RESUMO
BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. CONCLUSION: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Animais , Plexo Corióideo/metabolismo , Peróxido de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases , Membro 2 da Família 12 de Carreador de Soluto/metabolismoRESUMO
BACKGROUND: Chronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na+/H+ exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS). METHODS: Sham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR. RESULTS: Chronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP+ astrocytes and Iba1+ microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation. CONCLUSION: Our study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment.
Assuntos
Astrócitos/efeitos dos fármacos , Estenose das Carótidas/tratamento farmacológico , Cognição/efeitos dos fármacos , Gliose/tratamento farmacológico , Guanidinas/uso terapêutico , Sulfonas/uso terapêutico , Substância Branca/efeitos dos fármacos , Animais , Astrócitos/patologia , Estenose das Carótidas/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Disfunção Cognitiva/patologia , Gliose/patologia , Guanidinas/farmacologia , Inflamação/patologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/patologia , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonas/farmacologia , Substância Branca/patologiaRESUMO
Na+ /H+ exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-CreER ;Nhe1f/f mice on neuroinflammation and tissue repair after ischemic stroke. Infarct volume was similar in corn oil or tamoxifen (Tam)-treated mice at 48 hr and 14 days post-stroke. However, the Tam-treated mice showed significantly higher survival rate and faster neurological function recovery during day 1-14 post-stroke. Deletion of microglial Nhe1 prevented the elevation of CD11b+ /CD45low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-ß, IL-10, decreased expression of CD86 and IL-1ß, and reduced GFAP+ reactive astrocytes. Moreover, at day 14 post-stroke, enhanced white matter myelination was detected in the microglial Nhe1 deleted mice. In comparison, neuronal Nhe1-null mice (the CamKII-Cre+/- ;Nhe1f/f mice) showed a significant reduction in both acute and subacute infarct volume, along with increased survival rate and moderate neurological function recovery. However, these neuronal Nhe1-null mice did not exhibit reduced activation of CD11b+ /CD45low-med microglia or CD11b+ /CD45hi macrophages in the ischemic brains, and they exhibited no reductions in white matter lesions. Taken together, this study demonstrated that deletion of microglial and neuronal Nhe1 had differential effects on ischemic brain damage. Microglial NHE1 is involved in pro-inflammatory responses during post-stroke brain tissue repair. In contrast, neuronal NHE1 activation is directly associated with the acute ischemic neuronal injury but not inflammation. Our study reveals that NHE1 protein is a potential therapeutic target critical for differential regulation of ischemic neuronal injury, demyelination and tissue repair.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Doenças Desmielinizantes/etiologia , Infarto da Artéria Cerebral Média/complicações , Microglia/metabolismo , Recuperação de Função Fisiológica/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Substância Branca/patologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Receptor 1 de Quimiocina CX3C/genética , Proteínas de Ligação ao Cálcio/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Trocador 1 de Sódio-Hidrogênio/genética , Distúrbios Somatossensoriais/etiologia , Tamoxifeno/farmacologia , Substância Branca/diagnóstico por imagemRESUMO
Stimulation of Na+ /H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/- ;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/- ;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/patologia , Gliose/patologia , Infarto da Artéria Cerebral Média/complicações , Trocador 1 de Sódio-Hidrogênio/deficiência , Animais , Astrócitos/ultraestrutura , Barreira Hematoencefálica/ultraestrutura , Infarto Encefálico/diagnóstico , Infarto Encefálico/etiologia , Infarto Encefálico/genética , Circulação Cerebrovascular/genética , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/etiologia , Gliose/genética , Gliose/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Atividade Motora/genética , Exame Neurológico , Reperfusão , Trocador 1 de Sódio-Hidrogênio/genéticaRESUMO
Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na(+) signaling influences and regulates important glial activities, and plays a role in neuron-glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na(+) pumps and Na(+) -dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na(+) homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na(+) -dependent ion transporters, including Na(+) /K(+) ATPase, Na(+) /Ca(2+) exchangers, Na(+) /H(+) exchangers, Na(+) -K(+) -Cl(-) cotransporters, and Na(+) - HCO3- cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na(+) -dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na(+) dynamics in different neurological disorders. GLIA 2016;64:1677-1697.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Doenças do Sistema Nervoso/patologia , Neuroglia/fisiologia , Sódio/metabolismo , Animais , Humanos , Transporte de Íons/fisiologia , Transdução de Sinais/fisiologiaRESUMO
