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1.
Glia ; 2020 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-32525239

RESUMO

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.

2.
J Clin Invest ; 2020 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-32597830

RESUMO

Characterization of the key cellular targets contributing to sustained microglial activation in neurodegenerative diseases, including Parkinson's disease (PD), and optimal modulation of these targets can provide potential treatments to halt disease progression. Here, we demonstrated that microglial Kv1.3, a voltage-gated potassium channel, was transcriptionally upregulated in response to aggregated α-synuclein (αSynAgg) stimulation in primary microglial cultures and animal models of PD, as well as in postmortem human PD brains. Patch-clamp electrophysiological studies confirmed that the observed Kv1.3 upregulation translated to increased Kv1.3 channel activity. The kinase Fyn, a risk factor for PD, modulated transcriptional upregulation and posttranslational modification of microglial Kv1.3. Multiple state-of-the-art analyses, including Duolink proximity ligation assay imaging, revealed that Fyn directly bound to Kv1.3 and posttranslationally modified its channel activity. Furthermore, we demonstrated the functional relevance of Kv1.3 in augmenting the neuroinflammatory response by using Kv1.3-KO primary microglia and the Kv1.3-specific small-molecule inhibitor PAP-1, thus highlighting the importance of Kv1.3 in neuroinflammation. Administration of PAP-1 significantly inhibited neurodegeneration and neuroinflammation in multiple animal models of PD. Collectively, our results imply that Fyn-dependent regulation of Kv1.3 channels plays an obligatory role in accentuating the neuroinflammatory response in PD and identify Kv1.3 as a potential therapeutic target for PD.

3.
Ann Clin Transl Neurol ; 6(4): 723-738, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31019997

RESUMO

Objective: Microglia play a pivotal role in the initiation and progression of Alzheimer's disease (AD). We here tested the therapeutic hypothesis that the Ca2+-activated potassium channel KCa3.1 constitutes a potential target for treating AD by reducing neuroinflammation. Methods: To determine if KCa3.1 is relevant to AD, we tested if treating cultured microglia or hippocampal slices with Aß oligomer (AßO) activated KCa3.1 in microglia, and if microglial KCa3.1 was upregulated in 5xFAD mice and in human AD brains. The expression/activity of KCa3.1 was examined by qPCR, Western blotting, immunohistochemistry, and whole-cell patch-clamp. To investigate the role of KCa3.1 in AD pathology, we resynthesized senicapoc, a clinically tested KCa3.1 blocker, and determined its pharmacokinetic properties and its effect on microglial activation, Aß deposition and hippocampal long-term potentiation (hLTP) in 5xFAD mice. Results: We found markedly enhanced microglial KCa3.1 expression/activity in brains of both 5xFAD mice and AD patients. In hippocampal slices, microglial KCa3.1 expression/activity was increased by AßO treatment, and its inhibition diminished the proinflammatory and hLTP-impairing activities of AßO. Senicapoc exhibited excellent brain penetrance and oral availability, and in 5xFAD mice, reduced neuroinflammation, decreased cerebral amyloid load, and enhanced hippocampal neuronal plasticity. Interpretation: Our results prompt us to propose repurposing senicapoc for AD clinical trials, as senicapoc has excellent pharmacological properties and was safe and well-tolerated in a prior phase-3 clinical trial for sickle cell anemia. Such repurposing has the potential to expedite the urgently needed new drug discovery for AD.


Assuntos
Acetamidas/farmacologia , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Compostos de Tritil/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Reposicionamento de Medicamentos/métodos , Humanos , Camundongos Transgênicos , Microglia/efeitos dos fármacos
4.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-30755796

RESUMO

As their name implies, cation channels allow the regulated flow of cations such as sodium, potassium, calcium, and magnesium across cellular and intracellular membranes. Cation channels have long been known for their fundamental roles in controlling membrane potential and excitability in neurons and muscle. In this review, we provide an update on the recent advances in our understanding of the structure-function relationship and the physiological and pathophysiological role of cation channels. The most exciting developments in the last two years, in our opinion, have been the insights that cryoelectron microscopy has provided into the inner life and the gating of not only voltage-gated channels but also mechanosensitive and calcium- or sodium-activated channels. The mechanosensitive Piezo channels especially have delighted the field not only with a fascinating new type of structure but with important roles in blood pressure regulation and lung function.

