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1.
Artigo em Russo | MEDLINE | ID: mdl-31851173

RESUMO

AIM: To study an effect of cabergoline on dopamine and noradrenaline concentration and BDNF mRNA level in the rat midbrain and hypothalamus. MATERIAL AND METHODS: Twenty adult male Wistar rats were used in a single treatment paradigm: animals of the treatment group (n=10) received cabergoline (i.p., 0.5 mg/kg) and the control group (n=10) received an equivalent volume of the solvent. Quantitative analysis for the dopamine (DA) and noradrenaline (NA) was carried out using high-performance liquid chromatography (HPLC) coupled with electrochemical detection. BDNF mRNA levels were studied using quantitative RT-PCR. RESULTS AND CONCLUSION: Cabergoline significantly increases NA concentration in the midbrain 24 hours after injection: 639.2±64.5 ng/g in the treatment group versus 398.0±66.0 ng/g in the control group (p<0.05), while mean content of DA is not significantly changed (211.4±16.3 ng/g vs 169.7±54.6 ng/g, respectively). Cabergoline does not affect hypothalamic DA and NA levels. The drug increases BDNF mRNA levels by 2-times in the midbrain, but not in the hypothalamus, 24 hours after injection.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Cabergolina , Catecolaminas , Receptores de Dopamina D2 , Animais , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Cabergolina/farmacologia , Catecolaminas/metabolismo , Ergolinas , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Receptores de Dopamina D2/agonistas
2.
Elife ; 82019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486769

RESUMO

Action potentials trigger neurotransmitter release at active zones, specialized release sites in axons. Many neurons also secrete neurotransmitters or neuromodulators from their somata and dendrites. However, it is unclear whether somatodendritic release employs specialized sites for release, and the molecular machinery for somatodendritic release is not understood. Here, we identify an essential role for the active zone protein RIM in stimulated somatodendritic dopamine release in the midbrain. In mice in which RIMs are selectively removed from dopamine neurons, action potentials failed to evoke significant somatodendritic release detected via D2 receptor-mediated currents. Compellingly, spontaneous dopamine release was normal upon RIM knockout. Dopamine neuron morphology, excitability, and dopamine release evoked by amphetamine, which reverses dopamine transporters, were also unaffected. We conclude that somatodendritic release employs molecular scaffolds to establish secretory sites for rapid dopamine signaling during firing. In contrast, basal release that is independent of action potential firing does not require RIM.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dendritos/metabolismo , Dopamina/metabolismo , Mesencéfalo/metabolismo , Potenciais de Ação , Animais , Camundongos , Receptores de Dopamina D2/metabolismo , Transmissão Sináptica
3.
Mol Pharmacol ; 96(4): 493-504, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31409621

RESUMO

Methamphetamine (MA) is highly addictive and neurotoxic, causing cell death in humans and in rodent models. MA, along with many of its analogs, is an agonist at the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 activation protects against MA-induced degeneration of dopaminergic neurons, suggesting that TAAR1 plays a role in regulating MA-induced neurotoxicity. However, the mechanisms involved in TAAR1's role in neurotoxicity and cell death have not been described in detail. In this study, we investigated the apoptosis pathway in Taar1 wild-type (WT) and knockout (KO) mice and in cells expressing the recombinant receptor. Bcl-2, an antiapoptotic protein, was upregulated ∼3-fold in the midbrain area (substantial nigra and ventral tegmental area) in Taar1 KO compared with WT mice, and MA significantly increased Bcl-2 expression in WT mice but decreased Bcl-2 expression in KO mice. The proapoptotic protein Bax did not differ across genotype or in response to MA. Bcl-2 expression was significantly upregulated by the TAAR1 agonist RO5166017 ((S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine) in cells expressing the recombinant mouse TAAR1. Additionally, activation of TAAR1 by RO5166017 increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and protein kinase B (AKT), but only inhibition of ERK1/2 phosphorylation prevented TAAR1-induced increases in Bcl-2 levels, indicating that TAAR1 activation increases Bcl-2 through an ERK1/2-dependent pathway. All changes to ERK1/2 pathway intermediates were blocked by the TAAR1 antagonist, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl) benzamide. These findings suggest that TAAR1 activation protects against MA-induced cell apoptosis and TAAR1 may play a role in cell death in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Methamphetamine stimulates TAAR1, a G protein-coupled receptor. The role and mechanisms for TAAR1 in methamphetamine-induced neurotoxicity are not known. Here, we report that, in genetic mouse models and cells expressing the recombinant receptor, TAAR1 activates the ERK1/2 pathway but not the AKT pathway to upregulate the antiapoptotic protein Bcl-2, which protects cells from drug-induced toxicity.