In this study, we investigated the development of endoplasmic reticulum (ER) stress after traumatic brain injury (TBI) and the efficacy of post-TBI administration of docosahexaenoic acid (DHA) in reducing ER stress. TBI was induced by cortical contusion injury in Sprague-Dawley rats. Either DHA (16 mg/kg in DMSO) or vehicle DMSO (1 ml/kg) was administered intraperitoneally at 5 min after TBI, followed by a daily dose for 3-21 d. TBI triggered sustained expression of the ER stress marker proteins including phosphorylated eukaryotic initiation factor-2α, activating transcription factor 4, inositol requiring kinase 1, and C/EBP homologous protein in the ipsilateral cortex at 3-21 d after TBI. The prolonged ER stress was accompanied with an accumulation of abnormal ubiquitin aggregates and increased expression of amyloid precursor protein (APP) and phosphorylated tau (p-Tau) in the frontal cortex after TBI. The ER stress marker proteins were colocalized with APP accumulation in the soma. Interestingly, administration of DHA attenuated all ER stress marker proteins and reduced the accumulation of both ubiquitinated proteins and APP/p-Tau proteins. In addition, the DHA-treated animals exhibited early recovery of their sensorimotor function after TBI. In summary, our study demonstrated that TBI induces a prolonged ER stress, which is positively correlated with abnormal APP accumulation. The sustained ER stress may play a role in chronic neuronal damage after TBI. Our findings illustrate that post-TBI administration of DHA has therapeutic potentials in reducing ER stress, abnormal protein accumulation, and neurological deficits.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Lesões Encefálicas/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , Estresse do Retículo Endoplasmático/fisiologia , Neurônios/metabolismo , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Animais , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Ácidos Docosa-Hexaenoicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Proteínas tau/antagonistas & inibidoresRESUMO
BACKGROUND AND PURPOSE: WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. METHOD: Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. RESULTS: WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr(203)/Thr(207)/Thr(212), as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. CONCLUSIONS: These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.
Assuntos
Lesões Encefálicas/enzimologia , Doenças do Sistema Nervoso/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/biossíntese , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/enzimologia , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Gravidez , Proteínas Serina-Treonina Quinases/deficiência , Recuperação de Função Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes/métodos , Genoma Humano , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Forma Celular , Tamanho Celular , Proteínas Culina/genética , Proteínas Culina/metabolismo , Regulação para Baixo , Éxons , Genótipo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas dos Microfilamentos , Antígenos de Histocompatibilidade Menor , Pressão Osmótica , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
BACKGROUND: The bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown. METHODS: Expression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons. RESULTS: We show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment. CONCLUSION: Together, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.
Assuntos
Neoplasias Encefálicas/metabolismo , Movimento Celular , Glioma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Western Blotting , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glioma/patologia , Humanos , Imuno-Histoquímica , Antígenos de Histocompatibilidade Menor , Análise Serial de Tecidos , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Strict homeostatic control of pH in both intra- and extracellular compartments of the brain is fundamentally important, primarily due to the profound impact of free protons ([H+]) on neuronal activity and overall brain function. Astrocytes, crucial players in the homeostasis of various ions in the brain, actively regulate their intracellular [H+] (pHi) through multiple membrane transporters and carbonic anhydrases. The activation of astroglial pHi regulating mechanisms also leads to corresponding alterations in the acid-base status of the extracellular fluid. Notably, astrocyte pH regulators are modulated by various neuronal signals, suggesting their pivotal role in regulating brain acid-base balance in both health and disease. This review presents the mechanisms involved in pH regulation in astrocytes and discusses their potential impact on extracellular pH under physiological conditions and in brain disorders. Targeting astrocytic pH regulatory mechanisms represents a promising therapeutic approach for modulating brain acid-base balance in diseases, offering a potential critical contribution to neuroprotection.