5.
Glia ; 66(9): 1881-1895, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30043400

RESUMO

Microglia show a rich repertoire of activation patterns regulated by a complex ensemble of surface ion channels, receptors, and transporters. We and others have investigated whether microglia vary their K+ channel expression as a means to achieve functional diversity. However, most of the prior studies were conducted using in vitro models such as BV2 cells, primary microglia, or brain slices in culture, which may not accurately reflect microglia physiology in adult individuals. Here we employed an in vivo mouse model of selective innate immune activation by intracerebroventricular injection of lipopolysaccharides (ICV-LPS) to determine the role of the voltage-gated Kv1.3 channel in LPS-induced M1-like microglial activation. Using microglia acutely isolated from adult brains, we detected Kv1.3 and Kir2.1 currents, and found that ICV-LPS increased the current density and RNA expression of Kv1.3 but did not affect those of Kir2.1. Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators such as IL-1ß, TNF-α, IL-6, and iNOS. Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity. Pharmacological intervention using PAP-1, a small molecule that selectively blocks homotetrameric Kv1.3 channels, achieved anti-inflammatory and hLTP-recovery effects similar to Kv1.3 knockout. We conclude that Kv1.3 is required for microglial M1-like pro-inflammatory activation in vivo. A significant implication of our in vivo data is that Kv1.3 blockers could be therapeutic candidates for neurological diseases where microglia-mediated neurotoxicity is implicated in the pathogenesis.


Assuntos
Imunidade Inata , Inflamação/metabolismo , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Escherichia coli , Imunidade Inata/efeitos dos fármacos , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Lipopolissacarídeos , Potenciação de Longa Duração/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Técnicas de Cultura de Tecidos
6.
MAbs ; 10(4): 636-650, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29494279

RESUMO

Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.


Assuntos
Anticorpos Monoclonais , Descoberta de Drogas/métodos , Canal de Potássio Kv1.3/antagonistas & inibidores , Animais , Camelídeos Americanos , Galinhas , Humanos , Proteínas Recombinantes , Tetrahymena thermophila
7.
Ann Clin Transl Neurol ; 5(2): 147-161, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29468176

RESUMO

Objective: Inhibitors of the voltage-gated K+ channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up-regulated on "M1-like" microglia, we here tested the therapeutic hypothesis that the brain-penetrant small-molecule Kv1.3-inhibitor PAP-1 reduces secondary inflammatory damage after ischemia/reperfusion. Methods: We studied microglial Kv1.3 expression using electrophysiology and immunohistochemistry, and evaluated PAP-1 in hypoxia-exposed organotypic hippocampal slices and in middle cerebral artery occlusion (MCAO) with 8 days of reperfusion in both adult male C57BL/6J mice (60 min MCAO) and adult male Wistar rats (90 min MCAO). In both models, PAP-1 administration was started 12 h after reperfusion. Results: We observed Kv1.3 staining on activated microglia in ischemic infarcts in mice, rats, and humans and found higher Kv1.3 current densities in acutely isolated microglia from the infarcted hemisphere than in microglia isolated from the contralateral hemisphere of MCAO mice. PAP-1 reduced microglia activation and increased neuronal survival in hypoxia-exposed hippocampal slices as effectively as minocycline. In mouse MCAO, PAP-1 dose-dependently reduced infarct area, improved neurological deficit score, and reduced brain levels of IL-1ß and IFN-γ without affecting IL-10 and brain-derived nerve growth factor (BDNF) levels or inhibiting ongoing phagocytosis. The beneficial effects on infarct area and neurological deficit score were reproduced in rats providing confirmation in a second species. Interpretation: Our findings suggest that Kv1.3 constitutes a promising therapeutic target for preferentially inhibiting "M1-like" inflammatory microglia/macrophage functions in ischemic stroke.

