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1.
Psychoneuroendocrinology ; 133: 105393, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34481327

RESUMO

Maternal separation (MS) is a known chronic stressor in the postnatal period and when associated with another paradigm like the activity-based anorexia (ABA) rat model, causes different effects in the two sexes. In ABA females, the separation leads to increased hyperactivity and anxiety reduction, whereas, in males, the separation induces decreased locomotor activity without similar reduction of anxiety-like behaviors as observed in females. To understand the mechanisms altered by MS in synergy with the induction of the anorexic-like phenotype, we considered the reward system, which involves neurons synthesizing dopamine (DA) in the ventral tegmental area (VTA), substantia nigra pars compacta, and serotoninergic neurons in the dorsal raphe nucleus. Moreover, we analyzed the orexin circuit in the lateral hypothalamic area (LHA), which affects DA synthesis in the VTA and is also known to regulate food consumption and locomotor activity. Rats of both sexes were exposed to the two paradigms (MS and ABA), leading to four experimental groups for each sex: non-separated control (CON), non-separated ABA groups (ABA), MS control (MSCON), and MS plus ABA groups (MSABA). Immunohistochemistry analysis was performed to determine quantitative differences in the number of cells expressing DA, orexin, and serotonin (5-HT) among the experimental groups. The results showed that, in the DA system, the effect of MS was more evident in females than in males, with a substantial increase in DA cells in the VTA of MSABA. However, the analysis of the orexin system revealed a similar cellular increment in the LHA in the non-separated ABA groups of both sexes. Regarding 5-HT, there was an opposite effect in males and females of the MSABA groups, with only females showing a greater density of 5-HT cells. The changes in the reward system could partially explain the behavioral data: the hyperactivity, weight loss, and decreased anxiety levels of the MSABA females could be linked to an increase in DA and 5-HT cells, whereas in males, MS could mitigate the behavioral effects of the ABA protocol affecting the anxiety levels and locomotor activity through a lack of increased activation of the reward system.


Assuntos
Anorexia , Privação Materna , Recompensa , Animais , Anorexia/complicações , Ansiedade/complicações , Modelos Animais de Doenças , Dopamina , Núcleo Dorsal da Rafe/citologia , Feminino , Masculino , Neurônios , Orexinas , Parte Compacta da Substância Negra/citologia , Ratos , Serotonina , Área Tegmentar Ventral/citologia
2.
Nat Commun ; 12(1): 4409, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285209

RESUMO

Appetitive locomotion is essential for animals to approach rewards, such as food and prey. The neuronal circuitry controlling appetitive locomotion is unclear. In a goal-directed behavior-predatory hunting, we show an excitatory brain circuit from the superior colliculus (SC) to the substantia nigra pars compacta (SNc) to enhance appetitive locomotion in mice. This tectonigral pathway transmits locomotion-speed signals to dopamine neurons and triggers dopamine release in the dorsal striatum. Synaptic inactivation of this pathway impairs appetitive locomotion but not defensive locomotion. Conversely, activation of this pathway increases the speed and frequency of approach during predatory hunting, an effect that depends on the activities of SNc dopamine neurons. Together, these data reveal that the SC regulates locomotion-speed signals to SNc dopamine neurons to enhance appetitive locomotion in mice.


Assuntos
Comportamento Apetitivo/fisiologia , Locomoção/fisiologia , Parte Compacta da Substância Negra/fisiologia , Comportamento Predatório/fisiologia , Colículos Superiores/fisiologia , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Vias Neurais/fisiologia , Parte Compacta da Substância Negra/citologia , Técnicas Estereotáxicas , Colículos Superiores/citologia , Transmissão Sináptica/fisiologia
3.
N Engl J Med ; 382(20): 1926-1932, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32402162

RESUMO

We report the implantation of patient-derived midbrain dopaminergic progenitor cells, differentiated in vitro from autologous induced pluripotent stem cells (iPSCs), in a patient with idiopathic Parkinson's disease. The patient-specific progenitor cells were produced under Good Manufacturing Practice conditions and characterized as having the phenotypic properties of substantia nigra pars compacta neurons; testing in a humanized mouse model (involving peripheral-blood mononuclear cells) indicated an absence of immunogenicity to these cells. The cells were implanted into the putamen (left hemisphere followed by right hemisphere, 6 months apart) of a patient with Parkinson's disease, without the need for immunosuppression. Positron-emission tomography with the use of fluorine-18-L-dihydroxyphenylalanine suggested graft survival. Clinical measures of symptoms of Parkinson's disease after surgery stabilized or improved at 18 to 24 months after implantation. (Funded by the National Institutes of Health and others.).


