Decreased connexin 43 in astrocytes inhibits the neuroinflammatory reaction in an acute mouse model of neonatal sepsis

Neonatal sepsis is common in neonatal intensive care units,often complicated by injury to the immature brain. Previous studies have shown that the expression of the gap junction protein connexin 43(Cx43) in the brain decreases when stimulated by neuro-infl ammatory drugs such as lipopolysaccharide(LPS). Here we showed that partial deletion of Cx43 in astrocytes resulted in weakened inflammatory responses. The up-regulation of pro-inflammatory cytokines was significantly reduced in mice with partial deletion of Cx43 in astrocytes compared with wild-type littermates after systemic LPS injection. Moreover,microglial activation was inhibited in mice with partial deletion of Cx43. These results showed that Cx43 in astrocytes plays a critical role in neuro-infl ammatory responses. This work provides a potential therapeutic target for inhibiting neuroinfl ammatory responses in neonatal sepsis.     

Astrocyte cultures from conditional connexin43-deficient mice

Connexin43 (Cx43) mainly provides the molecular basis for astrocytic gap junctions. Interastrocytic coupling is thought to mediate extracellular ion homeostasis, long-range signaling, and neuroprotection in the brain. Cx43 has been implicated in astrocytic growth control and is also expressed in other cell types in the brain, such as leptomeningeal and vascular cells. Cx43 function has been studied in astrocyte cultures of Cx43-deficient mice, which lack Cx43 in all cell types. We have generated conditionally deficient mice with an astrocyte-directed inactivation of Cx43, which leaves expression in other cell types unaffected. Other connexins have been detected in astrocytes. For the study of astrocytes lacking Cx45 and Cx26 in vitro, which deficiencies are embryonic lethal, conditionally deficient astrocyte cultures are essential. In the present study, we describe the developmental kinetics of Cx43 inactivation and loss of intercellular communication in astrocyte cultures derived from conditional Cx43-deficient mice. Conditional ablation of Cx43 is efficient, reaches a plateau at 4 weeks in culture, but retains Cx43 expression in contaminating nonastrocytic cells. Our findings indicate that conditional knockout astrocytes are a promising tool for the study of embryonic lethal genes in astrocyte cultures.     

Proteomic analysis of astroglial connexin43 silencing uncovers a cytoskeletal platform involved in process formation and migration

Connexin43 (Cx43) is the most abundant gap junction protein of the brain, where it is predominantly expressed in astrocytes. Recent studies imply a role of Cx43 in the regulation of important cellular processes, including migration, proliferation, and shape formation. These processes are assumed to be reflected by the proteome of the Cx43 expressing cells. To analyze the influence of Cx43 on the astrocytic proteome, we used RNA interference to downregulate the expression of this connexin in cultures of mouse astrocytes. We applied difference gel electrophoresis (DIGE) to compare silenced astrocytes with control cells. The differential proteome analysis revealed 15 significantly regulated proteins (between 1.2‐ and 1.6‐fold), of which six are known to belong to a group of cytoskeletal proteins involved in cortical platform formation. Astrocytes treated with Cx43 small interfering (si)RNA showed an increased expression of the cytoskeletal proteins: actin, tropomyosin, microtubule‐associated protein RP/EB1, transgelin, and GFAP, and a decreased expression of cofilin‐1. Quantitative immunocytochemistry and Western blotting revealed similar results showing an upregulation of actin, tubulin, tropomyosin, EB1, transgelin and GFAP, and a downregulation of Ser‐3‐phosphorylated cofilin. Furthermore, Cx43 silencing led to phenotypical changes in cell morphology, migratory activity, and cell adhesion. Our results provide mechanistic clues for an understanding of Cx43 interaction with cellular motor activities such as migration and process formation in astrocytes. © 2009 Wiley‐Liss, Inc.     

Analysis of connexin expression during mouse Schwann cell development identifies connexin29 as a novel marker for the transition of neural crest to precursor cells

Connexins are transmembrane proteins forming gap junction channels for direct intercellular and, for example in myelinating glia cells, intracellular communication. In mature myelin-forming Schwann cells, expression of multiple connexins, i.e. connexin (Cx) 43, Cx29, Cx32, and Cx46 (after nerve injury) has been detected. However, little is known about connexin protein expression during Schwann cell development. Here we use histochemical methods on wildtype and Cx29lacZ transgenic mice to investigate the developmental expression of connexins in the Schwann cell lineage. Our data demonstrate that in the mouse Cx43, Cx29, and Cx32 protein expression is activated in a developmental sequence that is clearly correlated with major developmental steps in the lineage. Only Cx43 was expressed from neural crest cells onwards. Cx29 protein expression was absent from neural crest cells but appeared as neural crest cells generated precursors (embryonic day 12) both in vivo and in vitro. This identifies Cx29 as a novel marker for cells of the defined Schwann cell lineage. The only exception to this were dorsal roots, where the expression of Cx29 was delayed four days relative to ventral roots and spinal nerves. Expression of Cx32 commenced postnatally, coinciding with the onset of myelination. Thus, the coordinated expression of connexin proteins in cells of the embryonic and postnatal Schwann cell lineage might point to a potential role in peripheral nerve development and maturation.     

