Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.126
Filtrar
1.
Folia Neuropathol ; 57(2): 196-204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31556578

RESUMO

INTRODUCTION: Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons. MATERIAL AND METHODS: Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens. RESULTS: Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well. CONCLUSIONS: The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.


Assuntos
Acrilamida/toxicidade , Morte Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Mesencéfalo/efeitos dos fármacos , Animais , Células Cultivadas , Neurônios Dopaminérgicos/citologia , L-Lactato Desidrogenase/análise , Mesencéfalo/citologia , Camundongos
2.
Science ; 365(6454): 699-704, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31371562

RESUMO

Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.


Assuntos
Monitorização Fisiológica/métodos , Neuroimagem/métodos , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Comportamento Animal , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Engenharia Genética , Larva , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Camundongos , Optogenética , Domínios Proteicos , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Natação , Peixe-Zebra
3.
Nat Commun ; 10(1): 3674, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417077

RESUMO

Midbrain dopamine neurons are known to encode reward prediction errors (RPE) used to update value predictions. Here, we examine whether RPE signals coded by midbrain dopamine neurons are modulated by the cost paid to obtain rewards, by recording from dopamine neurons in awake behaving monkeys during performance of an effortful saccade task. Dopamine neuron responses to cues predicting reward and to the delivery of rewards were increased after the performance of a costly action compared to a less costly action, suggesting that RPEs are enhanced following the performance of a costly action. At the behavioral level, stimulus-reward associations are learned faster after performing a costly action compared to a less costly action. Thus, information about action cost is processed in the dopamine reward system in a manner that amplifies the following dopamine RPE signal, which in turn promotes more rapid learning under situations of high cost.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Aprendizagem/fisiologia , Recompensa , Animais , Sinais (Psicologia) , Mesencéfalo/citologia , Mesencéfalo/fisiologia , Movimentos Sacádicos
4.
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
5.
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
6.
Interdiscip Sci ; 11(2): 247-257, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177377

RESUMO

INTRODUCTION: GPCR share a common structural feature, i.e., the presence of seven trans-membrane helices having three intracellular and three extracellular loops. The carboxyl terminal is intracellular whereas amino terminal is extracellular. Various conformational changes are observed in structure of GPCR during the binding with ligand, coupling with G protein and interaction with other proteins. In Rhodopsin class of GPCR the basic structure of GPCR is resolved by X-ray crystallography. Ligand acts as an extracellular stimulus for GPCRs to bring physiological changes in organisms. GPR139 has been found to have effective physiological role in primary dopaminergic midbrain neurons and in central nervous system. Recent reports suggested that the ligand of GPR139 protein inhibits the growth of primary dopaminergic midbrain neurons in central nervous system. These discoveries indicated the potential involvement and influence of GPR139 protein in central nervous system METHOD: Therefore, we used multi-approach analysis to investigate the role of GPR139 in the molecular mechanisms of central nervous system. In silico screening was performed to study compound 1 binding with GPR139 protein in their predicted three-dimensional structures. Compound 1 was subjected to molecular dynamics (MD) simulation and stability analysis. RESULTS: The results of MD analysis suggested that the loop region in GPR139 protein structure could affect its binding with drugs. Finally, we cross-validated the predicted compound 1 through systems biology approach. Our results suggested that GPR139 might play an important role in primary dopaminergic midbrain neurons therapy.


Assuntos
Neurônios Dopaminérgicos/citologia , Avaliação Pré-Clínica de Medicamentos , Mesencéfalo/citologia , Simulação de Dinâmica Molecular , Neuroproteção , Receptores Acoplados a Proteínas-G/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Biologia de Sistemas , Sítios de Ligação , Diabetes Mellitus Tipo 2/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Neuroproteção/efeitos dos fármacos , Doença de Parkinson/metabolismo , Domínios Proteicos , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Tempo
7.
Nat Commun ; 10(1): 2508, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175277

RESUMO

Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Parte Compacta da Substância Negra/metabolismo , Fatores de Transcrição/genética , Tirosina 3-Mono-Oxigenase/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Mesencéfalo/citologia , Camundongos
8.
PLoS One ; 14(4): e0215277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31022188