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Doença de Parkinson/terapia , Parte Compacta da Substância Negra/citologia , Idoso , Animais , Gânglios da Base/diagnóstico por imagem , Gânglios da Base/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/transplante , Seguimentos , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Masculino , Camundongos , Camundongos SCID , Doença de Parkinson/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Putamen/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Transplante Autólogo , Transplante Homólogo
4.
PLoS Genet ; 15(8): e1008352, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31449520

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD.


Assuntos
Neurônios Dopaminérgicos/patologia , Plasticidade Neuronal/genética , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/patologia , Receptores de Dopamina D2/genética , Animais , Axônios/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Fosforilação Oxidativa , Doença de Parkinson/genética , Parte Compacta da Substância Negra/citologia , Receptores de Dopamina D2/metabolismo
5.
Neuropharmacology ; 158: 107705, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31301335

RESUMO

Dopamine neurons in the substantia nigra zona compacta (SNC) are well known to express D2 receptors. When dopamine is released from somatodendritic sites, activation of D2 autoreceptors suppresses dopamine neuronal activity through activation of G protein-coupled K+ channels. AMP-activated protein kinase (AMPK) is a master enzyme that acts in somatic tissues to suppress energy expenditure and encourage energy production. We hypothesize that AMPK may also conserve energy in central neurons by reducing desensitization of D2 autoreceptors. We used whole-cell patch-clamp recordings to study the effects of AMPK activators and inhibitors on D2 autoreceptor-mediated current in SNC neurons in midbrain slices from rat pups (11-23 days post-natal). Slices were superfused with 100 µM dopamine or 30 µM quinpirole for 25 min, which evoked outward currents that decayed slowly over time. Although the AMPK activators A769662 and ZLN024 significantly slowed rundown of dopamine-evoked current, slowing of quinpirole-evoked current required the presence of a D1-like agonist (SKF38393). Moreover, the D1-like agonist also slowed the rundown of quinpirole-induced current even in the absence of an AMPK activator. Pharmacological antagonist experiments showed that the D1-like agonist effect required activation of either protein kinase A (PKA) or exchange protein directly activated by cAMP 2 (Epac2) pathways. In contrast, the effect of AMPK on rundown of current evoked by quinpirole plus SKF38393 required PKA but not Epac2. We conclude that AMPK slows D2 autoreceptor desensitization by augmenting the effect of D1-like receptors.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autorreceptores/metabolismo , Agonistas de Dopamina/farmacologia , Dopamina/farmacologia , Neurônios/metabolismo , Parte Compacta da Substância Negra/citologia , Quimpirol/farmacologia , Receptores de Dopamina D2/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Animais , Autorreceptores/efeitos dos fármacos , Compostos de Bifenilo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativadores de Enzimas/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Pirimidinas/farmacologia , Pironas/farmacologia , Ratos , Receptores de Dopamina D2/efeitos dos fármacos , Tiofenos/farmacologia
6.
JCI Insight ; 52019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31120439

RESUMO

Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/genética , Parte Compacta da Substância Negra/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Dopamina D2/metabolismo , alfa-Sinucleína/metabolismo , Fatores Etários , Idade de Início , Animais , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/patologia , Humanos , Locomoção , Camundongos , Camundongos Knockout , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/patologia , Quimpirol/farmacologia , Transmissão Sináptica
7.
Int J Mol Sci ; 20(3)2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699944

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder defined by progressive deterioration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Dental pulp stem cells (DPSCs) have been proposed to replace the degenerated dopaminergic neurons due to its inherent neurogenic and regenerative potential. However, the effective delivery and homing of DPSCs within the lesioned brain has been one of the many obstacles faced in cell-based therapy of neurodegenerative disorders. We hypothesized that DPSCs, delivered intranasally, could circumvent these challenges. In the present study, we investigated the therapeutic efficacy of intranasally administered DPSCs in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Human deciduous DPSCs were cultured, pre-labelled with PKH 26, and intranasally delivered into PD mice following MPTP treatment. Behavioural analyses were performed to measure olfactory function and sensorimotor coordination, while tyrosine hydroxylase (TH) immunofluorescence was used to evaluate MPTP neurotoxicity in SNpc neurons. Upon intranasal delivery, degenerated TH-positive neurons were ameliorated, while deterioration in behavioural performances was significantly enhanced. Thus, the intranasal approach enriched cell delivery to the brain, optimizing its therapeutic potential through its efficacious delivery and protection against dopaminergic neuron degeneration.