Normobaric hyperoxia plays a neuroprotective role after cerebral ischemia by maintaining the redox homeostasis and the level of connexin43 in astrocytes

IntroductionAcute cerebral ischemia is caused by an insufficient blood supply to brain tissue. Oxygen therapy, which is able to aid diffusion to reach the ischemic region, has been regarded as a possible treatment for cerebral ischemia. Recent animal and pilot clinical studies have reported that normobaric hyperoxia (NBO) showed neuroprotective effects if started soon after the onset of stroke. However, little is known about the role and mechanism of NBO treatment in astrocytes. Connexin43, one of the main gap junction proteins in astrocytes, is extremely sensitive to hypoxia and oxidative stress after cerebral ischemia.AimsIn the present study, we used sutures to develop an ischemia/reperfusion model in rats to mimic clinical recanalization and investigated the role of connexin43 in NBO‐treated stroke rats, as well as the underlying mechanism of NBO therapy.ResultsNormobaric hyperoxia treatment maintained the homeostasis of oxidoreductases: glutathione peroxidase 4 (GPX4) and NADPH oxidase 4 (two important oxidoreductases) and rescued the ischemia/reperfusion‐induced downregulation of connexin43 protein in astrocytes. Furthermore, NBO treatment attenuated cerebral ischemia‐induced cytochrome c release from mitochondria and was involved in neuroprotective effects by regulating the GPX4 and connexin43 pathway, using Ferrostatin‐1 (an activator of GPX4) or Gap27 (an inhibitor of connexin43).ConclusionsThis study showed the neuroprotective effects of NBO treatment by reducing oxidative stress and maintaining the level of connexin43 in astrocytes, which could be used for the clinical treatment of ischemic stroke.     

Connexin43 immunoreactivity in the catfish retina

We used antibodies directed against rat heart connexin43 (Cx43) to perform immunoblot and immunohistochemical (IHC) analyses of the catfish retina. The antibodies recognized a retinal protein which co-migrated with mouse brain Cx43. IHC staining resulted in punctate labeling of the external limiting membrane and the outer nuclear layer. Although infrequent, labeling was also observed in the inner nuclear layer. These results suggest that a Cx43 isoform may be present in Müller glial cells and neurons of the distal catfish retina.     

Cx43/Cx45异型缝隙连接通道参与实验性蛛网膜下腔出血后脑血管痉挛的实验研究

目的探讨缝隙连接通道参与脑血管痉挛(CVS)的机制。方法选择健康新西兰大白兔36只,随机分为3组:正常对照组(每组n=12)、SAH-7d组(每组n=12)、SAH-7d+甘珀酸(CBX)组(每组n=12);用二次注血法制成兔SAH模型。应用脑血管造影技术观察造影前后各组基底动脉的血管直径,免疫共沉淀技术观察Cx43与Cx45在各实验组中相互作用的变化。结果脑血管造影显示单纯注血7d组较正常组基底动脉明显痉挛(P0.01),正常对照组及SAH-7d+CBX组基底动脉无明显痉挛,免疫共沉淀示Cx43与Cx45在体内的相互作用在SAH-7d组较正常组及SAH-7d+CBX组均明显增加(P0.01)。结论实验结果显示SAH后兔基底动脉较正常组明显痉挛,单纯出血组Cx43与Cx45组成的异型缝隙连接通道较正常组增加,而CBX能缓解SAH后的CVS及抑制Cx43/Cx45缝隙连接蛋白的高表达,即Cx43/Cx45异型通道的增加可能参与SAH后CVS的形成。     

兔骨髓间充质干细胞体外向心肌样细胞诱导分化：缝隙连接蛋白43的

背景：骨髓间充质干细胞经过诱导因素刺激后,能够分化为心肌细胞,同时表达缝隙连接蛋白43,移植后可改善心功能或修复受损的心脏起搏传导系统。而缝隙连接蛋白43在维持心脏正常功能中发挥着重要作用。 目的：观察兔骨髓间充质干细胞体外诱导分化为心肌样细胞过程中缝隙连接蛋白43的表达变化,及诱导后骨髓间充质干细胞间隙连接通讯功能的改变。 方法：采用Percoll非密度梯度离心法与贴壁法分离培养兔骨髓间充质干细胞,正常组不进行任何干预,诱导组加入5-氮胞苷向心肌样细胞诱导,免疫荧光及流式细胞仪检验细胞诱导前后缝隙连接蛋白43的表达。将原代培养1 d的乳鼠心肌细胞分别接种于上述两组细胞爬片上,免疫荧光观察兔骨髓间充质干细胞与心肌细胞形成间隙连接的情况。划痕试验设立正常组、诱导组以及添加甘草次酸的阻滞剂组,观察兔骨髓间充质干细胞诱导前后细胞间隙连接的功能变化。 结果与结论：正常组骨髓间充质干细胞缝隙连接蛋白43呈弱表达,5-氮胞苷诱导2,4周后缝隙连接蛋白43的表达均显著增加(P < 0.01),细胞间隙连接通讯功能明显增强(P < 0.001),且随诱导时间的延长呈依赖性增强(P < 0.05)。骨髓间充质干细胞与心肌细胞共培养后,诱导组缝隙连接蛋白43明显呈线状表达在两个相邻细胞的接触面。与诱导4周时比较,阻滞剂组细胞间隙连接通讯功能明显受到抑制(P < 0.001)。提示骨髓间充质干细胞能够自发表达缝隙连接蛋白43,在移植后早期能与心肌细胞形成间隙连接,从而有利于移植后心肌电传导,并发挥骨髓间充质干细胞的旁分泌效应。 关键词：缝隙连接蛋白43;间隙连接;5-氮胞苷;诱导;心肌细胞;骨髓间充质干细胞     

