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Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5-1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.
Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Aripiprazol/farmacologia , Cinética , Técnicas de Patch-Clamp , Miocárdio , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.
Assuntos
Mucina-5B , Doença Pulmonar Obstrutiva Crônica , Humanos , Camundongos , Animais , Suínos , Mucina-5AC , Pulmão/metabolismo , Vesículas Secretórias/metabolismo , Mamíferos/metabolismoRESUMO
Background: Tumor therapeutics are aimed to affect tumor cells selectively while sparing healthy ones. For this purpose, a huge variety of different drugs are in use. Recently, also blockers of voltage-gated sodium channels (VGSCs) have been recognized to possess potentially beneficial effects in tumor therapy. As these channels are a frequent target of numerous drugs, we hypothesized that currently used tumor therapeutics might have the potential to block VGSCs in addition to their classical anti-cancer activity. In the present work, we have analyzed the imipridone TIC10, which belongs to a novel class of anti-cancer compounds, for its potency to interact with VGSCs. Methods: Electrophysiological experiments were performed by means of the patch-clamp technique using heterologously expressed human heart muscle sodium channels (hNav1.5), which are among the most common subtypes of VGSCs occurring in tumor cells. Results: TIC10 angular inhibited the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state was weak with an extrapolated Kr of about 600 µM. TIC10 most probably did not interact with fast inactivation. In protocols for slow inactivation, a half-maximal inhibition occurred around 2 µM. This observation was confirmed by kinetic studies indicating that the interaction occurred with a slow time constant. Furthermore, TIC10 also interacted with the open channel with an affinity of approximately 4 µM. The binding site for local anesthetics or a closely related site is suggested as a possible target as the affinity for the well-characterized F1760K mutant was reduced more than 20-fold compared to wild type. Among the analyzed derivatives, ONC212 was similarly effective as TIC10 angular, while TIC10 linear more selectively interacted with the different states. Conclusion: The inhibition of VGSCs at low micromolar concentrations might add to the anti-tumor properties of TIC10.
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Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet-neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a human ex vivo whole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pH in vitro enhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected. Ex vivo exposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis.
Assuntos
Ativação de Neutrófilo/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Sepse/imunologia , Animais , Endotoxemia/imunologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Inflamação/imunologia , Masculino , Potenciais da Membrana , NADPH Oxidase 2/fisiologia , Ativação de Neutrófilo/fisiologia , SuínosRESUMO
Secretion of pulmonary surfactant in the alveoli of the lungs is essential to maintain lung function. Stretching of alveoli during lung inflation is the main trigger for surfactant secretion. Yet, the molecular mechanisms how mechanical distension of alveoli results in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar epithelial type I (ATI) and surfactant secreting type II (ATII) cells. ATI, but not ATII cells, express caveolae, small plasma membrane invaginations that can respond to plasma membrane stresses and serve mechanotransductive roles. Within this study, we investigated the role of caveolae as mechanosensors in the alveolus. We generated a human caveolin-1 knockout ATI cell (hAELVicav-/- ) using CRISPR/Cas9. Wildtype (hAELViwt ) and hAELVicav-/- cells grown on flexible membranes responded to increasing stretch amplitudes with rises in intracellular Ca2+ . The response was less frequent and started at higher stretch amplitudes in hAELVicav-/- cells. Stretch-induced Ca2+ -signals depended on Ca2+ -entry via piezo1 channels, localized within caveolae in hAELViwt and primary ATI cells. Ca2+ -entry via piezo1 activated pannexin-1 hemichannels resulting in ATP release from ATI cells. ATP release was reduced in hAELVicav-/- cells. In co-cultures resembling the alveolar epithelium, released ATP stimulated Ca2+ signals and surfactant secretion from neighboring ATII cells when co-cultured with hAELViwt but not hAELVicav-/- cells. In summary, we propose that caveolae in ATI cells are mechanosensors within alveoli regulating stretch-induced surfactant secretion from ATII cells.
Assuntos
Células Epiteliais Alveolares , Cavéolas/metabolismo , Caveolina 1/metabolismo , Canais Iônicos/metabolismo , Surfactantes Pulmonares/metabolismo , Estresse Mecânico , Trifosfato de Adenosina/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The distal lung provides an intricate structure for gas exchange in mammalian lungs. Efficient gas exchange depends on the functional integrity of lung alveoli. The cells in the alveolar tissue serve various functions to maintain alveolar structure, integrity and homeostasis. Alveolar epithelial cells secrete pulmonary surfactant, regulate the alveolar surface liquid (ASL) volume and, together with resident and infiltrating immune cells, provide a powerful host-defense system against a multitude of particles, microbes and toxicants. It is well established that all of these cells express purinergic P2 receptors and that purinergic signaling plays important roles in maintaining alveolar homeostasis. Therefore, it is not surprising that purinergic signaling also contributes to development and progression of severe pathological conditions like pulmonary inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and pulmonary fibrosis. Within this review we focus on the role of P2 purinergic signaling in the distal lung in health and disease. We recapitulate the expression of P2 receptors within the cells in the alveoli, the possible sources of ATP (adenosine triphosphate) within alveoli and the contribution of purinergic signaling to regulation of surfactant secretion, ASL volume and composition, as well as immune homeostasis. Finally, we summarize current knowledge of the role for P2 signaling in infectious pneumonia, ALI/ARDS and idiopathic pulmonary fibrosis (IPF).