Assuntos
Metanfetamina/efeitos adversos , Neurônios/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Acoplados a Proteínas-G/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mesencéfalo/metabolismo , Metanfetamina/farmacologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxazóis/farmacologia , Fenetilaminas/farmacologia , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Regulação para Cima
4.
Brain Struct Funct ; 224(7): 2525-2535, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31324969

RESUMO

The midbrain dopamine system via the dorsal and ventral striatum regulates a wide range of behaviors. To dissect the role of dopaminergic projections to the dorsal striatum (nigrostriatal projection) and ventral striatum (mesolimbic projection) in sleep-wake behavior, we selectively chemogenetically stimulated nigrostriatal or mesolimbic projections and examined the resulting effects on sleep in rats. Stimulation of nigrostriatal pathways increased sleep and EEG delta power, while stimulation of mesolimbic pathways decreased sleep and reduced cortical EEG power. These results indicate that midbrain dopamine signaling in the dorsal or ventral striatum promotes sleep or wake, respectively.


Assuntos
Comportamento Animal/fisiologia , Dopamina/metabolismo , Mesencéfalo/metabolismo , Sono/fisiologia , Animais , Corpo Estriado/metabolismo , Sistema Límbico/metabolismo , Vias Neurais/fisiologia , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo
5.
Biochemistry (Mosc) ; 84(3): 310-320, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221069

RESUMO

Ionotropic glutamate and GABA receptors regulate the differentiation and determine the functional properties of mature neurons. Both insufficient and excessive activity of these neurotransmission systems are associated with various nervous system diseases. Our knowledge regarding the expression profiles of these receptors and the mechanisms of their regulation during the differentiation of specialized human neuron subtypes is limited. Here the expression profiles of the NMDA and GABAA receptor subunits were explored during in vitro differentiation of human induced pluripotent stem cells (iPSCs) into ventral mesencephalic neurons. The correlation between the neuronal maturation and the expression dynamics of these genes was investigated, and the functional activity of these receptors was assessed by calcium imaging. The role of NMDA and GABAA receptors in neurite outgrowth and the development of spontaneous activity was analyzed using the viral transduction of neural progenitors with the reporter genes TagGFP and TagRFP. The data indicate that agonists of the investigated receptors can be employed for optimization of existing protocols for neural differentiation of iPSCs, in particular for acceleration of neuronal maturation.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Neurônios/citologia , Receptores de GABA-A/genética , Receptores de N-Metil-D-Aspartato/genética
6.
Genes Brain Behav ; 18(7): e12593, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31215739

RESUMO

In addition to gene expression differences in distinct cell types, there is substantial post-transcriptional regulation driven in part by RNA binding proteins (RBPs). Loss-of-function RBP mutations have been associated with neurodevelopmental disorders, such as Fragile-X syndrome and syndromic autism. Work performed in animal models to elucidate the influence of neurodevelopmental disorder-associated RBPs on distinct behaviors has showed a connection between normal post-transcriptional regulation and conditioned learning. We previously reported cognitive inflexibility in a mouse model null for the RBP CUG-BP, Elav-like factor 6 (CELF6), which we also found to be associated with human autism. Specifically, these mice failed to potentiate exploratory hole-poking behavior in response to familiarization to a rewarding stimuli. Characterization of Celf6 gene expression showed high levels in monoaminergic populations such as the dopaminergic midbrain populations. To better understand the underlying behavioral disruption mediating the resistance to change exploratory behavior in the holeboard task, we tested three hypotheses: Does Celf6 loss lead to global restricted patterns of behavior, failure of immediate response to reward or failure to alter behavior in response to reward (conditioning). We found the acute response to reward was intact, yet Celf6 mutant mice exhibited impaired conditioned learning to both reward and aversive stimuli. Thus, we found that the resistance to change by the Celf6 mutant in the holeboard was most parsimoniously explained as a failure of conditioning, as the mice had blunted responses even to potent rewarding stimuli such as cocaine. These findings further support the role of RBPs in conditioned learning.