8.
Brain ; 141(2): 596-612, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29272333

RESUMO

Microglia significantly contribute to the pathophysiology of Alzheimer's disease but an effective microglia-targeted therapeutic approach is not yet available clinically. The potassium channels Kv1.3 and Kir2.1 play important roles in regulating immune cell functions and have been implicated by in vitro studies in the 'M1-like pro-inflammatory' or 'M2-like anti-inflammatory' state of microglia, respectively. We here found that amyloid-ß oligomer-induced expression of Kv1.3 and Kir2.1 in cultured primary microglia. Likewise, ex vivo microglia acutely isolated from the Alzheimer's model 5xFAD mice co-expressed Kv1.3 and Kir2.1 as well as markers traditionally associated with M1 and M2 activation suggesting that amyloid-ß oligomer induces a microglial activation state that is more complex than previously thought. Using the orally available, brain penetrant small molecule Kv1.3 blocker PAP-1 as a tool, we showed that pro-inflammatory and neurotoxic microglial responses induced by amyloid-ß oligomer required Kv1.3 activity in vitro and in hippocampal slices. Since we further observed that Kv1.3 was highly expressed in microglia of transgenic Alzheimer's mouse models and human Alzheimer's disease brains, we hypothesized that pharmacological Kv1.3 inhibition could mitigate the pathology induced by amyloid-ß aggregates. Indeed, treating APP/PS1 transgenic mice with a 5-month oral regimen of PAP-1, starting at 9 months of age, when the animals already manifest cognitive deficits and amyloid pathology, reduced neuroinflammation, decreased cerebral amyloid load, enhanced hippocampal neuronal plasticity, and improved behavioural deficits. The observed decrease in cerebral amyloid deposition was consistent with the in vitro finding that PAP-1 enhanced amyloid-ß uptake by microglia. Collectively, these results provide proof-of-concept data to advance Kv1.3 blockers to Alzheimer's disease clinical trials.


Assuntos
Doença de Alzheimer , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Aprendizagem da Esquiva/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Ficusina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Canal de Potássio Kv1.3/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Mutação/genética , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Presenilina-1/genética , Canais de Potássio Shab/metabolismo
9.
Arch Toxicol ; 92(2): 833-844, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29038840

RESUMO

The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the United States Department of Homeland Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2ß3γ2 (IC50 480 nM, 95% CI 320-640 nM) and α6ß3γ2 (IC50 400 nM, 95% CI 290-510 nM). Introducing ß1 or ß2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/ß3 or α6/ß3 composition. Since α2ß3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggest that α2ß3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/metabolismo , Receptores de GABA-A/metabolismo , Rodenticidas/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Fibroblastos/efeitos dos fármacos , Humanos , Camundongos , Técnicas de Patch-Clamp , Picrotoxina/análogos & derivados , Ratos , Especificidade por Substrato , Ácido gama-Aminobutírico/metabolismo
10.
Science ; 358(6360)2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-29026015

RESUMO

Spaceborne measurements by NASA's Orbiting Carbon Observatory-2 (OCO-2) at the kilometer scale reveal distinct structures of atmospheric carbon dioxide (CO2) caused by known anthropogenic and natural point sources. OCO-2 transects across the Los Angeles megacity (USA) show that anthropogenic CO2 enhancements peak over the urban core and decrease through suburban areas to rural background values more than ~100 kilometers away, varying seasonally from ~4.4 to 6.1 parts per million. A transect passing directly downwind of the persistent isolated natural CO2 plume from Yasur volcano (Vanuatu) shows a narrow filament of enhanced CO2 values (~3.4 parts per million), consistent with a CO2 point source emitting 41.6 kilotons per day. These examples highlight the potential of the OCO-2 sensor, with its unprecedented resolution and sensitivity, to detect localized natural and anthropogenic CO2 sources.

11.
Chemistry ; 23(35): 8466-8472, 2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28411375

RESUMO

There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABAA ) receptors. General synthetic approach toward two groups of analogues was developed. Screening of the resulting library of compounds by FLIPR or whole-cell voltage-clamp revealed that, despite the structural differences, some of the TETS analogues retained GABAA receptor inhibition; however, their potency was an order of magnitude lower. Antibodies raised in rabbits against some of the TETS analogues conjugated to protein recognized free TETS and will be used for the development of an immunoassay for TETS.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/síntese química , Haptenos/química , Receptores de GABA-A/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Imunoensaio/métodos , Concentração Inibidora 50 , Estrutura Molecular , Neurônios , Coelhos , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
12.
Environ Int ; 103: 91-98, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28343720

RESUMO

Occupational medical surveillance is highly desirable in manufacturing facilities where exposure to chemicals is significant. The insecticide fipronil is generally considered safe for humans but with increasing use, exposure to fipronil is of concern. Identification of urinary metabolites of fipronil may allow development of affordable, cheap and rapid procedures for human exposure evaluation. In this study we developed a fast and easy approach for synthesis of hydroxy-fipronil, a potential urinary metabolite of fipronil. This standard was used to develop a sensitive analytical LC-MS/MS method with a limit of quantification (LOQ) of 0.4ng/mL. Fipronil sulfone, a known metabolite, and hydroxy-fipronil were quantified in urine samples from rats treated with a fipronil containing diet. Fipronil sulfone concentration centered around 20ng/mL, while the concentration of hydroxy-fipronil was dose-dependent ranging in 10-10,000ng/mL and thus being a more sensitive marker of fipronil exposure. A fipronil immunoassay with cross-reactivity to hydroxy-fipronil showed a good correlation in signal intensity with LC-MS data. It was also used to demonstrate the applicability of the method for sample screening in the evaluation of exposure levels.


Assuntos
Inseticidas/urina , Pirazóis/urina , Animais , Cromatografia Líquida , Inseticidas/farmacocinética , Inseticidas/normas , Limite de Detecção , Pirazóis/farmacocinética , Pirazóis/normas , Ratos , Espectrometria de Massas em Tandem
13.
Channels (Austin) ; 11(4): 305-315, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28277939

RESUMO

Potassium channels play important roles in microglia functions and thus constitute potential targets for the treatment of neurodegenerative diseases like Alzheimer, Parkinson and stroke. However, uncertainty still prevails as to which potassium channels are expressed and at what levels in different species, how the expression pattern changes upon activation with M1 or M2 polarizing stimuli compared with more complex exposure paradigms, and - most importantly - how these findings relate to the in vivo situation. In this mini-review we discuss the functional potassium channel expression pattern in cultured neonatal mouse microglia in the light of data obtained previously from animal disease models and immunohistochemical studies and compare it with a recent study of adult human microglia isolated from epilepsy patients. Overall, microglial potassium channel expression is very plastic and possibly shows species differences and therefore should be studied carefully in each disease setting and respective animal models.


Assuntos
Microglia/metabolismo , Plasticidade Neuronal , Canais de Potássio/metabolismo , Animais , Células Cultivadas , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Humanos , Camundongos , Mutação , Fenótipo , Especificidade da Espécie
14.
Mol Pharmacol ; 91(4): 392-402, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28126850

RESUMO

The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.


Assuntos
Modelos Moleculares , Bloqueadores dos Canais de Potássio/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/química , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Nifedipino/química , Nifedipino/farmacologia , Bloqueadores dos Canais de Potássio/química , Domínios Proteicos , Alinhamento de Sequência , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Tiazinas/química , Tiazinas/farmacologia
15.
Glia ; 65(1): 106-121, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27696527

RESUMO

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (∼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio Kv1.3/metabolismo , Microglia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Células Cultivadas , Interferon gama/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Potenciais da Membrana , Camundongos Endogâmicos C57BL
16.
J Cereb Blood Flow Metab ; 36(12): 2146-2161, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26661208