Assuntos
Astrócitos , Encéfalo , Astrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Animais , Encéfalo/metabolismo , Encefalopatias/metabolismo , Encefalopatias/patologia , HomeostaseRESUMO
The blood-brain barrier (BBB) plays a critical role in maintaining ion and fluid homeostasis, essential for brain metabolism and neuronal function. Regulation of nutrient, water, and ion transport across the BBB is tightly controlled by specialized ion transporters and channels located within its unique cellular components. These dynamic transport processes not only influence the BBB's structure but also impact vital signaling mechanisms, essential for its optimal function. Disruption in ion, pH, and fluid balance at the BBB is associated with brain pathology and has been implicated in various neurological conditions, including stroke, epilepsy, trauma, and neurodegenerative diseases such as Alzheimer's disease (AD). However, knowledge gaps exist regarding the impact of ion transport dysregulation on BBB function in neurodegenerative dementias. Several factors contribute to this gap: the complex nature of these conditions, historical research focus on neuronal mechanisms and technical challenges in studying the ion transport mechanisms in in vivo models and the lack of efficient in vitro BBB dementia models. This review provides an overview of current research on the roles of ion transporters and channels at the BBB and poses specific research questions: 1) How are the expression and activity of key ion transporters altered in AD and vascular dementia (VaD); 2) Do these changes contribute to BBB dysfunction and disease progression; and 3) Can restoring ion transport function mitigate BBB dysfunction and improve clinical outcomes. Addressing these gaps will provide a greater insight into the vascular pathology of neurodegenerative disorders.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Demência Vascular , Canais Iônicos , Humanos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Demência Vascular/metabolismo , Demência Vascular/fisiopatologia , Demência Vascular/patologia , Canais Iônicos/metabolismo , Animais , Transporte de Íons , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Neuroinflammation is a pathological event associated with many neurological disorders, including dementia and stroke. The choroid plexus (ChP) is a key structure in the ventricles of the brain that secretes cerebrospinal fluid (CSF), forms a blood-CSF barrier, and responds to disease conditions by recruiting immune cells and maintaining an immune microenvironment in the brain. Despite these critical roles, the exact structural and functional changes to the ChP over post-stroke time remain to be elucidated. We induced ischemic stroke in C57BL/6J mice via transient middle cerebral artery occlusion which led to reduction of cerebral blood flow and infarct stroke. At 1-7 days post-stroke, we detected time-dependent increase in the ChP blood-CSF barrier permeability to albumin, tight-junction damage, and dynamic changes of SPAK-NKCC1 protein complex, a key ion transport regulatory system for CSF production and clearance. A transient loss of SPAK protein complex but increased phosphorylation of the SPAK-NKCC1 complex was observed in both lateral ventricle ChPs. Most interestingly, stroke also triggered elevation of proinflammatory Lcn2 mRNA and its protein as well as infiltration of anti-inflammatory myeloid cells in ChP at day 5 post-stroke. These findings demonstrate that ischemic strokes cause significant damage to the ChP blood-CSF barrier, contributing to neuroinflammation in the subacute stage.
RESUMO
Docosahexaenoic acid (DHA) has neuroprotective effects in several neurodegenerative disease conditions. However, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on astrocyte Ca(2+) signaling under in vitro ischemic conditions (oxygen/glucose deprivation and reoxygenation, OGD/REOX). OGD (2h) triggered a Ca(2+) (ER) store overload (â¼1.9-fold). Ca(2+) uptake by the Ca(2+) (ER) stores was further augmented during REOX and Ca(2+) (ER) was elevated by â¼4.7-fold at 90min REOX. Interestingly, Ca(2+) (ER) stores abruptly released Ca(2+) at â¼120min REOX and emptied at 160min REOX. Depletion of Ca(2+) (ER) stores led to delayed elevation of intracellular Ca(2+) concentration (Ca(2+) (cyt) ) and cell death. Activation of the purinergic receptor P2Y1 was responsible for the release of Ca(2+) (ER) . Most importantly, DHA blocked the initial Ca(2+) (ER) store overload, the delayed depletion of Ca(2+) (ER) , and rise in Ca(2+) (cyt) , which was in part via inhibiting d-myo-inositol 1,4,5-triphosphate receptors. The DHA metabolite DiHDoHE exhibited similar effects. DHA also attenuated expression of phosphorylated eukaryotic initiation factor 2α and activating transcription factor-4, two ER stress markers, following in vitro ischemia. Taken together, these findings suggest that DHA has protective effects in astrocytes following in vitro ischemia, in part, by inhibiting Ca(2+) dysregulation and ER stress.
Assuntos
Astrócitos/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/antagonistas & inibidores , Ácidos Docosa-Hexaenoicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Isquemia/patologia , Fármacos Neuroprotetores/farmacologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Células Cultivadas , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/fisiologia , Isquemia/metabolismo , Camundongos , Oxirredução/efeitos dos fármacosRESUMO
The hallmark of apoptosis is a significant reduction in cell volume (AVD) resulting from loss of K(+)(i) and Cl(-)(i). Loss of cell volume and lowering of ionic strength of intracellular K(+) and Cl(-) occur before any other detectable characteristics of apoptosis. In the present study, temozolomide (TMZ) triggered loss of K(+)(i) and Cl(-)(i) and AVD in primary glioblastoma multiforme (GBM) cancer cells (GC) and GC cancer stem cells (GSC). We hypothesize that Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1) counteracts AVD during apoptosis in GBM cancer cells by regulating cell volume and Cl(-) homeostasis. NKCC1 protein was expressed in both GC and GSC and played an essential role in regulatory volume increase (RVI) in response to hypertonic cell shrinkage and isotonic cell shrinkage. Blocking NKCC1 activity with its potent inhibitor bumetanide abolished RVI. These cells maintained a basal [Cl(-)](i) (~ 68 mM) above the electrochemical equilibrium for Cl(-)(i). NKCC1 also functioned to replenish Cl(-)(i) levels following the loss of Cl(-)(i). TMZ-treated cells exhibited increased phosphorylation of NKCC1 and its up-stream novel Cl(-)/volume-sensitive regulatory kinase WNK1. Inhibition of NKCC1 activity with bumetanide accelerated AVD, early apoptosis, as well as activation of caspase-3 and caspase-8. Taken together, this study strongly suggests that NKCC1 is an essential mechanism in GBM cells to maintain K(+), Cl(-), and volume homeostasis to counteract TMZ-induced loss of K(+), Cl(-) and AVD. Therefore, blocking NKCC1 function augments TMZ-induced apoptosis in glioma cells.
Assuntos
Apoptose/efeitos dos fármacos , Bumetanida/farmacologia , Dacarbazina/análogos & derivados , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Tamanho Celular/efeitos dos fármacos , Cloretos/metabolismo , Dacarbazina/farmacologia , Sinergismo Farmacológico , Glioblastoma , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Temozolomida , Imagem com Lapso de Tempo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Reactive astrocytes (RA) secrete lipocalin-2 (LCN2) glycoprotein that regulates diverse cellular processes including cell death/survival, inflammation, iron delivery and cell differentiation. Elevated levels of LCN2 are considered as a biomarker of brain injury, however, the underlying regulatory mechanisms of its expression and release are not well understood. In this study, we investigated the role of astrocytic Na+/H+ exchanger 1 (NHE1) in regulating reactive astrocyte LCN2 secretion and neurodegeneration after stroke. Astrocyte specific deletion of Nhe1 in Gfap-CreER+/-;Nhe1f/f mice reduced astrogliosis and astrocytic LCN2 and GFAP expression, which was associated with reduced loss of NeuN+ and GRP78+ neurons in stroke brains. In vitro ischemia in astrocyte cultures triggered a significant increase of secreted LCN2 in astrocytic exosomes, which caused neuronal cell death and neurodegeneration. Inhibition of NHE1 activity during in vitro ischemia with its potent inhibitor HOE642 significantly reduced astrocytic LCN2+ exosome secretion. In elucidating the cellular mechanisms, we found that stroke triggered activation of NADPH oxidase (NOX)-NF-κB signaling and ROS-mediated LCN2 expression. Inhibition of astrocytic NHE1 activity attenuated NOX signaling and LCN2-mediated neuronal apoptosis and neurite degeneration. Our findings demonstrate for the first time that RA use NOX signaling to stimulate LCN2 expression and secretion. Blocking astrocytic NHE1 activity is beneficial to reduce LCN2-mediated neurotoxicity after stroke.