RESUMO

Most of the Parkinson's disease (PD) cases are sporadic, although several genes are directly related to PD. Several pathways are central in PD pathogenesis: protein aggregation linked to proteasomal impairments, mitochondrial dysfunctions and impairment in dopamine (DA) release. Here we studied the close crossing of mitochondrial dysfunction and aggregation of α-synuclein (α-syn) and in the extension in the dopaminergic neuronal death. Here, using rat primary cultures of mesencephalic neurons, we induced the mitochondrial impairments using "DA-toxins" (MPP+, 6OHDA, rotenone). We showed that the DA-Toxins induced dopaminergic cell death through different pathways: caspase-dependent cell death for 6OHDA; MPP+ stimulated caspase-independent cell death, and rotenone activated both pathways. In addition, a decrease in energy production and/or a development of oxidative stress were observed and were linked to α-syn aggregation with generation of Lewy body-like inclusions (found inside and outside the dopaminergic neurons). We demonstrated that any of induced mitochondrial disturbances and processes of death led to α-syn protein aggregation and finally to cell death. Our study depicts the cell death mechanisms taking place in in vitro models of Parkinson's disease and how mitochondrial dysfunctions is at the cross road of the pathologies of this disease.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurotoxinas/toxicidade , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/patologia , 1-Metil-4-fenilpiridínio/toxicidade , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/patologia , Embrião de Mamíferos , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Mesencéfalo/citologia , Mitocôndrias/metabolismo , Necrose/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Cultura Primária de Células , Agregação Patológica de Proteínas/etiologia , Ratos , Rotenona/toxicidade , alfa-Sinucleína/metabolismo
9.
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
10.
Neurosci Lett ; 704: 181-188, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-30954607

RESUMO

Alpha-synuclein (α-Syn) is a soluble protein primarily expressed in presynaptic terminals in the central nervous system (CNS). Aggregates of fibrillated α-Syn are the major component of Lewy bodies (LB), a pathologic hallmark of idiopathic Parkinson's disease (PD). Recently, naturally occurring autoantibodies against human α-Syn (nAbs α-Syn) were detected in the peripheral blood of PD patients and controls. Here, we investigated the inhibitory effects of nAbs α-Syn on distinct α-Syn fragments, as well as inflammatory responses and cytotoxicity evoked by nAbs α-Syn in primary microglia. All α-Syn fragments induced the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) from microglia in primary culture. Cotreatment with nAbs α-Syn alleviated the release of pro-inflammatory cytokines induced by α-Syn fragments α-Syn 1-95, α-Syn 61-140, α-Syn 96-140 and α-Syn 112. Treatment with the α-Syn fragments α-Syn 1-95, α-Syn 61-140 and α-Syn 112 impaired the viability of primary microglia. This effect could not be counteracted by cotreatment with nAbs α-Syn. Data suggest an important role of nAbs α-Syn in the α-Syn-induced inflammation cascade, and indicate the potential importance of nAbs in the pathogenesis of PD. This could provide an experimental therapeutic target for patients with PD.


Assuntos
Autoanticorpos/metabolismo , alfa-Sinucleína/imunologia , alfa-Sinucleína/metabolismo , Animais , Autoanticorpos/farmacologia , Sobrevivência Celular , Humanos , Interleucina-6/metabolismo , Mesencéfalo/citologia , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doença de Parkinson/patologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Cultura Primária de Células , Ligação Proteica , Fator de Necrose Tumoral alfa/metabolismo , alfa-Sinucleína/toxicidade
11.
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
12.
BMC Biol ; 17(1): 22, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30849972

RESUMO

BACKGROUND: Although the overall brain organization is shared in vertebrates, there are significant differences within subregions among different groups, notably between Sarcopterygii (lobe-finned fish) and Actinopterygii (ray-finned fish). Recent comparative studies focusing on the ventricular morphology have revealed a large diversity of the hypothalamus. Here, we study the development of the inferior lobe (IL), a prominent structure forming a bump on the ventral surface of the teleost brain. Based on its position, IL has been thought to be part of the hypothalamus (therefore forebrain). RESULTS: Taking advantage of genetic lineage-tracing techniques in zebrafish, we reveal that cells originating from her5-expressing progenitors in the midbrain-hindbrain boundary (MHB) participate in the formation of a large part of the IL. 3D visualization demonstrated how IL develops in relation to the ventricular system. We found that IL is constituted by two developmental components: the periventricular zone of hypothalamic origin and the external zone of mesencephalic origin. The mesencephalic external zone grows progressively until adulthood by adding new cells throughout development. CONCLUSION: Our results disprove a homology between the IL and the mammalian lateral hypothalamus. We suggest that the IL is likely to be involved in multimodal sensory integration rather than feeding motivation. The teleost brain is not a simpler version of the mammalian brain, and our study highlights the evolutionary plasticity of the brain which gives rise to novel structures.