Assuntos
Pulmão/metabolismo , Pulmão/patologia , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Pneumonia/metabolismo , Pneumonia/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Surfactantes Pulmonares/metabolismoRESUMO
Keratin filaments (KFs) comprise the intermediate filaments of epithelial cells and are well known for their cytoprotective properties and their mechanical resilience. Although, several studies have demonstrated KFs' remarkable tensile properties relatively little is known about acute implications of mechanical stretch on KFs in living cells. This includes structural effects on the KFs and their higher level assembly structures as well as posttranslational response mechanisms to possibly modify KF's properties. We subjected simple epithelial A549 lung cells to 30% unidirectional stretch and already after 10 seconds we observed morphological changes of the KF-network as well as structural effects on their desmosomal anchor sites-both apparently caused by the tensile strain. Interestingly, the effect on the desmosomes was attenuated after 30 seconds of cell stretch with a concomitant increase in phosphorylation of keratin8-S432, keratin18-S53, and keratin18-S34 without an apparent increase in keratin solubility. When mimicking the phosphorylation of keratin18-S34 the stretch-induced effect on the desmosomes could be diminished and probing the cell surface with atomic force microscopy showed a lowered elastic modulus. We conclude that the stretch-induced KF phosphorylation affects KF's tensile properties, probably to lower the mechanical load on strained desmosomal cell-cell contacts, and hence, preserve epithelial integrity.
Assuntos
Queratinas/metabolismo , Pulmão/metabolismo , Células A549 , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Fosforilação/fisiologiaRESUMO
The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.
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Neutrófilos/metabolismo , Toxinas Biológicas/farmacologia , ADP Ribose Transferases/metabolismo , Avidina/metabolismo , Biotinilação , Toxinas Botulínicas/metabolismo , Linhagem Celular , Citosol/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/químicaRESUMO
The antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication.
Assuntos
Antibacterianos/farmacologia , Bacitracina/farmacologia , Toxinas Bacterianas/metabolismo , Substâncias Protetoras/farmacologia , Animais , Antígenos de Bactérias/metabolismo , Bacillus anthracis/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Clostridioides difficile/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Exotoxinas/metabolismo , Células HeLa , Humanos , Bicamadas Lipídicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Células VeroRESUMO
KEY POINTS: Re-sensitization of P2X4 receptors depends on a protonation/de-protonation cycle Protonation and de-protonation of the receptors is achieved by internalization and recycling of P2X4 receptors via acidic compartments Protonation and de-protonation occurs at critical histidine residues within the extracellular loop of P2X4 receptors Re-sensitization is blocked in the presence of the receptor agonist ATP ABSTRACT: P2X4 receptors are members of the P2X receptor family of cation-permeable, ligand-gated ion channels that open in response to the binding of extracellular ATP. P2X4 receptors are implicated in a variety of biological processes, including cardiac function, cell death, pain sensation and immune responses. These physiological functions depend on receptor activation on the cell surface. Receptor activation is followed by receptor desensitization and deactivation upon removal of ATP. Subsequent re-sensitization is required to return the receptor into its resting state. Desensitization and re-sensitization are therefore crucial determinants of P2X receptor signal transduction and responsiveness to ATP. However, the molecular mechanisms controlling desensitization and re-sensitization are not fully understood. In the present study, we provide evidence that internalization and recycling via acidic compartments is essential for P2X4 receptor re-sensitization. Re-sensitization depends on a protonation/de-protonation cycle of critical histidine residues within the extracellular loop of P2X4 receptors that is mediated by receptor internalization and recycling. Interestingly, re-sensitization under acidic conditions is completely revoked by receptor agonist ATP. Our data support the physiological importance of the unique subcellular distribution of P2X4 receptors that is predominantly found within acidic compartments. Based on these findings, we suggest that recycling of P2X4 receptors regulates the cellular responsiveness in the sustained presence of ATP.
Assuntos
Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Células HEK293 , Células HeLa , Humanos , Transporte Proteico , Prótons , Receptores Purinérgicos P2X4/química , Transdução de SinaisRESUMO
BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer related deaths worldwide and prognosis in advanced tumor stage still remains poor. Since CK1 isoforms have been reported to be deregulated in several tumor entities CK1 has emerged as a novel drug target in cancer therapy. In this study we set out to investigate whether CK1α might have the potential to serve as prognostic marker. METHODS: CK1α RNA and protein expression levels in healthy and tumor tissue of CRC patients were analyzed using quantitative real-time PCR and Western Blot analysis, respectively. Prognostic relevance was investigated by correlating obtained CK1α expression levels with patients' survival rate generating Kaplan-Meier survival plots. RESULTS: It could be shown that CK1α is overexpressed in colorectal tumor tissue compared to normal tissue and CK1α overexpression in tumor tissue correlates with poor survival in CRC patients. Results become more significant when only considering patients with high-grade tumors, as well as patients assigned to UICC II and UICC III stage. Furthermore, Cox regression analysis revealed that CK1α is an independent prognostic factor. In addition, tumors expressing decreased levels of the kinase reveal positive effects on overall survival when localized in the right colon compared to those in the left side. CONCLUSION: In summary, this study provides evidence for the first time that CK1α RNA levels might serve as prognostic marker for CRC.
Assuntos
Biomarcadores Tumorais/genética , Caseína Quinase Ialfa/genética , Neoplasias Colorretais/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Caseína Quinase Ialfa/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
Background and Purpose IC261 (3-[(2,4,6-trimethoxyphenyl)methylidenyl]-indolin-2-one) has previously been introduced as an isoform specific inhibitor of casein kinase 1 (CK1) causing cell cycle arrest or cell death of established tumor cell lines. However, it is reasonable to assume that not all antitumor activities of IC261 are mediated by the inhibition of CK1. Meanwhile there is growing evidence that functional voltage-gated sodium channels are also implicated in the progression of tumors as their blockage suppresses tumor migration and invasion of different tumor cell lines. Thus, we asked whether IC261 functionally inhibits voltage-gated sodium channels. Experimental Approach Electrophysiological experiments were performed using the patch-clamp technique at human heart muscle sodium channels heterologously expressed in human TsA cells. Key Results IC261 inhibits sodium channels in a state-dependent manner. IC261 does not interact with the open channel and has only a low affinity for the resting state of the hNav1.5 (human voltage-gated sodium channel; Kr: 120 µM). The efficacy of IC261 strongly increases with membrane depolarisation, indicating that the inactivated state is an important target. The results of different experimental approaches finally revealed an affinity of IC261 to the inactivated state between 1 and 2 µM. Conclusion and Implications IC261 inhibits sodium channels at a similar concentration necessary to reduce CK1δ/ε activity by 50% (IC50 value 1 µM). Thus, inhibition of sodium channels might contribute to the antitumor activity of IC261.
Assuntos
Caseína Quinase I/antagonistas & inibidores , Indóis/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.5/fisiologia , Floroglucinol/análogos & derivados , Bloqueadores dos Canais de Sódio/farmacologia , Linhagem Celular , Humanos , Floroglucinol/farmacologiaRESUMO
The apical surface liquid (ASL) layer covers the airways and forms a first line of defense against pathogens. Maintenance of ASL volume by airway epithelia is essential for maintaining lung function. The proteolytic activation of epithelial Na+ channels is believed to be the dominating mechanism to cope with increases in ASL volumes. Alternative mechanisms, in particular increases in epithelial osmotic water permeability (Posm), have so far been regarded as rather less important. However, most studies mainly addressed immediate effects upon apical volume expansion (AVE) and increases in ASL. This study addresses the response of lung epithelia to long-term AVE. NCI-H441 cells and primary human tracheal epithelial cells, both cultivated in air-liquid interface conditions, were used as models for the lung epithelium. AVE was established by adding isotonic solution to the apical surface of differentiated lung epithelia, and time course of ASL volume restoration was assessed by the deuterium oxide dilution method. Concomitant ion transport was investigated in Ussing chambers. We identified a low resorptive state immediately after AVE, which coincided with proteolytic ion transport activation within 10-15 minutes after AVE. The main clearance of excess ASL occurred during a delayed (hours after AVE) high resorptive state, which did not correlate with ion transport activation. Instead, high resorptive state onset coincided with an increase in Posm, which depended on aquaporin up-regulation. In summary, our data demonstrate that, aside from ion transport activation, modulation of Posm is a major mechanism to compensate for long-term AVE in lung epithelia.