Assuntos
Astrócitos , Lipocalina-2 , Acidente Vascular Cerebral , Animais , Astrócitos/metabolismo , Isquemia/metabolismo , Lipocalina-2/genética , Lipocalina-2/metabolismo , Camundongos , NADPH Oxidases/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na+/H+ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that â¼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/ß-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.
Assuntos
Barreira Hematoencefálica , Isquemia Encefálica , AVC Isquêmico , Animais , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Camundongos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
In the present study, we investigated changes of cytosolic Ca2+([Ca2+](cyt)), endoplasmic reticulum Ca2+([Ca2+](ER)) and mitochondrial Ca2+(Ca2+(m)) in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Two hours OGD did not cause changes in [Ca2+](cyt), but led to a significant increase in [Ca2+](ER). The elevation in [Ca2+](ER) continued and reached a peak level (130 +/- 2 microM) by 90 min REOX. An abrupt release of Ca2+(ER) occurred during 1.5-2.5 h REOX, which was accompanied with a delayed and sustained rise in [Ca2+](cyt). Moreover, Ca2+(m) content was increased significantly within 15 min REOX followed by a secondary rise (approximately 4.5-fold) and a release of mitochondrial cytochrome c (Cyt c). Astrocytes exhibited translocation of Cyt c from mitochondria to endoplasmic reticulum (ER) and up regulation of ER stress protein p-eIF2alpha. Blocking Na+-K+-Cl(-) cotransporter isoform 1 activity, either by its potent inhibitor bumetanide or genetic ablation, abolished release of ER Ca2+, delayed rise in [Ca2+](cyt) and Ca2+(m). Inhibition of the reverse mode operation of the Na+/Ca2+ exchanger significantly attenuated OGD/REOX-mediated Cyt c release. In summary, this study illustrates that OGD/REOX triggers a time-dependent loss of Ca2+ homeostasis in cytosol and organelles (ER and mitochondria) in astrocytes. Collective stimulation of Na+-K+-Cl(-) cotransporter isoform 1 and reverse mode function of Na+/Ca2+ exchanger contributes to these changes.
Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Citocromos c/metabolismo , Retículo Endoplasmático/metabolismo , Glucose/deficiência , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Animais , Astrócitos/enzimologia , Hipóxia Celular/fisiologia , Células Cultivadas , Retículo Endoplasmático/enzimologia , Camundongos , Camundongos Mutantes , Mitocôndrias/enzimologia , Consumo de Oxigênio/fisiologiaRESUMO
BACKGROUND: The role and dose of anticoagulants in thromboprophylaxis for patients with cancer receiving chemotherapy through central venous catheters (CVCs) is controversial. We therefore assessed whether warfarin reduces catheter-related thrombosis compared with no warfarin and whether the dose of warfarin determines the thromboprophylactic effect. METHODS: In 68 clinical centres in the UK, we randomly assigned 1590 patients aged at least 16 years with cancer who were receiving chemotherapy through CVCs to no warfarin, fixed-dose warfarin 1 mg per day, or dose-adjusted warfarin per day to maintain an international normalised ratio between 1.5 and 2.0. Clinicians who were certain of the benefit of warfarin randomly assigned patients to fixed-dose or dose-adjusted warfarin groups. The primary outcome was the rate of radiologically proven, symptomatic catheter-related thrombosis. Analysis was by intention to treat. This trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN 50312145. FINDINGS: Compared with no warfarin (n=404), warfarin (n=408; 324 [79%] on fixed-dose and 84 [21%] on dose-adjusted) did not reduce the rate of catheter-related thromboses (24 [6%] vs 24 [6%]; relative risk 0.99, 95% CI 0.57-1.72, p=0.98). However, compared with fixed-dose warfarin (n=471), dose-adjusted warfarin (n=473) was superior in the prevention of catheter-related thromboses (13 [3%] vs 34 [7%]; 0.38, 0.20-0.71, p=0.002). Major bleeding events were rare; an excess was noted with warfarin compared with no warfarin (7 vs 1, p=0.07) and with dose-adjusted warfarin compared with fixed-dose warfarin (16 vs 7, p=0.09). A combined endpoint of thromboses and major bleeding showed no difference between comparisons. We did not note a survival benefit in either comparison. INTERPRETATION: The findings show that prophylactic warfarin compared with no warfarin is not associated with a reduction in symptomatic catheter-related or other thromboses in patients with cancer and therefore we should consider newer treatments. FUNDING: Medical Research Council and Cancer Research UK.
