RESUMO
Recent studies indicate that connexin hemichannels do not act as freely permeable non-selective pores, but they select permeants in an isoform-specific manner with cooperative, competitive and saturable kinetics. The aim of this study was to investigate whether the treatment with a mixture of IL-1ß plus TNF-α, a well-known pro-inflammatory condition that activates astroglial connexin 43 (Cx43) hemichannels, could alter their permeability to molecules. We found that IL-1ß plus TNF-α left-shifted the dye uptake rate vs. dye concentration relationship for Etd and 2-NBDG, but the opposite took place for DAPI or YO-PRO-1, whereas no alterations were observed for Prd. The latter modifications were accompanied of changes in Kd (Etd, DAPI, YO-PRO-1 or 2-NBDG) and Hill coefficients (Etd and YO-PRO-1), but not in alterations of Vmax. We speculate that IL-1ß plus TNF-α may distinctively affect the binding sites to permeants in astroglial Cx43 hemichannels rather than their number in the cell surface. Alternatively, IL-1ß plus TNF-α could induce the production of endogenous permeants that may favor or compete for in the pore-lining residues of Cx43 hemichannels. Future studies shall elucidate whether the differential ionic/molecule permeation of Cx43 hemichannels in astrocytes could impact their communication with neurons in the normal and inflamed nervous system.
Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Citocinas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacocinética , Animais , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacocinética , Corantes Fluorescentes/farmacocinética , Junções Comunicantes , Inflamação , Interleucina-1beta/farmacologia , Íons , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Permeabilidade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Neuronal excitotoxicity induced by glutamate leads to cell death and functional impairment in a variety of central nervous system pathologies. Glutamate-mediated excitotoxicity triggers neuronal apoptosis in the cell soma as well as degeneration of axons and dendrites by a process associated with Ca2+ increase and mitochondrial dysfunction. Importantly, degeneration of axons initiated by diverse stimuli, including excitotoxicity, has been proposed as an important pathological event leading to functional impairment in neurodegenerative conditions. Here, we demonstrate that excitotoxicity-induced axonal degeneration proceeds by a mechanism dependent on the necroptotic kinases RIPK1 and RIPK3, and the necroptotic mediator MLKL. Inhibition of RIPK1, RIPK3 or MLKL prevents key steps in the axonal degeneration cascade, including mitochondrial depolarization, the opening of the permeability transition pore and Ca2+ dysregulation in the axon. Interestingly, the same excitotoxic stimuli lead to apoptosis in the cell soma, demonstrating the co-activation of two independent degenerative mechanisms in different compartments of the same cell. The identification of necroptosis as a key mechanism of axonal degeneration after excitotoxicity is an important initial step in the development of novel therapeutic strategies for nervous system disorders.
Assuntos
Axônios/metabolismo , Ácido Glutâmico/metabolismo , Necrose/metabolismo , Degeneração Neural/metabolismo , HumanosRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1(G93A) (ACM-hSOD1(G93A)) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1(G93A), but not ACM-hSOD1(WT), increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1(G93A)-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1(G86R) or TDP43(A315T). We further find that co-application of ACM-SOD1(G93A) with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1(G93A) induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.
RESUMO
Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1â»/â») showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1â»/â» hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1â»/â» astrocytes also showed more intracellular Ca²âº signal oscillations mediated by functional connexin 43 hemichannels and P2Y1 receptors. Therefore, Npc1â»/â» astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.
Assuntos
Astrócitos/metabolismo , Cálcio/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Sinalização do Cálcio , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Conexinas/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Junções Comunicantes/efeitos dos fármacos , Deleção de Genes , Regulação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/patologia , Proteínas/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Técnicas de Cultura de TecidosRESUMO
Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell-cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration-dependent manner (and by zero glucose) and were not observed in connexin 43(-/-) astrocytes. The responses were transient and persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La(3+) or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia-reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Conexina 43/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Hipóxia Encefálica/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Comunicação Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Córtex Cerebral/fisiopatologia , Conexina 43/efeitos dos fármacos , Corantes Fluorescentes/farmacocinética , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Glucose/toxicidade , Células HeLa , Humanos , Hiperglicemia/fisiopatologia , Hipóxia Encefálica/fisiopatologia , Lantânio/farmacologia , Camundongos , Oxigênio/metabolismo , Oxigênio/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologiaRESUMO
There is evidence that amyloid beta-protein (Abeta) deposits or Abeta intermediates trigger pathogenic factors in Alzheimer's disease patients. We have previously reported that c-Abl kinase activation involved in cell signalling regulates the neuronal death response to Abeta fibrils (Abeta(f)). In the present study we investigated the therapeutic potential of the selective c-Abl inhibitor STI571 on both the intrahippocampal injection of Abeta(f) and APPsw/PSEN1DeltaE9 transgenic mice Alzheimer's disease models. Injection of Abeta(f) induced an increase in the numbers of p73 and c-Abl immunoreactive cells in the hippocampal area near to the lesion. Chronic intraperitoneal administration of STI571 reduced the rat behavioural deficit induced by Abeta(f), as well as apoptosis and tau phosphorylation. Our in vitro studies suggest that inhibition of the c-Abl/p73 signalling pathway is the mechanism underlying of the effects of STI571 on Abeta-induced apoptosis for the following reasons: (i) Abeta(f) induces p73 phosphorylation, the TAp73 isoform levels increase so as to enhance its proapoptotic function, and all these effects where reduced by STI571; (ii) c-Abl kinase activity is required for neuronal apoptosis and (iii) STI571 prevents the Abeta-induced increase in the expression of apoptotic genes. Furthermore, in the Abeta-injected area there was a huge increase in phosphorylated p73 and a larger number of TAp73-positive cells, with these changes being prevented by STI571 coinjection. Moreover, the intraperitoneal administration of STI571 rescued the cognitive decline in APPsw/PSEN1DeltaE9 mice, p73 phosphorylation, tau phosphorylation and caspase-3 activation in neurons around Abeta deposits. Besides, we observed a decrease in the number and size of Abeta deposits in the APPsw/PSEN1DeltaE9-STI571-treated mice. These results are consistent with the role of the c-Abl/p73 signalling pathway in Abeta neurodegeneration, and suggest that STI571-like compounds would be effective in therapeutic treatments of Alzheimer disease.