RESUMO

Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K+ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K+ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1-/- mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Inflamação Neurogênica/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Animais , Encéfalo/metabolismo , Isquemia Encefálica/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Infarto da Artéria Cerebral Média , Ativação de Macrófagos , Camundongos , Terapia de Alvo Molecular , Pirazóis/farmacologia , Pirazóis/uso terapêutico
17.
Neurotherapeutics ; 12(1): 234-49, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25256961

RESUMO

Inhibitors of voltage-gated sodium channels (Na(v)) have been used as anticonvulsants since the 1940s, while potassium channel activators have only been investigated more recently. We here describe the discovery of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a thioanalog of riluzole, as a potent, novel anticonvulsant, which combines the two mechanisms. SKA-19 is a use-dependent NaV channel blocker and an activator of small-conductance Ca(2+)-activated K(+) channels. SKA-19 reduces action potential firing and increases medium afterhyperpolarization in CA1 pyramidal neurons in hippocampal slices. SKA-19 is orally bioavailable and shows activity in a broad range of rodent seizure models. SKA-19 protects against maximal electroshock-induced seizures in both rats (ED50 1.6 mg/kg i.p.; 2.3 mg/kg p.o.) and mice (ED50 4.3 mg/kg p.o.), and is also effective in the 6-Hz model in mice (ED50 12.2 mg/kg), Frings audiogenic seizure-susceptible mice (ED50 2.2 mg/kg), and the hippocampal kindled rat model of complex partial seizures (ED50 5.5 mg/kg). Toxicity tests for abnormal neurological status revealed a therapeutic index (TD50/ED50) of 6-9 following intraperitoneal and of 33 following oral administration. SKA-19 further reduced acute pain in the formalin pain model and raised allodynic threshold in a sciatic nerve ligation model. The anticonvulsant profile of SKA-19 is comparable to riluzole, which similarly affects Na(V) and KCa2 channels, except that SKA-19 has a ~4-fold greater duration of action owing to more prolonged brain levels. Based on these findings we propose that compounds combining KCa2 channel-activating and Na(v) channel-blocking activity exert broad-spectrum anticonvulsant and analgesic effects.


Assuntos
Anticonvulsivantes/farmacologia , Riluzol/análogos & derivados , Riluzol/farmacologia , Convulsões/tratamento farmacológico , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Canais de Sódio Disparados por Voltagem/metabolismo
18.
FASEB J ; 28(9): 3952-64, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24891519

RESUMO

The voltage-gated potassium (Kv) 1.3 channel is widely regarded as a therapeutic target for immunomodulation in autoimmune diseases. ShK-186, a selective inhibitor of Kv1.3 channels, ameliorates autoimmune diseases in rodent models, and human phase 1 trials of this agent in healthy volunteers have been completed. In this study, we identified and characterized a large family of Stichodactyla helianthus toxin (ShK)-related peptides in parasitic worms. Based on phylogenetic analysis, 2 worm peptides were selected for study: AcK1, a 51-residue peptide expressed in the anterior secretory glands of the dog-infecting hookworm Ancylostoma caninum and the human-infecting hookworm Ancylostoma ceylanicum, and BmK1, the C-terminal domain of a metalloprotease from the filarial worm Brugia malayi. These peptides in solution adopt helical structures closely resembling that of ShK. At doses in the nanomolar-micromolar range, they block native Kv1.3 in human T cells and cloned Kv1.3 stably expressed in L929 mouse fibroblasts. They preferentially suppress the proliferation of rat CCR7(-) effector memory T cells without affecting naive and central memory subsets and inhibit the delayed-type hypersensitivity (DTH) response caused by skin-homing effector memory T cells in rats. Further, they suppress IFNγ production by human T lymphocytes. ShK-related peptides in parasitic worms may contribute to the potential beneficial effects of probiotic parasitic worm therapy in human autoimmune diseases.