Assuntos
Polpa Dentária/citologia , Intoxicação por MPTP/terapia , Doença de Parkinson/terapia , Parte Compacta da Substância Negra/citologia , Células-Tronco/fisiologia , Animais , Comportamento Animal , Diferenciação Celular/fisiologia , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Humanos , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Degeneração Neural/metabolismo , Degeneração Neural/terapia , Doença de Parkinson/metabolismo , Parte Compacta da Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Eur J Neurosci ; 46(11): 2746-2753, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29057540

RESUMO

ATP-sensitive K+ (K-ATP) channels play significant roles in regulating the excitability of dopamine neurons in the substantia nigra zona compacta (SNC). We showed previously that K-ATP channel function is up-regulated by AMP-activated protein kinase (AMPK). This study extended these studies to the neurons adjacent to the SNC in the ventral tegmental area (VTA). Using patch pipettes to record whole-cell currents in slices of rat midbrain, we found that the AMPK activator A769662 increased the amplitude of currents evoked by the K-ATP channel opener diazoxide in presumed dopamine-containing VTA neurons. However, current evoked by diazoxide with A769662 was significantly smaller in VTA neurons compared to SNC neurons. Moreover, a significantly lower proportion of VTA neurons responded to diazoxide with outward current. However, A769662 was able to increase the incidence of diazoxide-responsive neurons in the VTA. In contrast, A769662 did not potentiate diazoxide-evoked currents in presumed non-dopamine VTA neurons. These results show that AMPK activation augments K-ATP currents in presumed dopamine neurons in the VTA and SNC, although diazoxide-evoked currents remain less robust in the VTA. We conclude that K-ATP channels may play important physiological roles in VTA and SNC dopamine neurons.


Assuntos
Adenilato Quinase/metabolismo , Canais KATP/metabolismo , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo , Animais , Compostos de Bifenilo , Diazóxido/farmacologia , Neurônios Dopaminérgicos/fisiologia , Sinergismo Farmacológico , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Pironas/farmacologia , Ratos , Tiofenos/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos
9.
Mol Pharmacol ; 92(6): 640-652, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29025968

RESUMO

The autophagy-lysosome pathway (ALP) plays a critical role in the pathology of Parkinson's disease (PD). Clk1 (coq7) is a mitochondrial hydroxylase that is essential for coenzyme Q (ubiquinone) biosynthesis. We have reported previously that Clk1 regulates microglia activation via modulating microglia metabolic reprogramming, which contributes to dopaminergic neuronal survival. This study explores the direct effect of Clk1 on dopaminergic neuronal survival. We demonstrate that Clk1 deficiency inhibited dopaminergic neuronal autophagy in cultured MN9D dopaminergic neurons and in the substantia nigra pars compacta of Clk+/- mutant mice and consequently sensitized dopaminergic neuron damage and behavioral defects. These mechanistic studies indicate that Clk1 regulates the AMP-activated protein kinase (AMPK)/rapamycin complex 1 pathway, which in turn impairs the ALP and TFEB nuclear translocation. As a result, Clk1 deficiency promotes dopaminergic neuronal damage in vivo and in vitro, which ultimately contributes to sensitizing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal death and behavioral impairments in Clk1-deficient mice. Moreover, we found that activation of autophagy by the AMPK activator metformin increases dopaminergic neuronal survival in vitro and in the MPTP-induced PD model in Clk1 mutant mice. These results reveal that Clk1 plays a direct role in dopaminergic neuronal survival via regulating ALPs that may contribute to the pathologic development of PD. Modulation of Clk1 activity may represent a potential therapeutic target for PD.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Neurônios Dopaminérgicos/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Membrana/metabolismo , Metformina/farmacologia , Proteínas Mitocondriais/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Sobrevivência Celular , Células Cultivadas , Dopaminérgicos/farmacologia , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/genética , Oxigenases de Função Mista , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/efeitos dos fármacos
10.
Endocr Regul ; 51(2): 73-83, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28609288

RESUMO

OBJECTIVE: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning. METHODS: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 µl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination. RESULTS: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA. CONCLUSIONS: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.