Assuntos
Proteínas CELF/genética , Condicionamento Clássico , Medo , Mutação , Recompensa , Animais , Proteínas CELF/metabolismo , Cocaína/administração & dosagem , Neurônios Dopaminérgicos/metabolismo , Comportamento Exploratório , Feminino , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Mesencéfalo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Drug Resist Updat ; 44: 15-25, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31202081

RESUMO

Diffuse midline gliomas (DMG) are rapidly fatal tumors of the midbrain in children, characterized by a diffuse growing pattern and high levels of intrinsic resistance to therapy. The location of these tumors, residing behind the blood-brain barrier (BBB), and the limited knowledge about the biology of these tumors, has hindered the development of effective treatment strategies. However, the introduction of diagnostic biopsies and the implementation of autopsy protocols in several large centers world-wide has allowed for a detailed characterization of these rare tumors. This has resulted in the identification of novel therapeutic targets, as well as major advances in understanding the biology of DMG in relation to therapy resistance. We here provide an overview of the cellular pathways and tumor-specific aberrations that have been targeted in preclinical DMG research, and discuss the advantages and limitations of these therapeutic strategies in relation to therapy resistance and BBB-penetration. Therewith, we aim to provide researchers with a framework for successful preclinical therapy development.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioma/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/genética , Antineoplásicos/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Criança , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Transdução de Sinais
8.
Psychopharmacology (Berl) ; 236(9): 2687-2697, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31053935

RESUMO

RATIONALE: Antipsychotics exert therapeutic effects by modulating various cellular signalling pathways and several types of receptors, including PKA- and GSK3ß-mediated signalling pathways, and NMDA receptors. The ventral midbrain, mainly containing the ventral tegmental area (VTA) and substantia nigra (SN), are the nuclei with dopamine origins in the brain, which are also involved in the actions of antipsychotics. Whether antipsychotics can modulate these cellular pathways in the ventral midbrain is unknown. OBJECTIVE: This study aims to investigate the effects of antipsychotics, including aripiprazole (a dopamine D2 receptor (D2R) partial agonist), bifeprunox (a D2R partial agonist), and haloperidol (a D2R antagonist) on the PKA- and GSK3ß-mediated pathways and NMDA receptors in the ventral midbrain. METHODS: Male rats were orally administered aripiprazole (0.75 mg/kg, t.i.d. (ter in die)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for either 1 week or 10 weeks. The levels of PKA, p-PKA, Akt, p-Akt, GSK3ß, p-GSK3ß, Dvl-3, ß-catenin, and NMDA receptor subunits in the ventral midbrain were assessed by Western Blots. RESULTS: The results showed that chronic antipsychotic treatment with aripiprazole selectively increased PKA activity in the VTA. Additionally, all three drugs elevated the activity of the Akt-GSK3ß signalling pathway in a time-dependent manner, while only aripiprazole stimulated the Dvl-3-GSK3ß-ß-catenin signalling pathway in the SN. Furthermore, chronic administration with both aripiprazole and haloperidol decreased the expression of NMDA receptors. CONCLUSION: This study suggests that activating PKA- and GSK3ß-mediated pathways and downregulating NMDA receptor expression in the ventral midbrain might contribute to the clinical effects of antipsychotics.


Assuntos
Antipsicóticos/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Glicogênio Sintase Quinase 3 beta/biossíntese , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/biossíntese , Animais , Antipsicóticos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo
9.
Brain Struct Funct ; 224(6): 2061-2078, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31144035