Assuntos
Epitélio/metabolismo , Pulmão/metabolismo , Reologia , Água/metabolismo , Amilorida/farmacologia , Aquaporinas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Epitélio/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Pulmão/efeitos dos fármacos , Osmose/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Reologia/efeitos dos fármacos , Propriedades de Superfície , Fatores de TempoRESUMO
OBJECTIVE: Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC-1α in mitochondria, no studies have addressed whether PGC-1α directly influences oligomerization of α-syn or whether α-syn oligomers impact PGC-1α expression. MATERIALS AND METHODS: We tested whether pharmacological or genetic activation of PGC-1α or PGC-11α knockdown could modulate the oligomerization of α-syn in vitro by using an α-syn -fragment complementation assay. RESULTS: In this study, we found that both PGC-1α reference gene (RG-PGC-1α) and the central nervous system (CNS)-specific PGC-1α (CNS-PGC-1α) are downregulated in human PD brain, in A30P α-syn transgenic animals, and in a cell culture model for α-syn oligomerization. Importantly, downregulation of both RG-PGC-1α and CNS-PGC-1α in cell culture or neurons from RG-PGC-1α-deficient mice leads to a strong induction of α-syn oligomerization and toxicity. In contrast, pharmacological activation or genetic overexpression of RG-PGC-1α reduced α-syn oligomerization and rescued α-syn-mediated toxicity. INTERPRETATION: Based on our results, we propose that PGC-1α downregulation and α-syn oligomerization form a vicious circle, thereby influencing and/or potentiating each other. Our data indicate that restoration of PGC-1α is a promising approach for development of effective drugs for the treatment of PD and related synucleinopathies.
Assuntos
Regulação da Expressão Gênica/genética , PPAR gama/genética , PPAR gama/metabolismo , Substância Negra/metabolismo , Fatores de Transcrição/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Humanos , Macrolídeos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/metabolismo , Doença de Parkinson/patologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Resveratrol , Estilbenos/farmacologia , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição/genética , alfa-Sinucleína/genéticaRESUMO
High-frequency tetanic stimulation is associated with an increase in extracellular and T-tubular K(+) and changes of Na(+) and Cl(-) concentrations, membrane depolarization as well as inactivation of voltage-gated Na(+) channels. These alterations are expected to lead to fiber inexcitability, which is largely prevented by mechanisms intrinsic or extrinsic to muscle fibers. They act by adapting electrical membrane properties or by accelerating the reconstitution of ionic homeostasis. The high Cl(-) conductance of muscle fibers supports the K(+) conductance in fast and complete repolarization and creates a mechanism for the fast reuptake of K(+), thereby reducing the T-tubular K(+) accumulation. Excitability is increased by a Ca(2+) and proteinkinase C dependent inhibition of the Cl(-) conductance which is efficient especially in the T-tubular system. Several mediators activate the Na(+)/K(+)-ATPase and thus enhance the restoration of ionic homeostasis. Examples are purines (ATP, ADP), calcitonin-gene related peptide and adrenaline. It is also necessary to adapt the strength of the sarcoplasmic Ca(2+) concentration to the requirements of tetanic contractions. An overwhelming Ca(2+) signal leads to enzymatically driven excitation-contraction uncoupling. This process is most likely driven by the Ca(2+) dependent protease µ-calpain and might lead to the long-lasting fatigue observed after excessive physical activity.
Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Potenciais da Membrana/fisiologia , Contração Muscular , Fadiga Muscular/fisiologia , Potássio/metabolismo , Humanos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Normal resting potential (P1) of myofibers follows the Nernst equation, exhibiting about -85 mV at a normal extracellular K(+) concentration ([K(+)](o)) of 4 mM. Hyperpolarization occurs with decreased [K(+)](o), although at [K(+)](o) < 1.0 mM, myofibers paradoxically depolarize to a second stable potential of -60 mV (P2). In rat myofiber bundles, P2 also was found at more physiological [K(+)](o) and was associated with inexcitability. To increase the relative frequency of P2 to 50%, [K(+)](o) needed to be lowered to 1.5 mM. In the presence of the ionophore gramicidin, [K(+)](o) reduction to only 2.5 mM yielded the same effect. Acetazolamide normalized this increased frequency of P2 fibers. The findings mimic hypokalemic periodic paralysis (HypoPP), a channelopathy characterized by hypokalemia-induced weakness. Of myofibers from 7 HypoPP patients, up to 25% were in P2 at a [K(+)](o) of 4 mM, in accordance with their permanent weakness, and up to 99% were in P2 at a [K(+)](o) of 1.5 mM, in accordance with their paralytic attacks. Of 36 HypoPP patients, 25 had permanent weakness and myoplasmic intracellular Na(+) ([Na(+)](i)) overload (up to 24 mM) as shown by in vivo (23)Na-MRI. Acetazolamide normalized [Na(+)](i) and increased muscle strength. HypoPP myofibers showed a nonselective cation leak of 12-19.5 microS/cm(2), which may explain the Na(+) overload. The leak sensitizes myofibers to reduced serum K(+), and the resulting membrane depolarization causes the weakness. We postulate that the principle of paradoxical depolarization and loss of function upon [K(+)](o) reduction may apply to other tissues, such as heart or brain, when they become leaky (e.g., because of ischemia).