Assuntos
Doenças Autoimunes/prevenção & controle , Venenos de Cnidários/química , Helmintos/metabolismo , Memória Imunológica/efeitos dos fármacos , Canal de Potássio Kv1.3/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Linfócitos T/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Eletrofisiologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Hipersensibilidade Tardia/prevenção & controle , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Filogenia , Conformação Proteica , Ratos , Ratos Endogâmicos Lew , Receptores CCR7/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismo
19.
Front Behav Neurosci ; 8: 66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24624067

RESUMO

Previous studies have reported that multiple brain regions are activated during spatial navigation. However, it is unclear whether these activated brain regions are specifically associated with spatial updating or whether some regions are recruited for parallel cognitive processes. The present study aimed to localize current sources of event related potentials (ERPs) associated with spatial updating specifically. In the control phase of the experiment, electroencephalograms (EEGs) were recorded while subjects sequentially traced 10 blue checkpoints on the streets of a virtual town, which were sequentially connected by a green line, by manipulating a joystick. In the test phase of the experiment, the checkpoints and green line were not indicated. Instead, a tone was presented when the subjects entered the reference points where they were then required to trace the 10 invisible spatial reference points corresponding to the checkpoints. The vertex-positive ERPs with latencies of approximately 340 ms from the moment when the subjects entered the unmarked reference points were significantly larger in the test than in the control phases. Current source density analysis of the ERPs by standardized low-resolution brain electromagnetic tomography (sLORETA) indicated activation of brain regions in the test phase that are associated with place and landmark recognition (entorhinal cortex/hippocampus, parahippocampal and retrosplenial cortices, fusiform, and lingual gyri), detecting self-motion (posterior cingulate and posterior insular cortices), motor planning (superior frontal gyrus, including the medial frontal cortex), and regions that process spatial attention (inferior parietal lobule). The present results provide the first identification of the current sources of ERPs associated with spatial updating, and suggest that multiple systems are active in parallel during spatial updating.

20.
Mol Pharmacol ; 85(4): 630-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24482397

RESUMO

Bifenthrin, a relatively stable type I pyrethroid that causes tremors and impairs motor activity in rodents, is broadly used. We investigated whether nanomolar bifenthrin alters synchronous Ca(2+) oscillations (SCOs) necessary for activity-dependent dendritic development. Primary mouse cortical neurons were cultured 8 or 9 days in vitro (DIV), loaded with the Ca(2+) indicator Fluo-4, and imaged using a Fluorescence Imaging Plate Reader Tetra. Acute exposure to bifenthrin rapidly increased the frequency of SCOs by 2.7-fold (EC50 = 58 nM) and decreased SCO amplitude by 36%. Changes in SCO properties were independent of modifications in voltage-gated sodium channels since 100 nM bifenthrin had no effect on the whole-cell Na(+) current, nor did it influence neuronal resting membrane potential. The L-type Ca(2+) channel blocker nifedipine failed to ameliorate bifenthrin-triggered SCO activity. By contrast, the metabotropic glutamate receptor (mGluR)5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine] normalized bifenthrin-triggered increase in SCO frequency without altering baseline SCO activity, indicating that bifenthrin amplifies mGluR5 signaling independent of Na(+) channel modification. Competitive [AP-5; (-)-2-amino-5-phosphonopentanoic acid] and noncompetitive (dizocilpine, or MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]) N-methyl-d-aspartate antagonists partially decreased both basal and bifenthrin-triggered SCO frequency increase. Bifenthrin-modified SCO rapidly enhanced the phosphorylation of cAMP response element-binding protein (CREB). Subacute (48 hours) exposure to bifenthrin commencing 2 DIV-enhanced neurite outgrowth and persistently increased SCO frequency and reduced SCO amplitude. Bifenthrin-stimulated neurite outgrowth and CREB phosphorylation were dependent on mGluR5 activity since MPEP normalized both responses. Collectively these data identify a new mechanism by which bifenthrin potently alters Ca(2+) dynamics and Ca(2+)-dependent signaling in cortical neurons that have long term impacts on activity driven neuronal plasticity.


Assuntos
Cálcio/metabolismo , Córtex Cerebral/citologia , Inseticidas/toxicidade , Neurônios/efeitos dos fármacos , Piretrinas/toxicidade , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/metabolismo , Fosforilação , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de Sinais
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