RESUMO

Calcium-binding proteins are widely used to distinguish neuronal subsets in the brain. This study focuses on secretagogin, an EF-hand calcium sensor, to identify distinct neuronal populations in the brainstem of several vertebrate species. By using neural tube whole mounts of mouse embryos, we show that secretagogin is already expressed during the early ontogeny of brainstem noradrenaline cells. In adults, secretagogin-expressing neurons typically populate relay centres of special senses and vegetative regulatory centres of the medulla oblongata, pons and midbrain. Notably, secretagogin expression overlapped with the brainstem column of noradrenergic cell bodies, including the locus coeruleus (A6) and the A1, A5 and A7 fields. Secretagogin expression in avian, mouse, rat and human samples showed quasi-equivalent patterns, suggesting conservation throughout vertebrate phylogeny. We found reduced secretagogin expression in locus coeruleus from subjects with Alzheimer's disease, and this reduction paralleled the loss of tyrosine hydroxylase, the enzyme rate limiting noradrenaline synthesis. Residual secretagogin immunoreactivity was confined to small submembrane domains associated with initial aberrant tau phosphorylation. In conclusion, we provide evidence that secretagogin is a useful marker to distinguish neuronal subsets in the brainstem, conserved throughout several species, and its altered expression may reflect cellular dysfunction of locus coeruleus neurons in Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Tronco Encefálico/metabolismo , Norepinefrina/metabolismo , Secretagoguinas/metabolismo , Animais , Masculino , Mesencéfalo/metabolismo , Neurônios/metabolismo , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismo , Vertebrados/metabolismo
10.
Am J Psychiatry ; 176(6): 468-476, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31055968

RESUMO

OBJECTIVE: Nociceptin/orphanin FQ (N/OFQ) is an antistress neuropeptide transmitter in the brain that counteracts corticotropin-releasing factor (CRF)-mediated stress and anxiety symptoms during drug and alcohol withdrawal. It also inhibits the release of a wide array of neurotransmitters, including dopamine and glutamate, which allows for it to block the rewarding properties of cocaine. Chronic cocaine administration in rodents has been shown to decrease N/OFQ and increase nociceptive opioid peptide (NOP) receptors in the nucleus accumbens. No previous studies have reported on the in vivo status of NOP in chronic cocaine-abusing humans. METHODS: [11C]NOP-1A and positron emission tomography (PET) were used to measure in vivo NOP binding in 24 individuals with cocaine use disorder and 26 healthy control subjects matched for age, sex, and smoking status. Participants with cocaine use disorder with no comorbid psychiatric or medical disorders were scanned after 2 weeks of outpatient-monitored abstinence. [11C]NOP-1A distribution volume (VT) was measured with kinetic analysis using the arterial input function in brain regions that mediate reward and stress behaviors. Participants with cocaine use disorder were followed up for 12 weeks after PET scanning to document relapse and relate it to VT. RESULTS: A significant increase in [11C]NOP-1A VT was observed in the cocaine use disorder group compared with the healthy control group. This increase, which was generalized across all regions of interest (approximately 10%), was most prominent in the midbrain, ventral striatum, and cerebellum. However, increased VT in these regions did not predict relapse. CONCLUSIONS: Increased NOP in cocaine use disorder suggests an adaptive response to decreased N/OFQ, or increased CRF transmission, or both. Future studies should examine the interactions between CRF and NOP to elucidate their role in negative reinforcement and relapse. NOP agonist medications to enhance N/OFQ should be explored as a therapeutic to treat cocaine use disorder.


Assuntos
Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Adulto , Encéfalo/diagnóstico por imagem , Compostos Bicíclicos Heterocíclicos com Pontes , Estudos de Casos e Controles , Cerebelo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/diagnóstico por imagem , Dopamina/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Mesencéfalo/metabolismo , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Compostos de Espiro , Estriado Ventral/metabolismo
11.
Cell ; 177(5): 1293-1307.e16, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31031008

RESUMO

The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.


Assuntos
Hipotálamo/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Fases do Sono/fisiologia , Animais , Colecistocinina/metabolismo , Hipotálamo/citologia , Mesencéfalo/citologia , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Optogenética
12.
Artigo em Inglês | MEDLINE | ID: mdl-30928412

RESUMO

Both sphingomyelinase and Toll-Like Receptor 4 (TLR4) are implicated in neurodegenerative diseases. However, the relationship between the two molecules remains unclear. In this study, using WT and TLR4-deficient mice, treated or not with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we aimed to investigate the relation between TLR4 and neutral sphingomyelinase (nSMase) in the midbrain. We found that the lack of TLR4 caused increase in nSMase protein expression and enzyme activity in the midbrain, as well as a marked delocalization from the cell membranes. This provoked a decrease in sphingomyelin (SM) species and an increase in ceramide levels. We found that exposure of TLR4-deficient mice to MPTP reduces unsaturated SM species by increasing saturated/unsaturated SM ratio. Saturated fatty acid make SM more rigid and could contribute to reducing neural plasticity. In this study we showed that the absence of TLR4 also induced reduction of both heavy neurofilaments and glial fibrillary acidic protein (GFAP) and mice exhibited higher sensitivity to MPTP administration. We speculated about the possible association between nSMase-TLR4 complex and MPTP midbrain damage. Taken together, our findings provide for the first time indications about the role of TLR4 in change of SM metabolism in MPTP neurotoxicity.