Assuntos
Mesencéfalo/embriologia , Prosencéfalo/embriologia , Peixe-Zebra/embriologia , Animais , Evolução Biológica , Linhagem da Célula/fisiologia , Mesencéfalo/citologia , Células-Tronco Neurais/citologia , Prosencéfalo/citologia
13.
Int J Mol Sci ; 20(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704073

RESUMO

In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Oxidopamina/farmacologia , Animais , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
14.
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
15.
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
16.
Methods Mol Biol ; 1880: 257-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610703

RESUMO

To appreciate the positive or negative impact of autophagy during the initiation and progression of human diseases, the isolation or de novo generation of appropriate cell types is required to support focused in vitro assays. In human neurodegenerative diseases such as Parkinson's disease (PD), specific subsets of acutely sensitive neurons become susceptible to stress-associated operational decline and eventual cell death, emphasizing the need for functional studies in those vulnerable groups of neurons. In PD, a class of dopaminergic neurons in the ventral midbrain (mDANs) is affected. To study these, human-induced pluripotent stem cells (hiPSCs) have emerged as a valuable tool, as they enable the establishment and study of mDAN biology in vitro. In this chapter, we describe a stepwise protocol for the generation of mDANs from hiPSCs using a monolayer culture system. We then outline how imaging-based autophagy assessment methodologies can be applied to these neurons, thereby providing a detailed account of the application of imaging-based autophagy assays to human iPSC-derived mDANs.


Assuntos
Autofagia , Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia , Microscopia de Fluorescência/métodos , Neurogênese , Técnicas de Cultura de Células/métodos , Células Cultivadas , Neurônios Dopaminérgicos/patologia , Imunofluorescência/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Mesencéfalo/patologia , Doença de Parkinson/patologia , Fixação de Tecidos/métodos
17.
Neuropsychopharmacology ; 44(2): 344-355, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30054584

RESUMO

Astrocytes are ubiquitous CNS cells that support tissue homeostasis through ion buffering, neurotransmitter recycling, and regulation of CNS vasculature. Yet, despite the essential functional roles they fill, very little is known about the physiology of astrocytes in the ventral midbrain, a region that houses dopamine-releasing neurons and is critical for reward learning and motivated behaviors. Here, using a combination of whole-transcriptome sequencing, histology, slice electrophysiology, and calcium imaging, we performed the first functional and molecular profiling of ventral midbrain astrocytes and observed numerous differences between these cells and their telencephalic counterparts, both in their gene expression profile and in their physiological properties. Ventral midbrain astrocytes have very low membrane resistance and inward-rectifying potassium channel-mediated current, and are extensively coupled to surrounding oligodendrocytes through gap junctions. They exhibit calcium responses to glutamate but are relatively insensitive to norepinephrine. In addition, their calcium activity can be dynamically modulated by dopamine D2 receptor signaling. Taken together, these data indicate that ventral midbrain astrocytes are physiologically distinct from astrocytes in cortex and hippocampus. This work provides new insights into the extent of functional astrocyte heterogeneity within the adult brain and establishes the foundation for examining the impact of regional astrocyte differences on dopamine neuron function and susceptibility to degeneration.


Assuntos
Astrócitos/fisiologia , Córtex Cerebral/metabolismo , Mesencéfalo/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Cálcio/metabolismo , Forma Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feminino , Junções Comunicantes/metabolismo , Ácido Glutâmico/farmacologia , Masculino , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Camundongos , Norepinefrina/farmacologia
18.
Neuroscience ; 396: 108-118, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439538

RESUMO

Panic disorder (PD), a prevalent anxiety disorder, is characterized by unexpected panic attacks, persistent anxiety and avoidance of panic contexts. Selective serotonin reuptake inhibitors (SSRIs) are effective in treating PD; however, the mechanisms underlying SSRI efficacy are poorly understood. Using CO2-inhalation, a PD-relevant translational paradigm, we examined the effect of chronic SSRI (fluoxetine) treatment on unconditioned and context-conditioned defensive behaviors, as well as respiratory responses, in mice. In addition, cFos expression was evaluated as a measure of the functional activity and interregional correlation matrices were used to explore the neurocircuitry recruited in CO2-conditioned behavior and SSRI treatment response. Chronic fluoxetine attenuated CO2-induced passive (freezing) behavior during inhalation and active (rearing) behavior on re-exposure to context, in addition to reducing CO2-evoked respiratory responses. Brain mapping in CO2-context-conditioned mice revealed altered regional neuronal activation within and correlations across midbrain regions subserving defensive behaviors (periaqueductal gray (PAG) and raphe nuclei) and forebrain emotional and contextual processing loci (medial prefrontal cortex, insular cortex and hippocampus). Importantly, fluoxetine treatment normalized these alterations. Collectively, our results provide novel information on fluoxetine modulation of panic-relevant defensive behaviors and neurocircuitry, facilitating increased understanding of panic neurobiology in the context of treatment response.