Assuntos
Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Condicionamento Psicológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Estresse Psicológico/metabolismo , Tirosina 3-Mono-Oxigenase/efeitos dos fármacos , Animais , Área Postrema/citologia , Área Postrema/efeitos dos fármacos , Área Postrema/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Catecolaminas/biossíntese , Dibenzocicloeptenos , Imuno-Histoquímica , Locus Cerúleo/citologia , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/metabolismo , Masculino , Bulbo/citologia , Bulbo/efeitos dos fármacos , Bulbo/metabolismo , Microscopia de Fluorescência , Neurônios/metabolismo , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/metabolismo , Parte Reticular da Substância Negra/citologia , Parte Reticular da Substância Negra/efeitos dos fármacos , Parte Reticular da Substância Negra/metabolismo , Ponte/citologia , Ponte/efeitos dos fármacos , Ponte/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Núcleo Solitário/citologia , Núcleo Solitário/efeitos dos fármacos , Núcleo Solitário/metabolismo , Estresse Psicológico/psicologia , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismo
11.
Exp Neurol ; 287(Pt 1): 34-43, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27771354

RESUMO

Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01-5.0µM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1µM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Inseticidas/farmacologia , Locus Cerúleo/citologia , Neurônios/efeitos dos fármacos , Parte Compacta da Substância Negra/citologia , Rotenona/farmacologia , Animais , Animais Recém-Nascidos , Anti-Hipertensivos/farmacologia , Cálcio/metabolismo , Diazóxido/farmacologia , Peróxido de Hidrogênio/metabolismo , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Neurônios/classificação , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/metabolismo , Tolbutamida/farmacologia
12.
PLoS One ; 11(10): e0164094, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27788145

RESUMO

The neurotoxicity of paraquat dichloride (PQ) was assessed in two inbred strains of 9- or 16-week old male C57BL/6 mice housed in two different laboratories and compared to the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PQ was administered by intraperitoneal injections; either once (20 mg/kg) or twice (10 mg/kg) weekly for 3 weeks, while MPTP-HCl was injected 4 times on a single day (20 mg/kg/dose). Brains were collected 8, 16, 24, 48, 96 or 168 hours after the last PQ treatment, and 48 or 168 hours after MPTP treatment. Dopamine neurons in the substantia nigra pars compacta (SNpc) were identified by antibodies to tyrosine hydroxylase (TH+) and microglia were identified using Iba-1 immunoreactivity. The total number of TH+ neurons and the number of resting and activated microglia in the SNpc at 168 hours after the last dose were estimated using model- or design-based stereology, with investigators blinded to treatment. In a further analysis, a pathologist, also blinded to treatment, evaluated the SNpc and/or striatum for loss of TH+ neurons (SNpc) or terminals (striatum), cell death (as indicated by amino cupric silver uptake, TUNEL and/or caspase 3 staining) and neuroinflammation (as indicated by Iba-1 and/or GFAP staining). PQ, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of TH+ neurons or microglia in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. PQ did not induce neuronal cell loss or degeneration in the SNpc or striatum. Additionally, there was no evidence of apoptosis, microgliosis or astrogliosis. In MPTP-treated mice, the number of TH+ neurons in the SNpc was significantly decreased and the number of activated microglia increased. Histopathological assessment found degenerating neurons/terminals in the SNpc and striatum but no evidence of apoptotic cell death. MPTP activated microglia in the SNpc and increased the number of astrocytes in the SNpc and striatum.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Intoxicação por MPTP/patologia , Microglia/efeitos dos fármacos , Paraquat/toxicidade , Parte Compacta da Substância Negra/citologia , Animais , Peso Corporal/efeitos dos fármacos , Contagem de Células , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Ingestão de Alimentos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/patologia , Parte Compacta da Substância Negra/patologia , Análise de Sobrevida , Tirosina 3-Mono-Oxigenase/metabolismo
13.
Sci Rep ; 6: 30615, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27477243