Assuntos
Intoxicação por MPTP/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Receptor 4 Toll-Like/deficiência , Animais , Intoxicação por MPTP/enzimologia , Intoxicação por MPTP/patologia , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Camundongos , Esfingomielinas/metabolismo
13.
J Biol Chem ; 294(17): 6957-6971, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824538

RESUMO

The dopamine transporter (DAT) regulates dopamine neurotransmission via reuptake of dopamine released into the extracellular space. Interactions with partner proteins alter DAT function and thereby dynamically shape dopaminergic tone important for normal brain function. However, the extent and nature of these interactions are incompletely understood. Here, we describe a novel physical and functional interaction between DAT and the voltage-gated K+ channel Kv2.1 (potassium voltage-gated channel subfamily B member 1 or KCNB1). To examine the functional consequences of this interaction, we employed a combination of immunohistochemistry, immunofluorescence live-cell microscopy, co-immunoprecipitation, and electrophysiological approaches. Consistent with previous reports, we found Kv2.1 is trafficked to membrane-bound clusters observed both in vivo and in vitro in rodent dopamine neurons. Our data provide evidence that clustered Kv2.1 channels decrease DAT's lateral mobility and inhibit its internalization, while also decreasing canonical transporter activity by altering DAT's conformational equilibrium. These results suggest that Kv2.1 clusters exert a spatially discrete homeostatic braking mechanism on DAT by inducing a relative increase in inward-facing transporters. Given recent reports of Kv2.1 dysregulation in neurological disorders, it is possible that alterations in the functional interaction between DAT and Kv2.1 affect dopamine neuron activity.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Endocitose , Canais de Potássio Shab/metabolismo , Animais , Dopamina/metabolismo , Feminino , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
14.
Neuroscience ; 406: 278-289, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30885640

RESUMO

Methamphetamine (MA), neurotoxic drug of abuse, causes cell death in vitro and in vivo via several mechanisms such as mitochondrial dysfunction. In this study we evaluated the effect of MA on cell viability and mitochondrial biogenesis in primary midbrain culture. Primary mesencephalon cells prepared from E14.5 rat embryo were treated with 0.2-5 mM MA concentrations for 24, 48, and 72 h. Morphological changes of the cells were observed under light microscope. Cell viability and cell death following MA were assessed using MTT assay and immunocytochemistry. Gene expressions of mitochondrial biogenesis-involved factors (PGC1α, NRF1 and TFAM), and neuronal and glial markers were measured by qPCR. Low to moderate MA concentrations elevated cell viability in all time points, while higher concentrations and longer incubation times (48 and 72 h) decreased it. Sphered cell bodies and neurites degeneration were observed following exposure to high MA concentrations. MA at 5 mM concentration decreased the number of ß3-tubulin-, TH-, GFAP- and Iba1-positive cells, and their corresponding mRNA levels; however, 1 mM MA reduced α-synuclein mRNA. Unexpectedly, gene expression of PGC1α, NRF1 and TFAM was increased in response to 5 mM MA, with no changes following 1 mM MA. The results indicated that MA effect on cell viability occurs in a dose-dependent manner. While moderate concentrations increased cell viability, the higher ones reduced it and caused cell death. Mitochondrial biogenesis activation, as a compensatory mechanism, did not prevent neuronal and glial cell death following high MA concentration.