Assuntos
Dióxido de Carbono/metabolismo , Fluoxetina/farmacologia , Mesencéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Respiração/efeitos dos fármacos , Administração por Inalação , Animais , Resposta de Imobilidade Tônica/efeitos dos fármacos , Masculino , Mesencéfalo/citologia , Camundongos , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Prosencéfalo/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo
19.
Neuron ; 101(1): 133-151.e7, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30503173

RESUMO

Ventral tegmental area (VTA) dopamine (DA) neurons play a central role in mediating motivated behaviors, but the circuitry through which they signal positive and negative motivational stimuli is incompletely understood. Using in vivo fiber photometry, we simultaneously recorded activity in DA terminals in different nucleus accumbens (NAc) subnuclei during an aversive and reward conditioning task. We find that DA terminals in the ventral NAc medial shell (vNAcMed) are excited by unexpected aversive outcomes and to cues that predict them, whereas DA terminals in other NAc subregions are persistently depressed. Excitation to reward-predictive cues dominated in the NAc lateral shell and was largely absent in the vNAcMed. Moreover, we demonstrate that glutamatergic (VGLUT2-expressing) neurons in the lateral hypothalamus represent a key afferent input for providing information about aversive outcomes to vNAcMed-projecting DA neurons. Collectively, we reveal the distinct functional contributions of separate mesolimbic DA subsystems and their afferent pathways underlying motivated behaviors. VIDEO ABSTRACT.


Assuntos
Aprendizagem da Esquiva/fisiologia , Neurônios Dopaminérgicos/metabolismo , Sistema Límbico/metabolismo , Rede Nervosa/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Sistema Límbico/citologia , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/citologia , Técnicas de Cultura de Órgãos , Fotometria/métodos , Área Tegmentar Ventral/citologia , Proteína Vesicular 2 de Transporte de Glutamato/biossíntese
20.
Pharmacol Res ; 139: 452-459, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30527894

RESUMO

Astroglia serve as a critical role in metabolic and neurotrophic support to neurons. The loss of astroglia-derived neurotrophic effects could be a primary contributor to Parkinson's disease (PD). Thus, understanding astroglia functions is an important strategy for enhancing neuronal survival. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key role in neuronal resistance to oxidative stress and glutamate-induced excitotoxicity. Balancing oxidative stress by up-regulation of Nrf2 has been demonstrated to be effective in neurodegenerative disease treatment. Naringenin (NAR), a dietary flavonoid, displays anti-oxidant, cardioprotective, anti-inflammatory and neuroprotective activities. However, the molecular mechanisms underlying NAR-mediated neuroprotection against neurodegeneration remain unelucidated. Here, the present study investigated whether NAR promoted astroglial neurotrophic effects to support neurons and the underlying mechanisms as well. In primary rat midbrain neuron-glia co-cultures, NAR conferred neurotrophic effects to support dopaminergic (DA) neurons survival in the concentration- and time-dependent manners. Furtherly, astroglia were essential for NAR-mediated neurotrophic actions. Also, NAR elicited astrogliosis and neurotrophic factors release in primary neuron-glia co-cultures and astroglia-enriched cultures. Mechanistically, astroglial Nrf2 activation participated in NAR-mediated neurotrophic actions to support DA neurons evidenced by the following observations: 1) NAR increased Nrf2 mRNA and protein expressions both in neuron-glia and astroglia-enriched cultures; 2) Nrf2-siRNA inhibited NAR-mediated astrogliosis and neurotrophic factors release; 3) astroglial Nrf2-siRNA abolished NAR-mediated neurotrophic effects on DA neurons. Together, this study demonstrates NAR enhanced astroglial neurotrophic effects on DA neurons through the regulation of Nrf2 activation, and these findings might open new potential promising avenues for neurotrophic factor-based treatment of PD.


Assuntos
Astrócitos/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Flavanonas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Astrócitos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Neurônios Dopaminérgicos/metabolismo , Feminino , Mesencéfalo/citologia , Fator 2 Relacionado a NF-E2/genética , Ratos Wistar
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA