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1.
Mol Ther Methods Clin Dev ; 31: 101106, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-37766790

RESUMO

Despite its established neuroprotective effect on dopaminergic neurons and encouraging phase I results, intraputaminal GDNF administration failed to demonstrate significant clinical benefits in Parkinson's disease patients. Different human GDNF doses were delivered in the striatum of rats with a progressive 6-hydroxydopamine lesion using a sensitive doxycycline-regulated AAV vector. GDNF treatment was applied either continuously or intermittently (2 weeks on/2 weeks off) during 17 weeks. Stable reduction of motor impairments as well as increased number of dopaminergic neurons and striatal innervation were obtained with a GDNF dose equivalent to 3- and 10-fold the rat endogenous level. In contrast, a 20-fold increased GDNF level only temporarily provided motor benefits and neurons were not spared. Strikingly, oxidized DNA in the substantia nigra increased by 50% with 20-fold, but not 3-fold GDNF treatment. In addition, only low-dose GDNF allowed to preserve dopaminergic neuron cell size. Finally, aberrant dopaminergic fiber sprouting was observed with 20-fold GDNF but not at lower doses. Intermittent 20-fold GDNF treatment allowed to avoid toxicity and spare dopaminergic neurons but did not restore their cell size. Our data suggest that maintaining GDNF concentration under a threshold generating oxidative stress is a pre-requisite to obtain significant symptomatic relief and neuroprotection.

2.
Sensors (Basel) ; 21(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073127

RESUMO

Huntington's disease (HD) is an autosomal dominant, incurable neurodegenerative disease caused by mutation in the huntingtin gene (HTT). HTT mutation leads to protein misfolding and aggregation, which affect cells' functions and structural features. Because these changes might modify the scattering strength of affected cells, we propose that random lasing (RL) is an appropriate technique for detecting cells that express mutated HTT. To explore this hypothesis, we used a cell model of HD based on the expression of two different forms-pathogenic and non-pathogenic-of HTT. The RL signals from both cell profiles were compared. A multivariate statistical analysis of the RL signals based on the principal component analysis (PCA) and linear discriminant analysis (LDA) techniques revealed substantial differences between cells that expressed the pathogenic and the non-pathogenic forms of HTT.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Mutação
3.
Pharmacol Res ; 165: 105434, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33484816

RESUMO

The dopamine transporter (DAT) is a membrane glycoprotein in dopaminergic neurons, which modulates extracellular and intracellular dopamine levels. DAT is regulated by different presynaptic proteins, including dopamine D2 (D2R) and D3 (D3R) receptors. While D2R signalling enhances DAT activity, some data suggest that D3R has a biphasic effect. However, despite the extensive therapeutic use of D2R/D3R agonists in neuropsychiatric disorders, this phenomenon has been little studied. In order to shed light on this issue, DAT activity, expression and posttranslational modifications were studied in mice and DAT-D3R-transfected HEK cells. Consistent with previous reports, acute treatment with D2R/D3R agonists promoted DAT recruitment to the plasma membrane and an increase in DA uptake. However, when the treatment was prolonged, DA uptake and total striatal DAT protein declined below basal levels. These effects were inhibited in mice by genetic and pharmacological inactivation of D3R, but not D2R, indicating that they are D3R-dependent. No changes were detected in mesostriatal tyrosine hydroxylase (TH) protein expression and midbrain TH and DAT mRNAs, suggesting that the dopaminergic system is intact and DAT is posttranslationally regulated. The use of immunoprecipitation and cell surface biotinylation revealed that DAT is phosphorylated at serine residues, ubiquitinated and released into late endosomes through a PKCß-dependent mechanism. In sum, the results indicate that long-term D3R activation promotes DAT down-regulation, an effect that may underlie neuroprotective and antidepressant actions described for some D2R/D3R agonists.


Assuntos
Agonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Receptores de Dopamina D3/metabolismo , Ubiquitinação/fisiologia , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pramipexol/farmacologia , Receptores de Dopamina D3/agonistas , Ubiquitinação/efeitos dos fármacos
4.
Autophagy ; 16(7): 1279-1295, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31538542

RESUMO

Growing evidence shows that autophagy is deficient in neurodegenerative and psychiatric diseases, and that its induction may have beneficial effects in these conditions. However, as autophagy shares signaling pathways with cell death and interferes with protein synthesis, prolonged use of autophagy inducers available nowadays is considered unwise. The search for novel autophagy inducers indicates that DRD2 (dopamine receptor 2)-DRD3 ligands may also activate autophagy, though critical aspects of the action mechanisms and effects of dopamine ligands on autophagy are still unknown. In order to shed light on this issue, DRD2- and DRD3-overexpressing cells and drd2 KO, drd3 KO and wild-type mice were treated with the DRD2-DRD3 agonist pramipexole. The results revealed that pramipexole induces autophagy through MTOR inhibition and a DRD3-dependent but DRD2-independent mechanism. DRD3 activated AMPK followed by inhibitory phosphorylation of RPTOR, MTORC1 and RPS6KB1 inhibition and ULK1 activation. Interestingly, despite RPS6KB1 inhibition, the activity of RPS6 was maintained through activation of the MAPK1/3-RPS6KA pathway, and the activity of MTORC1 kinase target EIF4EBP1 along with protein synthesis and cell viability, were also preserved. This pattern of autophagy through MTORC1 inhibition without suppression of protein synthesis, contrasts with that of direct allosteric and catalytic MTOR inhibitors and opens up new opportunities for G protein-coupled receptor ligands as autophagy inducers in the treatment of neurodegenerative and psychiatric diseases. ABBREVIATIONS: AKT/Protein kinase B: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; BECN1: beclin 1; EGFP: enhanced green fluorescent protein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GPCR; G protein-coupled receptor; GFP: green fluorescent protein; HEK: human embryonic kidney; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MDA: malonildialdehyde; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPX: pramipexole; RPTOR/raptor: regulatory associated protein of MTOR, complex 1; RPS6: ribosomal protein S6; RPS6KA/p90S6K: ribosomal protein S6 kinase A; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.


Assuntos
Autofagia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Biossíntese de Proteínas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Pramipexol/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proto-Oncogene Mas , Proteína S6 Ribossômica/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia
5.
Exp Neurol ; 299(Pt A): 137-147, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29056363

RESUMO

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.


Assuntos
Benzotiazóis/uso terapêutico , Agonistas de Dopamina/uso terapêutico , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Neostriado/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Receptores de Dopamina D3/efeitos dos fármacos , Animais , Autofagia , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Movimento , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Pramipexol , Complexo de Endopeptidases do Proteassoma
6.
Mol Ther Methods Clin Dev ; 5: 16027, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27069954

RESUMO

Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.

7.
Neurobiol Dis ; 88: 44-54, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26777664

RESUMO

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.


Assuntos
Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Análise de Variância , Animais , Membrana Celular/metabolismo , Corpo Estriado/citologia , Dopamina/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Ligadura , Masculino , Ratos , Ratos Sprague-Dawley , Transdução Genética , Trítio/metabolismo , alfa-Sinucleína/metabolismo
8.
Neurobiol Dis ; 74: 325-35, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25511804

RESUMO

The dopamine (DA) transporter (DAT), a membrane glycoprotein expressed in dopaminergic neurons, clears DA from extracellular space and is regulated by diverse presynaptic proteins like protein kinases, α-synuclein, D2 and D3 autoreceptors. DAT dysfunction is implicated in Parkinson's disease and depression, which are therapeutically treated by dopaminergic D2/D3 receptor (D2/D3R) agonists. It is, then, important to improve our understanding of interactions between D3R and DAT. We show that prolonged administration of pramipexole (0.1mg/kg/day, 6 to 21 days), a preferential D3R agonist, leads to a decrease in DA uptake in mouse striatum that reflects a reduction in DAT affinity for DA in the absence of any change in DAT density or subcellular distribution. The effect of pramipexole was absent in mice with genetically-deleted D3R (D3R(-/-)), yet unaffected in mice genetically deprived of D2R (D2R(-/-)). Pramipexole treatment induced a physical interaction between D3R and DAT, as assessed by co-immunoprecipitation and in situ proximity ligation assay. Furthermore, it promoted the formation of DAT dimers and DAT association with both D2R and α-synuclein, effects that were abolished in D3R(-/-) mice, yet unaffected in D2R(-/-) mice, indicating dependence upon D3R. Collectively, these data suggest that prolonged treatment with dopaminergic D3 agonists provokes a reduction in DA reuptake by dopaminergic neurons related to a hitherto-unsuspected modification of the DAT interactome. These observations provide novel insights into the long-term antiparkinson, antidepressant and additional clinical actions of pramipexole and other D3R agonists.


Assuntos
Autorreceptores/metabolismo , Benzotiazóis/farmacologia , Corpo Estriado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Dopamina/metabolismo , Receptores de Dopamina D3/metabolismo , Animais , Antidepressivos/farmacologia , Antiparkinsonianos/farmacologia , Corpo Estriado/metabolismo , Dimerização , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pramipexol , Receptores de Dopamina D3/agonistas , Receptores de Dopamina D3/genética , alfa-Sinucleína/metabolismo
9.
Front Neuroanat ; 8: 84, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25206324

RESUMO

Nowadays it is assumed that besides its roles in neuronal processing, dopamine (DA) is also involved in the regulation of cerebral blood flow. However, studies on the hemodynamic actions of DA have been mainly focused on the cerebral cortex, but the possibility that vessels in deeper brain structures receive dopaminergic axons and the origin of these axons have not been investigated. Bearing in mind the evidence of changes in the blood flow of basal ganglia in Parkinson's disease (PD), and the pivotal role of the dopaminergic mesostriatal pathway in the pathophysiology of this disease, here we studied whether striatal vessels receive inputs from midbrain dopaminergic neurons. The injection of an anterograde neuronal tracer in combination with immunohistochemistry for dopaminergic, vascular and astroglial markers, and dopaminergic lesions, revealed that midbrain dopaminergic axons are in close apposition to striatal vessels and perivascular astrocytes. These axons form dense perivascular plexuses restricted to striatal regions in rats and monkeys. Interestingly, they are intensely immunoreactive for tyrosine hydroxylase (TH) phosphorylated at Ser19 and Ser40 residues. The presence of phosphorylated TH in vessel terminals indicates they are probably the main source of basal TH activity in the striatum, and that after activation of midbrain dopaminergic neurons, DA release onto vessels precedes that onto neurons. Furthermore, the relative weight of this "vascular component" within the mesostriatal pathway suggests that it plays a relevant role in the pathophysiology of PD.

10.
J Neurosci ; 33(6): 2684-96, 2013 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-23392695

RESUMO

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K(+) conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP(2)) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP(2) binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk-SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.


Assuntos
Proteínas Imediatamente Precoces/biossíntese , Neurônios/enzimologia , Proteínas Serina-Treonina Quinases/biossíntese , Convulsões/enzimologia , Convulsões/prevenção & controle , Regulação para Cima/fisiologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Xenopus laevis
11.
Pediatrics ; 129(6): e1616-20, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22641764

RESUMO

We present 2 term newborn infants with apneic seizure originating in the occipital lobe that was diagnosed by video-EEG. One infant had ischemic infarction in the distribution of the posterior cerebral artery, extending to the cingulate gyrus. In the other infant, only transient occipital hyperechogenicity was observed by using neurosonography. In both cases, although the critical EEG discharge was observed at the occipital level, the infants presented no clinical manifestations. In patient 1, the discharge extended to the temporal lobe first, with subtle motor manifestations and tachycardia, then synchronously to both hemispheres (with bradypnea/hypopnea), and the background EEG activity became suppressed, at which point the infant experienced apnea. In patient 2, background EEG activity became suppressed right at the end of the focal discharge, coinciding with the appearance of apnea. In neither case did the clinical description by observers coincide with video-EEG findings. The existence of connections between the posterior limbic cortex and the temporal lobe and midbrain respiratory centers may explain the clinical symptoms recorded in these 2 cases. The novel features reported here include video-EEG capture of apneic seizure, ischemic lesion in the territory of the posterior cerebral artery as the cause of apneic seizure, and the appearance of apnea when the epileptiform ictal discharge extended to other cerebral areas or when EEG activity became suppressed. To date, none of these clinical findings have been previously reported. We believe this pathology may in fact be fairly common, but that video-EEG monitoring is essential for diagnosis.


Assuntos
Apneia/fisiopatologia , Eletroencefalografia/métodos , Lobo Occipital/fisiopatologia , Convulsões/fisiopatologia , Gravação em Vídeo/métodos , Apneia/complicações , Apneia/diagnóstico , Eletroencefalografia/instrumentação , Feminino , Humanos , Recém-Nascido , Masculino , Convulsões/complicações , Convulsões/diagnóstico
12.
Am J Physiol Cell Physiol ; 302(6): C868-79, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22159085

RESUMO

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, ß, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of ß- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αßγ-channels.


Assuntos
Endocitose/fisiologia , Canais Epiteliais de Sódio/metabolismo , Neurônios/metabolismo , Idoso , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Córtex Cerebral/citologia , Clonagem Molecular , Dinaminas/antagonistas & inibidores , Feminino , Células HEK293 , Humanos , Hidrazonas/farmacologia , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oócitos , Técnicas de Patch-Clamp/métodos , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , Xenopus laevis
13.
Front Neuroanat ; 4: 140, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21079748

RESUMO

The term vulnerability was first associated with the midbrain dopaminergic neurons 85 years ago, before they were identified as monoaminergic neurons, when Foix and Nicolesco (1925) reported the loss of neuromelanin containing neurons in the midbrain of patients with post-encephalitic Parkinson's disease (PD). A few years later, Hassler (1938) showed that degeneration is more intense in the ventral tier of the substantia nigra compacta than in its dorsal tier and the ventral tegmental area (VTA), outlining the concept of differential vulnerability of midbrain dopaminergic (DA-) neurons. Nowadays, we know that other neuronal groups degenerate in PD, but the massive loss of nigral DA-cells is its pathological hallmark, having a pivotal position in the pathophysiology of the disease as it is responsible for the motor symptoms. Data from humans as well as cellular and animal models indicate that DA-cell degeneration is a complex process, probably precipitated by the convergence of different risk factors, mediated by oxidative stress, and involving pathogenic factors arising within the DA-neuron (intrinsic factors), and from its environment and distant interconnected brain regions (extrinsic factors). In light of current data, intrinsic factors seem to be preferentially involved in the first steps of the degenerative process, and extrinsic factors in its progression. A controversial issue is the relative weight of the impairment of common cell functions, such as energy metabolism and proteostasis, and specific dopaminergic functions, such as pacemaking activity and DA handling, in the pathogenesis of DA-cell degeneration. Here we will review the current knowledge about the relevance of these factors at the beginning and during the progression of PD, and in the differential vulnerability of midbrain DA-cells.

14.
Neurobiol Dis ; 40(3): 518-30, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20688165

RESUMO

The dopamine transporter (DAT) is a transmembrane glycoprotein responsible for dopamine (DA) uptake, which has been shown to be involved in DA-cell degeneration in Parkinson's disease (PD). At the same time, some studies suggest that DAT may be regulated in response to dopaminergic injury. We have investigated the mechanisms underlying DAT regulation after different degrees of dopaminergic lesion. DAT is persistently down-regulated in surviving midbrain DA-neurons after substantial (62%) loss of striatal DA-terminals, and transiently after slight (11%) loss of DA-terminals in rats. Transient DAT down-regulation consisted of a decrease of glycosylated (mature) DAT in the plasma membrane with accumulation of non-glycosylated (immature) DAT in the endoplasmic reticulum-Golgi (ERG) compartment, and recovery of the normal expression pattern 5 days after lesion. DAT redistribution to the ERG was also observed in HEK cells expressing rat DAT exposed to MPP(+), but not after exposure to DAT-unrelated neurotoxins. In contrast to other midbrain DA-cells, those in the ventrolateral region of the substantia nigra do not regulate DAT and degenerate shortly after slight DA-lesion. These data suggest that DAT down-regulation is a post-translational event induced by DA-analogue toxins, consisting of a stop in its glycosylation and trafficking to the plasma membrane. Its persistence after substantial DA-lesion may act as a compensatory mechanism helping maintain striatal DA levels. The fact that neurons which do not regulate DAT die shortly after lesion suggests a relationship between DAT down-regulation and neuroprotection.


Assuntos
Adrenérgicos/toxicidade , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação da Expressão Gênica , Oxidopamina/toxicidade , Adrenérgicos/administração & dosagem , Animais , Western Blotting , Regulação para Baixo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Imuno-Histoquímica , Hibridização In Situ , Injeções Intraventriculares , Oxidopamina/administração & dosagem , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
15.
Am J Physiol Cell Physiol ; 299(4): C779-90, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20631247

RESUMO

The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δßγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Neurônios/enzimologia , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Córtex Cerebral/citologia , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Humanos , Proteínas Imediatamente Precoces/genética , Macaca fascicularis , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurônios/citologia , Oócitos/citologia , Oócitos/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Fosfolipases Tipo C/genética , Xenopus laevis
16.
Neurobiol Dis ; 36(3): 494-508, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19766189

RESUMO

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Mesencéfalo/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Idoso , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Feminino , Glicosilação , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
17.
Exp Neurol ; 220(2): 335-40, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19766632

RESUMO

Previous studies have suggested the increase of extracellular glutamate (GLU) in the substantia nigra (SN) as a cause of dopamine-cell degeneration (excitotoxicity) in Parkinson's disease (PD). However, the mechanisms involved in this increase remain unknown. The present work studied osmoregulation as a cause of GLU release in the SN. Microdialysis was used to change extracellular osmolarity, to administer drugs and to quantify the extracellular non-synaptic GLU (EnS-GLU). Two osmolarity modifications were performed, a moderate decrease (5%) resembling physiological modifications and a substantial decrease (>or=20% decrease) similar to that observed under pathological conditions. Hypo-osmolarity induced a dose-response (285-80 mOsm) increase of EnS-GLU which was detected after small osmolarity modifications (15 mOsm) and which was very marked (>1000%) after more intense osmolarity changes. This response disappeared after pre-treating rats with a P2 purinergic-receptor antagonist (pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid; 1 mM) suggesting ATP involvement in the osmosensitive EnS-GLU response. The EnS-GLU increase observed after administration of ATP (0.1-100 microM) and 2-methylthioadenosine triphosphate tetrasodium (P2-receptor agonist; 100 microM) and the lack of effects of adenosine administration (1 mM) suggest that the ATP action on P2 receptors is an amplificatory mechanism in the osmosensitive EnS-GLU response. The marked action of osmolarity on extracellular Glu suggests osmolarity regulation as a possible source for excitotoxicity in the SN.


Assuntos
Ácido Glutâmico/toxicidade , Concentração Osmolar , Substância Negra/efeitos dos fármacos , Trifosfato de Adenosina/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Masculino , Microdiálise , Antagonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/fisiologia
18.
Neurobiol Aging ; 30(6): 973-86, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17976862

RESUMO

Several studies report that the striatal dopamine (DA) uptake declines with age, but the underlying mechanisms are still unclear. The use of molecular, biochemical and morphological techniques, and antibodies which detect the glycosylated (80 kDa) and non-glycosylated (50 kDa) DA transporter (DAT) forms in the rat mesostriatal system, reveals that DAT is pre- and post-translationally damaged during aging. In middle age (18 months), the glycosylated DAT form decreases in the plasma membrane of striatal terminals, and the non-glycosylated form is accumulated in the endoplasmic reticulum-Golgi complex. Thereafter, in aged rats (24 months), DAT synthesis is also affected as the decrease in both DATmRNA and total DAT protein levels suggests. However, the evidence of a decrease in both DAT expression in the endosomal (vesicle-enriched) compartment and the phosphorylated DAT fraction from middle age, as well as its compartmental redistribution towards the terminal plasma membrane, with an increase in the membrane DAT/total DAT ratio in striatal synapotosomes, in aged rats, indicate that DA-cells activate compensatory mechanisms directed at maintaining DAT function during normal aging.


Assuntos
Envelhecimento/metabolismo , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Frações Subcelulares/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
19.
J Neural Transm Suppl ; (73): 21-37, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20411765

RESUMO

The degeneration of nigral dopaminergic (DA-) neurons is the histopathologic hallmark of Parkinson's disease (PD), but not all nigral DA-cells show the same susceptibility to degeneration. This starts in DA-cells in the ventrolateral and caudal regions of the susbtantia nigra (SN) and progresses to DA-cells in the dorsomedial and rostral regions of the SN and the ventral tegmental area, where many neurons remain intact until the final stages of the disease. This fact indicates a relationship between the topographic distribution of midbrain DA-cells and their differential vulnerability, and the possibility that this differential vulnerability is associated with phenotypic differences between different subpopulations of nigral DA-cells. Studies carried out during the last two decades have contributed to establishing the existence of different compartments of nigral DA-cells according to their neurochemical profile, and a possible relationship between the expression of some factors and the relative vulnerability or resistance of DA-cell subpopulations to degeneration. These aspects are reviewed and discussed here.


Assuntos
Dopamina/metabolismo , Regulação da Expressão Gênica/fisiologia , Neurônios/fisiologia , Fenótipo , Substância Negra/citologia , Substância Negra/fisiologia , Animais , Humanos
20.
Neurobiol Aging ; 29(11): 1702-11, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17582657

RESUMO

The vesicular monoamine transporter type 2 (VMAT2) is a transmembrane glycoprotein responsible for the vesicular monoamine uptake in the brain. This function declines in the dopaminergic mesostriatal system during normal aging, but the mechanisms responsible for this deficit are unknown. We investigated possible age-related changes in the expression and subcellular distribution of VMAT2 in the rat mesostriatal system. VMAT2 is constitutively expressed as glycosylated (75 kDa), partially glycosylated (55 kDa) and native (45 kDa) forms, they are all present in both synaptosomal compartments (synaptosomal membrane and synaptic vesicle-enriched fractions) of the striatal terminals in young rats. In aged rats, no changes were found in midbrain VMAT2mRNA and VMAT2 total protein levels in whole striatal extracts. However, its subcellular distribution and glycosylation pattern were severely modified. The three VMAT2 forms virtually disappeared from the synaptic vesicle-enriched fraction, while the 55 kDa form was accumulated in the soluble compartment. These changes may be responsible for the loss of VMAT2 activity during aging and may contribute to the high susceptibility of aged midbrain dopaminergic cells to degeneration.


Assuntos
Envelhecimento/metabolismo , Corpo Estriado/metabolismo , Frações Subcelulares/metabolismo , Sinapses/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Envelhecimento/patologia , Animais , Glicosilação , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
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