH immunoreactivity of the rats were measured. Chronic administration of HAL caused significant increase in locomotor activity and lower levels of TH immunoreactivity in the caudate putamen of the striatum. When the long-term haloperidol treatment was removed, the change in TH immunoreactivity was normalized, while the HAL induced high level of locomotor activity was returned to normal level only in the rats that stopped HAL consumption and received QTP treatment. In the substantia nigra and ventral tegmental areas, all rats showed comparable numbers of TH-positive cell bodies, which had no shrinkage. These results support a previously proposed relationship between EPS and TH levels in the striatum and provide valuable preclinical information towards understanding why QTP produces a lowest incidence of EPS among antipsychotics and has been used to treat EPS caused by other antipsychotics, and eventually establish a principle of treating EPS.
Assuntos
Antipsicóticos/farmacologia , Núcleo Caudado/enzimologia , Dibenzotiazepinas/farmacologia , Haloperidol/farmacologia , Atividade Motora/efeitos dos fármacos , Putamen/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Núcleo Caudado/efeitos dos fármacos , Discinesia Induzida por Medicamentos/psicologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismo , Putamen/efeitos dos fármacos , Fumarato de Quetiapina , Ratos , Ratos Sprague-Dawley , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Substância Negra/metabolismoRESUMO
In this work, two oximes for the treatment of tabun-inhibited acetylcholinesterase (AChE; EC 3.1.1.7), K074 (1,4-bis(4-hydroxyiminomethylpyridinium)butane dibromide) and K075 ((E)-1,4-bis(4-hydroxyiminomethylpyridinium)but-2-en dibromide), were tested in vitro as reactivators of AChE. Comparison was made with currently used AChE reactivators (pralidoxime, HI-6, methoxime and obidoxime). Human brain homogenate was taken as an appropriate source of the cholinesterases. As resulted, oxime K074 appears to be the most potent reactivator of tabun-inhibited AChE, with reactivation potency comparable to that of obidoxime. A second AChE reactivator, K075, does not attain as great a reactivation potency as K074, although its maximal reactivation (17%) was achieved at relevant concentrations for humans.
Assuntos
Acetilcolinesterase/metabolismo , Butanos/farmacologia , Núcleo Caudado/enzimologia , Inibidores da Colinesterase/toxicidade , Reativadores da Colinesterase/farmacologia , Organofosfatos/toxicidade , Oximas/farmacologia , Compostos de Piridínio/farmacologia , Butanos/química , Núcleo Caudado/efeitos dos fármacos , Reativadores da Colinesterase/química , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cloreto de Obidoxima/química , Cloreto de Obidoxima/farmacologia , Oximas/química , Compostos de Pralidoxima/química , Compostos de Pralidoxima/farmacologia , Compostos de Piridínio/químicaRESUMO
Methamphetamine (METH), leading to striatal dopamine (DA) nerve terminal toxicity in mammals, is also thought to induce apoptosis of striatal neurons in rodents. We investigated the acute effects induced by multiple injections of METH (4 x 5 mg/kg, i.p.) at 2-h intervals or a single injection of METH (20 mg/kg, i.p.) on terminal dopaminergic toxicity markers, including DA levels, DA turnover, and tyrosine hydroxylase (TH) immunoreactivity in rat caudate-putamen (CPu). We further investigated whether both treatment paradigms would change Bax and activate caspase-3 expression, thus triggering striatal apoptotic mitochondria-dependent biochemical cascades. The first injection of METH (5 mg/kg, i.p.) produced a significant release of DA that peaked 30 min and stayed above control levels up to 1.5 h within CPu. In another set of experiments, rats were killed 1 and 24 h following the last injection, for tissue DA and metabolite content measurement and Western blot analysis (24 h). Multiple doses induced DA depletion and increased turnover at both endpoints. Single-dose METH reproduced these effects at 24 h; however, turnover was significantly higher than that evoked by the multiple doses at 24 h. Although both paradigms evoked similar DA depletion, however, none of the dosing regimens induced changes in TH expression at 24 h. The former paradigm produced an increase in Bax expression in CPu not sufficient to induce cleavage of caspase-3 proenzyme at 24 h. This study suggests that both paradigm induced changes in striatal dopaminergic markers that are independent of terminal degeneration and striatal apoptotic mitochondria-dependent caspase-3 driven cascade within 24 h.