Assuntos
Mesencéfalo/efeitos dos fármacos , Metanfetamina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Feminino , Mesencéfalo/metabolismo , Mitocôndrias/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/metabolismo , Síndromes Neurotóxicas/tratamento farmacológico , Biogênese de Organelas , Ratos Wistar , alfa-Sinucleína/efeitos dos fármacos , alfa-Sinucleína/metabolismo
15.
Proc Natl Acad Sci U S A ; 116(9): 3817-3826, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808767

RESUMO

Midbrain dopamine neurons, which can be regulated by neuropeptides and hormones, play a fundamental role in controlling cognitive processes, reward mechanisms, and motor functions. The hormonal actions of insulin-like growth factor 1 (IGF-1) produced by the liver have been well described, but the role of neuronally derived IGF-1 remains largely unexplored. We discovered that dopamine neurons secrete IGF-1 from the cell bodies following depolarization, and that IGF-1 controls release of dopamine in the ventral midbrain. In addition, conditional deletion of dopamine neuron-derived IGF-1 in adult mice leads to decrease of dopamine content in the striatum and deficits in dopamine neuron firing and causes reduced spontaneous locomotion and impairments in explorative and learning behaviors. These data identify that dopamine neuron-derived IGF-1 acts as a regulator of dopamine neurons and regulates dopamine-mediated behaviors.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Fator de Crescimento Insulin-Like I/genética , Locomoção/genética , Mesencéfalo/fisiologia , Animais , Cognição/fisiologia , Corpo Estriado/metabolismo , Corpo Estriado/fisiologia , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/fisiologia , Comportamento Exploratório/fisiologia , Hormônios/metabolismo , Fator de Crescimento Insulin-Like I/biossíntese , Aprendizagem/fisiologia , Locomoção/fisiologia , Mesencéfalo/metabolismo , Camundongos , Neuropeptídeos/genética
16.
Proc Natl Acad Sci U S A ; 116(11): 5108-5117, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796187

RESUMO

Neuromelanin-sensitive MRI (NM-MRI) purports to detect the content of neuromelanin (NM), a product of dopamine metabolism that accumulates with age in dopamine neurons of the substantia nigra (SN). Interindividual variability in dopamine function may result in varying levels of NM accumulation in the SN; however, the ability of NM-MRI to measure dopamine function in nonneurodegenerative conditions has not been established. Here, we validated that NM-MRI signal intensity in postmortem midbrain specimens correlated with regional NM concentration even in the absence of neurodegeneration, a prerequisite for its use as a proxy for dopamine function. We then validated a voxelwise NM-MRI approach with sufficient anatomical sensitivity to resolve SN subregions. Using this approach and a multimodal dataset of molecular PET and fMRI data, we further showed the NM-MRI signal was related to both dopamine release in the dorsal striatum and resting blood flow within the SN. These results suggest that NM-MRI signal in the SN is a proxy for function of dopamine neurons in the nigrostriatal pathway. As a proof of concept for its clinical utility, we show that the NM-MRI signal correlated to severity of psychosis in schizophrenia and individuals at risk for schizophrenia, consistent with the well-established dysfunction of the nigrostriatal pathway in psychosis. Our results indicate that noninvasive NM-MRI is a promising tool that could have diverse research and clinical applications to investigate in vivo the role of dopamine in neuropsychiatric illness.


Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Imagem por Ressonância Magnética , Melaninas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Meios de Contraste , Feminino , Humanos , Masculino , Mesencéfalo/metabolismo , Pessoa de Meia-Idade , Mudanças Depois da Morte , Transtornos Psicóticos/diagnóstico por imagem , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Substância Negra/metabolismo
17.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1436-1450, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30796971

RESUMO

Mitochondrial impairment and calcium (Ca++) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca++-ATPase pumps are impaired causing cytoplasmic Ca++ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser65, and reduced calpain-cleavage of Parkin. Treatment with the Ca++ ionophore A23187, which facilitates Ca++ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca++ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca++-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.