RESUMO

The rodent ventral tegmental area (VTA) and substantia nigra pars compacta (SNC) contain dopamine neurons intermixed with glutamate neurons (expressing vesicular glutamate transporter 2; VGluT2), which play roles in reward and aversion. However, identifying the neuronal compositions of the VTA and SNC in higher mammals has remained challenging. Here, we revealed VGluT2 neurons within the VTA and SNC of nonhuman primates and humans by simultaneous detection of VGluT2 mRNA and tyrosine hydroxylase (TH; for identification of dopamine neurons). We found that several VTA subdivisions share similar cellular compositions in nonhuman primates and humans; their rostral linear nuclei have a high prevalence of VGluT2 neurons lacking TH; their paranigral and parabrachial pigmented nuclei have mostly TH neurons, and their parabrachial pigmented nuclei have dual VGluT2-TH neurons. Within nonhuman primates and humans SNC, the vast majority of neurons are TH neurons but VGluT2 neurons were detected in the pars lateralis subdivision. The demonstration that midbrain dopamine neurons are intermixed with glutamate or glutamate-dopamine neurons from rodents to humans offers new opportunities for translational studies towards analyzing the roles that each of these neurons play in human behavior and in midbrain-associated illnesses such as addiction, depression, schizophrenia, and Parkinson's disease.


Assuntos
Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Mesencéfalo/citologia , Neurônios/metabolismo , Animais , Callithrix , Humanos , Masculino , Mesencéfalo/metabolismo , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
14.
An Acad Bras Cienc ; 88(3): 1439-50, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27508995

RESUMO

The Portulaca oleracea L. (Portulacaceae) is a cosmopolitan species with a wide range of biological activities, including antioxidant and neuroprotective actions. We investigated the effects of P. oleracea extracts in a 6-hydroxydopamine rat model of Parkinson's disease, a debilitating disorder without effective treatments. Chemical profiles of aqueous and ethanolic extracts of whole plant were analyzed by thin layer chromatography and the antioxidant activity was assessed by 2,2-diphenyl-1-picrilhidrazila method. Male Wistar rats received intrastriatal 6-hydroxydopamine and were treated with vehicle or extracts (oral, 200 and 400 mg/kg) daily for two weeks. The behavioral open field test was conducted at days 1 and 15. Immunohistochemical analysis was performed 4 weeks after surgery to quantify tyrosine-hydroxylase cell counts in the substantia nigra pars compacta. Extracts presented antioxidant activity in concentrations above 300 µg/kg. The chromatographic analysis revealed the presence of Levodopa, alkaloids, flavonoids, saponins, tannins, terpenoids and polysaccharides. Both extracts improved motor recovery 15 days after lesion and protected from tyrosine-hydroxylase cell loss after 4 weeks, but these effects were more evident for the aqueous extract. Because the dopamine precursor is present, in addition to antioxidant compounds and neuroprotective effects, P. oleracea can be considered as potential strategy for treating Parkinson's disease.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oxidopamina/toxicidade , Portulaca/química , Simpatolíticos/toxicidade , Animais , Neurônios Dopaminérgicos/enzimologia , Masculino , Oxidopamina/antagonistas & inibidores , Doença de Parkinson/tratamento farmacológico , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/enzimologia , Ratos , Ratos Wistar , Simpatolíticos/antagonistas & inibidores , Tirosina 3-Mono-Oxigenase/análise
15.
Cell Death Dis ; 7: e2217, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27148690

RESUMO

Glial cell line-derived neurotrophic factor (GDNF) has strong neuroprotective and neurorestorative effects on dopaminergic (DA) neurons in the substantia nigra (SN); however, the underlying molecular mechanisms remain to be fully elucidated. In this study, we found that the expression level of transcription factor Six2 was increased in damaged DA neurons after GDNF rescue in vivo and in vitro. Knockdown of Six2 resulted in decreased cell viability and increased the apoptosis of damaged DA neurons after GDNF treatment in vitro. In contrast, Six2 overexpression increased cell viability and decreased cell apoptosis. Furthermore, genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) indicated that Six2 directly bound to the promoter CAGCTG sequence of smad ubiquitylation regulatory factor 1 (Smurf1). ChIP-quantitative polymerase chain reaction (qPCR) analysis showed that Smurf1 expression was significantly upregulated after GDNF rescue. Moreover, knockdown of Six2 decreased Smurf1 expression, whereas overexpression of Six2 increased Smurf1 expression in damaged DA neurons after GDNF rescue. Meanwhile, knockdown and overexpression of Smurf1 increased and decreased p53 expression, respectively. Taken together, our results from in vitro and in vivo analysis indicate that Six2 mediates the protective effects of GDNF on damaged DA neurons by regulating Smurf1 expression, which could be useful in identifying potential drug targets for injured DA neurons.


Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Proteínas de Homeodomínio/genética , Fármacos Neuroprotetores/farmacologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Motivos de Nucleotídeos , Oxidopamina/antagonistas & inibidores , Oxidopamina/toxicidade , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Técnicas Estereotáxicas , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
16.
Elife ; 52016 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-27074662

RESUMO

G protein gated inward rectifier K(+) (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gßγ subunits bind cooperatively to open GIRK2, and that intracellular Na(+) - which enters neurons during action potentials - further amplifies opening mostly by increasing Gßγ affinity. A Na(+) amplification function is characterized and used to estimate the concentration of Gßγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gßγ and Na(+), mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Potenciais de Ação/fisiologia , Animais , Bioensaio , Neurônios Dopaminérgicos/citologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/metabolismo , Técnicas de Patch-Clamp , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Pichia/genética , Pichia/metabolismo , Cultura Primária de Células , Multimerização Proteica , Subunidades Proteicas/genética , Proteolipídeos/química , Proteolipídeos/metabolismo , Receptores de GABA-B/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Sódio/metabolismo
17.
Neurobiol Aging ; 36(12): 3321-3333, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26433682

RESUMO

Age being a risk factor for Parkinson's disease, assessment of age-related changes in the human substantia nigra may elucidate its pathogenesis. Increase in Marinesco bodies, α-synuclein, free radicals and so forth in the aging nigral neurons are clear indicators of neurodegeneration. Here, we report the glial responses in aging human nigra. The glial numbers were determined on Nissl-stained sections. The expression of glial fibrillary acidic protein, S100ß, 2', 3'-cyclic nucleotide 3' phosphodiesterase, and Iba1 was assessed on cryosections of autopsied midbrains by immunohistochemistry and densitometry. The glial counts showed a biphasic increase, of which, the first prominent phase from fetal age to birth could be physiological gliogenesis whereas the second one after middle age may reflect mild age-related gliosis. Astrocytic morphology was altered, but glial fibrillary acidic protein expression increased only mildly. Presence of type-4 microglia suggests possibility of neuroinflammation. Mild reduction in 2', 3'-cyclic nucleotide 3' phosphodiesterase-labeled area denotes subtle demyelination. Stable age-related S100ß expression indicates absence of calcium overload. Against the expected prominent gliosis, subtle age-related morphological alterations in human nigral glia attribute them a participatory role in aging.


Assuntos
Envelhecimento/patologia , Astrócitos/patologia , Microglia/patologia , Degeneração Neural , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/patologia , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrócitos/metabolismo , Cálcio/metabolismo , Criança , Pré-Escolar , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Lactente , Masculino , Microglia/metabolismo , Pessoa de Meia-Idade , Inflamação Neurogênica , Doença de Parkinson , Fatores de Risco , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Adulto Jovem
18.
Brain Res ; 1622: 240-51, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26141374

RESUMO

A pathological hallmark of Parkinson׳s disease (PD) is progressive degeneration of nigrostriatal dopamine (NSDA) neurons, which underlies the motor symptoms of PD. While there is severe loss of midbrain NSDA neurons, tuberoinfundibular (TI) DA neurons in the mediobasal hypothalamus (MBH) remain intact. In the present study, confocal microscopic analysis revealed that mitochondrial content and numbers of mitophagosomes were lower in NSDA neuronal cell bodies in the substantia nigra pars compacta (SNpc) compared to TIDA neuronal cell bodies in the arcuate nucleus (ARC) of C57BL/6J male mice. Mitochondrial respiration, mass, membrane potential and morphology were determined using bioenergetic, flow cytometric and transmission electron microscopic analyses of synaptosomes isolated from discrete brain regions containing axon terminals of NSDA and TIDA neurons. Maximum and spare respiratory capacities, and mitochondrial mass were lower in synaptosomal mitochondria derived from the striatum (ST) as compared with the MBH, which correlated with lower numbers of mitochondria per synaptosome in these brain regions. In contrast, there was no regional difference in mitochondrial basal, maximum or spare respirations following inhibition of Complex I activity with rotenone. These results reveal that higher numbers of viable mitochondria are correlated with more extensive autophagic mitochondrial quality maintenance in TIDA neurons as compared with NSDA neurons.


Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Autofagia/fisiologia , Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/metabolismo , Parte Compacta da Substância Negra/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/citologia , Respiração Celular/fisiologia , Corpo Estriado/citologia , Neurônios Dopaminérgicos/citologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Vias Neurais/citologia , Vias Neurais/metabolismo , Parte Compacta da Substância Negra/citologia , Rotenona/farmacologia , Sinaptossomos/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Desacopladores/farmacologia
19.
PLoS One ; 10(7): e0133957, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26222442

RESUMO

Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson's disease (PD). Whereas the globus pallidus interna (GPi) has been less commonly targeted than the subthalamic nucleus (STN), a recent clinical trial suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Several laboratories have demonstrated that DBS of the STN provides neuroprotection of substantia nigra pars compacta (SNpc) dopamine neurons in preclinical neurotoxin models of PD and increases brain-derived neurotrophic factor (BDNF). However, whether DBS of the entopeduncular nucleus (EP), the homologous structure to the GPi in the rat, has similar neuroprotective potential in preclinical models has not been investigated. We investigated the impact of EP DBS on forelimb use asymmetry and SNpc degeneration induced by 6-hydroxydopamine (6-OHDA) and on BDNF levels. EP DBS in male rats received unilateral, intrastriatal 6-OHDA and ACTIVE or INACTIVE stimulation continuously for two weeks. Outcome measures included quantification of contralateral forelimb use, stereological assessment of SNpc neurons and BDNF levels. EP DBS 1) did not ameliorate forelimb impairments induced by 6-OHDA, 2) did not provide neuroprotection for SNpc neurons and 3) did not significantly increase BDNF levels in any of the structures examined. These results are in sharp contrast to the functional improvement, neuroprotection and BDNF-enhancing effects of STN DBS under identical experimental parameters in the rat. The lack of functional response to EP DBS suggests that stimulation of the rat EP may not represent an accurate model of clinical GPi stimulation.


Assuntos
Estimulação Encefálica Profunda , Núcleo Entopeduncular/efeitos dos fármacos , Núcleo Entopeduncular/fisiologia , Neuroproteção , Oxidopamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Núcleo Entopeduncular/citologia , Núcleo Entopeduncular/metabolismo , Masculino , Neuroproteção/efeitos dos fármacos , Parte Compacta da Substância Negra/citologia , Parte Compacta da Substância Negra/efeitos dos fármacos , Parte Compacta da Substância Negra/fisiologia , Ratos , Ratos Sprague-Dawley
20.
Neuroscience ; 295: 23-38, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25796140

RESUMO

Exercise reduces the risk of developing a number of neurological disorders and increases the efficiency of cellular energy production. However, overly strenuous exercise produces oxidative stress. Proper oxygenation is crucial for the health of all tissues, and tight regulation of cellular oxygen is critical to balance O2 levels and redox homeostasis in the brain. Hypoxia Inducible Factor (HIF)1α and HIF2α are transcription factors regulated by cellular oxygen concentration that initiate gene regulation of vascular development, redox homeostasis, and cell cycle control. HIF1α and HIF2α contribute to important adaptive mechanisms that occur when oxygen and ROS homeostasis become unbalanced. It has been shown that preconditioning by exposure to a stressor prior to a hypoxic event reduces damage that would otherwise occur. Previously we reported that 3 months of exercise protects SNpc dopaminergic (DA) neurons from toxicity caused by Complex I inhibition. Here, we identify the cells in the SNpc that express HIF1α and HIF2α and show that running exercise produces hypoxia in SNpc DA neurons, and alters the expression of HIF1α and HIF2α. In mice carrying a conditional knockout of Hif1α in postnatal neurons we observe that exercise alone produces SNpc TH+ DA neuron loss. Loss of HIF1α also abolishes exercise-induced neuroprotection. In mice lacking Hif2α in postnatal neurons, the number of TH+ DA neurons in the adult SNpc is diminished, but 3months of exercise rescues this loss. We conclude that HIF1α is necessary for exercise-induced neuroprotection and both HIF1α and HIF2α are necessary for the survival and function of adult SNpc DA neurons.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Intoxicação por MPTP/reabilitação , Parte Compacta da Substância Negra/citologia , Condicionamento Físico Animal/métodos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , beta-Galactosidase/metabolismo
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