Assuntos
Caspases/metabolismo , Núcleo Caudado/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Dopamina/metabolismo , Metanfetamina/farmacologia , Putamen/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Western Blotting , Caspase 3 , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Estimulantes do Sistema Nervoso Central/administração & dosagem , Cromatografia Líquida de Alta Pressão , Inibidores da Captação de Dopamina/administração & dosagem , Eletroquímica , Ácido Homovanílico/metabolismo , Masculino , Metanfetamina/administração & dosagem , Microdiálise , Putamen/efeitos dos fármacos , Putamen/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
GADD34, a stress response protein associated with cell rescue, DNA repair and apoptosis, is expressed in the ischaemic brain. The C-terminal region of GADD34 has homology with the Herpes Simplex Virus protein, ICP34.5, which overcomes the protein synthesis block after viral infection by actively dephosphorylating eukaryotic translation initiation factor 2alpha (eIF2alpha). The carboxy terminus of GADD34 is also capable of dephosphorylating eIF2alpha and therefore has the capacity to restore the protein synthesis shutoff associated with ischaemia. This study examines the distribution and time course of GADD34 expression after focal cerebral ischaemia. Focal ischaemia or sham procedure was carried out on Sprague-Dawley rats with survival times of 4, 12, 24 h, 7 and 30 days. Brains were processed for histology and immunohistochemistry. Ischaemic damage was mapped onto line diagrams and GADD34 positive cells counted in selected regions of cortex and caudate. GADD34 immunopositive cells (mainly neurones), expressed as cells/mm2, were present in ischaemic brains at 4 h (e.g., peri-infarct cortex 20 +/- 5; contralateral cortex 3 +/- 1, P < 0.05). Of the time points examined, numbers of GADD34 positive cells were highest 24 h after ischaemia (peri-infarct cortex 31 +/- 7.3, contralateral cortex 0.1 +/- 0.1, P < 0.05). Immunopositive cells, following a similar time course, were identified within the peri-infarct zone in the caudate nucleus and in ipsilateral cingulate cortex (possibly as a consequence of cortical spreading depression). GADD34 positive cells did not co-localise with a marker of irreversible cell death (TUNEL). Taken together, GADD34 positive cells in key neuroanatomical locations pertinent to the evolving ischaemic lesion, the lack of co-localisation with TUNEL and the protein's known effects on restoring protein synthesis, repairing DNA and involvement in ischaemic pre-conditioning suggests that it has the potential to influence cell survival in ischaemically compromised tissue.
Assuntos
Isquemia Encefálica/enzimologia , Encéfalo/enzimologia , Infarto Cerebral/enzimologia , Degeneração Neural/enzimologia , Neurônios/enzimologia , Proteínas/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Mapeamento Encefálico , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Núcleo Caudado/fisiopatologia , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Reparo do DNA/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Giro do Cíngulo/enzimologia , Giro do Cíngulo/patologia , Giro do Cíngulo/fisiopatologia , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Neocórtex/enzimologia , Neocórtex/patologia , Neocórtex/fisiopatologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para Cima/fisiologiaRESUMO
Types of NADPH-d+ neurons (Vincent et al., 1983) were identified in the striatum and basolateral nuclei of the amygdala; striocortical neurons were detected in the striatum using the DiI marker (Belichenko and Dahlström, 1995). NADPH-d+ cells were numerous. Staining of these cells and all their processes, along with our previous studies of the neurons in these formations in the human brain using the Golgi method, allowed us to identify their shapes and identify them as sparsely or extensively branched cells. The main efferent neurons of the striatum and basolateral amygdala (extensively branched medium spiny cells and bushy spiny cells respectively) and their extensively branched interneurons did not contain NADPH-d. Efferent NADPH-d+ neurons included reticular, sparsely branched cells with long dendrites, which were the most numerous cells in both formations, as well as occasional large multipolar branched neurons; the striatum also contained numerous sparsely branched short-dendrite cells (a neuron type most represented in the brainstem and especially the reticular formation). Projections of reticular cells from the striatum to the cortex were demonstrated. NADPH-d+ interneurons were sparsely branched: in the striatum, these were slender, long-dendrite, bipolar cells (numerous), ordinary bipolar cells, twisted and large dendrite-poor cells; the amygdala contained the same bipolar cells along with radial neurons. Thus, NADPH-d+ neurons in these formations were more ancient, i.e., structurally less complex, cell types.