Assuntos
Anticoagulantes/uso terapêutico , Antineoplásicos/administração & dosagem , Cateterismo Venoso Central/efeitos adversos , Neoplasias/tratamento farmacológico , Trombose Venosa/etiologia , Trombose Venosa/prevenção & controle , Varfarina/uso terapêutico , Idoso , Anticoagulantes/efeitos adversos , Antineoplásicos/uso terapêutico , Feminino , Hemorragia/induzido quimicamente , Humanos , Coeficiente Internacional Normatizado , Masculino , Metanálise como Assunto , Pessoa de Meia-Idade , Neoplasias/mortalidade , Varfarina/efeitos adversosRESUMO
Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Gliose/prevenção & controle , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Tamanho Celular , Feminino , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Gliose/genética , Gliose/metabolismo , Gliose/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Membro 2 da Família 12 de Carreador de Soluto/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Sodium/hydrogen exchanger 1 (NHE1), encoded by the SLC9A1 gene (SoLute Carrier family 9A1) in humans, is the main H+ efflux mechanism in maintaining alkaline intracellular pH (pHi) and Warburg effects in glioma. However, to date, there are no clinical studies exploring pharmacological inhibition of NHE1 protein in cancer treatment. In this study, we investigated NHE1 expression in gliomas and its relationship with glioma clinical outcome. METHODS: The Chinese Glioma Genome Atlas (CGGA) dataset containing transcriptome sequencing data of 325 glioma samples and the Cancer Genome Atlas (TCGA) with 698 glioma mRNAseq data were analyzed in this study. Mouse SB28 and GL26 intracranial syngeneic glioma models in C57BL/6 J mice were established to investigate NHE1 expression and impact of NHE1 protein inhibition with its inhibitor HOE642 on tumorigenesis and anti-PD1 therapy. Tumor angiogenesis, immunogenicity, and progression were assessed by immunofluorescence staining and flow cytometric profiling. RESULTS: Analysis of SLC9A1 mRNA expression in two data sets, CGGA and TCGA, reveals significantly higher SLC9A1 mRNA levels in higher grade gliomas. The SLC9A1 mRNA expression was especially enriched in isocitrate dehydrogenase (IDH)1/2 wild-type glioblastoma (GBM) and in mesenchymal glioma subtypes. Worsened survival probabilities were correlated with the elevated SLC9A1 mRNA levels in gliomas. The underlying mechanisms include promoting angiogenesis, and extracellular matrix remodeling. Increased SLC9A1 mRNA expression was also associated with tumor-associated macrophage accumulation. NHE1 inhibitor HOE642 reduced glioma volume, invasion, and prolonged overall survival in mouse glioma models. Blockade of NHE1 protein also stimulated immunogenic tumor microenvironment via activating CD8 T-cell accumulation, increasing expression of interferon-gamma (Ifng), and sensitized animals to anti-PD-1 therapy. CONCLUSION: Our findings strongly suggest that NHE1 protein emerges as a marker for tumorigenesis and prognosis in glioma. Blocking NHE1 protein is a novel strategy for adjuvant anti-cancer therapies.