Assuntos
Cálcio/metabolismo , Calpaína/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Calcimicina/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Creatinina/análogos & derivados , Creatinina/farmacologia , Embrião de Mamíferos , Regulação da Expressão Gênica , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Mitocôndrias/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ácido Okadáico/farmacologia , Oligomicinas/farmacologia , Oligopeptídeos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Cultura Primária de Células , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Rotenona/farmacologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética
18.
J Biol Chem ; 294(11): 4169-4176, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30655290

RESUMO

The liver X receptors Lxrα/NR1H3 and Lxrß/NR1H2 are ligand-dependent nuclear receptors critical for midbrain dopaminergic (mDA) neuron development. We found previously that 24(S),25-epoxycholesterol (24,25-EC), the most potent and abundant Lxr ligand in the developing mouse midbrain, promotes mDA neurogenesis in vitro In this study, we demonstrate that 24,25-EC promotes mDA neurogenesis in an Lxr-dependent manner in the developing mouse midbrain in vivo and also prevents toxicity induced by the Lxr inhibitor geranylgeranyl pyrophosphate. Furthermore, using MS, we show that overexpression of human cholesterol 24S-hydroxylase (CYP46A1) increases the levels of both 24(S)-hydroxycholesterol (24-HC) and 24,25-EC in the developing midbrain, resulting in a specific increase in mDA neurogenesis in vitro and in vivo, but has no effect on oculomotor or red nucleus neurogenesis. 24-HC, unlike 24,25-EC, did not affect in vitro neurogenesis, indicating that the neurogenic effect of 24,25-EC on mDA neurons is specific. Combined, our results indicate that increased levels of 24,25-EC in vivo, by intracerebroventricular delivery in WT mice or by overexpression of its biosynthetic enzyme CYP46A1, specifically promote mDA neurogenesis. We propose that increasing the levels of 24,25-EC in vivo may be a useful strategy to combat the loss of mDA neurons in Parkinson's disease.


Assuntos
Colesterol 24-Hidroxilase/biossíntese , Colesterol/análogos & derivados , Dopamina/metabolismo , Mesencéfalo/metabolismo , Neurogênese , Animais , Células Cultivadas , Colesterol/biossíntese , Feminino , Humanos , Camundongos , Camundongos Transgênicos
19.
Methods Mol Biol ; 1919: 97-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30656624

RESUMO

The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20-50%), we chose to follow a mix of four main protocols based on Kriks and colleagues' protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson's disease. We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening.


Assuntos
Diferenciação Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Animais , Biomarcadores , Cálcio/metabolismo , Sinalização do Cálcio , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesencéfalo/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de Parkinson
20.
Neurosci Lett ; 699: 91-96, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30685409

RESUMO

Reduction of natural illumination in fall/winter months causes seasonal affective disorders (SAD) in vulnerable individuals. Neurotransmitter serotonin (5-HT) is involved in the mechanism of SAD. Tryptophan hydroxylase-2 (TPH2) is the key enzyme of 5-HT synthesis in the brain. C1473 G polymorphism in the Tph2 gene is a key factor defining the enzyme activity in the mouse brain. The main aims of the study were to investigate the effects of C1473 G polymorphism on behavior and brain 5-HT system responses to photoperiod alterations. The experiment was carried out on adult mouse males of B6-1473C and B6-1473 G congenic lines with normal and low TPH2 activities, respectively. B6-1473C and B6-1473 G mice were divided into four groups of 8 each and exposed for 28 days to standard-day (14 h light and 10 h darkness) or short-day (4 h light and 20 h darkness) conditions. No effect of photoperiod on locomotor, exploratory activities and anxiety in the open field test was observed. At the same time, photoperiod alterations affected depressive-like immobility in the forced swim test, the 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) levels, 5-HIAA/5-HT ratio and the Htr2a mRNA level in hippocampus and midbrain. The effect of the interaction between C1473 G polymorphism and photoperiod on 5-HT level and 5-HIAA/5-HT ratio in hippocampus was revealed. Short-day conditions reduced the level and increased 5-HIAA/5-HT ratio in this structure only in B6-1473 G mice. At the same time, C1473 G polymorphism does not alter effects of short-day conditions on immobility time in the forced swim test and the Htr2a mRNA level in the brain.


Assuntos
Depressão/fisiopatologia , Fotoperíodo , Serotonina/metabolismo , Triptofano Hidroxilase/genética , Animais , Depressão/genética , Comportamento Exploratório/fisiologia , Hipocampo/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Resposta de Imobilidade Tônica/fisiologia , Masculino , Mesencéfalo/metabolismo , Camundongos , Atividade Motora/fisiologia , Polimorfismo de Nucleotídeo Único , Receptor 5-HT2A de Serotonina/biossíntese , Triptofano Hidroxilase/metabolismo
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