Assuntos
Tonsila do Cerebelo/enzimologia , Núcleo Caudado/enzimologia , Interneurônios/enzimologia , NADPH Desidrogenase/metabolismo , Neurônios/enzimologia , Óxido Nítrico/metabolismo , Adulto , Idoso , Tonsila do Cerebelo/citologia , Gânglios da Base/citologia , Gânglios da Base/enzimologia , Núcleo Caudado/citologia , Corpo Estriado/citologia , Corpo Estriado/enzimologia , Humanos , Interneurônios/classificação , Pessoa de Meia-Idade , Vias Neurais/enzimologia , Neurônios/classificaçãoRESUMO
Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes alpha-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/-) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/- mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/- mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/- mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Di-Hidrolipoamida Desidrogenase/deficiência , Predisposição Genética para Doença , Malonatos , Doenças Neurodegenerativas/enzimologia , Propionatos , Animais , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Núcleo Caudado/patologia , Contagem de Células , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Di-Hidrolipoamida Desidrogenase/genética , Modelos Animais de Doenças , Heterozigoto , Doença de Huntington/induzido quimicamente , Doença de Huntington/enzimologia , Doença de Huntington/patologia , Complexo Cetoglutarato Desidrogenase/efeitos dos fármacos , Complexo Cetoglutarato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Nitrocompostos , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/enzimologia , Transtornos Parkinsonianos/patologia , Putamen/efeitos dos fármacos , Putamen/enzimologia , Putamen/patologia , Complexo Piruvato Desidrogenase/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Substância Negra/patologiaRESUMO
Enzymes that hydrolyze extracellular ATP, i.e. ecto-ATPase and ecto-ATP diphosphohydrolase (ATPDase), can be differentiated by ability of the latter to hydrolyze ADP and by slightly different kinetic properties of the two enzymes. Synaptic plasma membrane fractions isolated from rat hippocampus and caudate nucleus exhibit ADP-hydrolyzing activity, as revealed by the enzyme assay, and the presence of ecto-ATPase protein, as revealed by immunological identification on Western blot. These findings indicate that both enzymes are co-expressed in the synaptic membrane compartment of hippocampal and caudate nucleus neurons. Kinetic analysis was performed to determine the relative contribution of each enzyme to the total ATP-hydrolyzing activity, while an inhibition study was carried out in order to exclude the interference of other nonspecific ATPase and phosphatase activities. Based on the kinetic properties, sensitivity to inhibitors and V(ATP)/V(ADP) ratio of about 2, we concluded that a substantial portion of ATP-hydrolyzing activity in both synaptic membrane preparations can be ascribed to the catalytic action of ATPDase. On the other hand, the highest catalytic efficacy when ATP is the substrate and the greater abundance of ecto-ATPase protein in caudate nucleus preparation suggest that the relative contribution of ecto-ATPase to the total ATP-hydrolyzing activity in the caudate nucleus is higher than in the hippocampus.
Assuntos
Adenosina Trifosfatases/metabolismo , Apirase/metabolismo , Hipocampo/enzimologia , Membranas Sinápticas/enzimologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD , Apirase/antagonistas & inibidores , Núcleo Caudado/efeitos dos fármacos , Núcleo Caudado/enzimologia , Núcleo Caudado/metabolismo , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hidrólise/efeitos dos fármacos , Immunoblotting , Cinética , Ratos , Ratos Wistar , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismoRESUMO
OBJECTIVE: It has been assumed that some behavioral changes associated with repeated exposure to dopaminergic psychostimulant drugs might be explained by changes in activity of dopamine receptors, including the dopamine D(1) receptor, which is linked by a stimulatory G protein to the effector enzyme adenylyl cyclase. To establish whether dopamine D(1) receptor function might be altered in human methamphetamine users, the authors measured dopamine-stimulated adenylyl cyclase activity in the brain of chronic human users of the drug. METHOD: Adenylyl cyclase activity stimulated by dopamine and by guanylyl-imidodiphosphate (to assess G protein and adenylyl cyclase coupling) was determined in the postmortem brain tissue of 16 methamphetamine users who had used the drug both recently and chronically (i.e., at least 1 year) as well as 21 matched comparison subjects. RESULTS: A 25%-30% decrease in the maximal extent of dopamine stimulation of adenylyl cyclase activity was seen in the striatum (nucleus accumbens, caudate, and putamen) of the methamphetamine users. No changes were found in basal or guanylyl-imidodiphosphate-stimulated enzyme activity. CONCLUSIONS: These data suggest that dopamine receptor function linked to adenylyl cyclase is partially desensitized in the striatum of human methamphetamine users. Decreased dopamine D(1) receptor function might underlie part of the known (drug withdrawal syndrome) or expected (drug tolerance) consequences of methamphetamine exposure in humans.