Deletion of oligodendrocyte Cx32 and astrocyte Cx43 causes white matter vacuolation, astrocyte loss and early mortality

CNS glia exhibit a variety of gap junctional interactions: between neighboring astrocytes, between neighboring oligodendrocytes, between astrocytes and oligodendrocytes, and as 'reflexive' structures between layers of myelin in oligodendrocytes. Together, these junctions are thought to form a network facilitating absorption and removal of extracellular K(+) released during neuronal activity. In mice, loss of the two major oligodendrocyte connexins causes severe demyelination and early mortality, while loss of the two major astrocyte connexins causes mild dysmyelination and sensorimotor impairment, suggesting that reflexive and/or oligo-oligo coupling may be more important for the maintenance of myelin than other forms. To further explore the functional relationships between glial connexins, we generated double knockout mice lacking one oligodendrocyte and one astrocyte connexin. Cx32-Cx43 dKO animals develop white matter vacuolation without obvious ultrastructural abnormalities in myelin. Progressive loss of astrocytes but not oligodendrocytes or microglia accompanies sensorimotor impairment, seizure activity and early mortality at around 16 weeks of age. Our data reveal an unexpected role for connexins in the survival of white matter astrocytes, requiring the expression of particular isoforms in both oligodendrocytes and astrocytes.     

Ischemia-induced cellular redistribution of the astrocytic gap junctional protein connexin43 in rat brain

The distribution and levels of the astrocytic gap junction protein, connexin43 (Cx43) was analyzed in various regions of brain as a function of time after neuronal loss and consequent reactive gliosis induced by bilateral carotid occlusion in rats. In the striatum 2 days after induction of ischemia, immunostaining intensity for Cx43 increased in animals exhibiting mild to moderate striatal damage, whereas areas of reduced staining surrounded by elevated levels of Cx43 immunoreactivity were observed in animals with severe ischemic damage. Immunolabelling of glial cell bodies was evident in ischemic, but not normal, striatum. Similar, though less dramatic, changes were seen at 7 days post-ischemia. Compared with the fine punctate pattern of Cx43 staining seen in normal striatum, ischemic striatal areas contained large aggregates of punctate profiles. In the hippocampus, increased immunostaining was seen at 2 and 7 days post-ischemia and, unlike normal hippocampus, neurons in the CA3 pyramidal cell layer were surrounded by a network of Cx43-immunoreactive puncta at the latter survival time. Immuno-EM analysis of ischemic tissue revealed numerous immunolabelled gap junctions among astrocytic processes in the vicinity of degenerating neurons and elevated levels of intracellular Cx43 immunoreactivity in astrocytic processes and cell bodies. No differences in protein levels or phosphorylation states of Cx43 were detected in either hippocampus or striatum by Western blot analyses of ischemic and control tissue. These results suggest that astrocytes respond to an ischemic insult by reorganizing their gap junctions, that the qualitative nature of their response is dependent on the severity of neuronal damage or loss, and that a pool of Cx43 normally undetectable by immunohistochemistry may contribute to the ischemia-induced elevations of immunolabelling for this protein.     

Quantitative immunohistochemical and biochemical correlates of connexin43 localization in rat brain.

We have shown by immunohistochemical methods that the gap junction protein connexin43 is heterogeneously distributed in rat brain (Yamamoto et al: J Comp Neurol 302:853, 1990). Here we have compared quantitatively the relative amount of connexin43 detected on Western blots of seven central nervous system (CNS) regions with the density of connexin43-immunoperoxidase reactivity in these regions. As has been observed on Western blots of several cell types, homogenates of these CNS regions contained two forms of connexin43, its dephospho form with an apparent mobility of approximately 41 kDa and its approximately 43 kDa phosphorylated form. While the relative quantities of connexin43 varied considerably among the brain regions, the ratio of the 43/41 kDa forms, 0.71, was relatively uniform (correlation coefficient, r = 0.92). Sections of brain processed for connexin43-immunolocalization by the peroxidase-antiperoxidase (PAP) method showed that chromogen deposition was linear with incubation time in reaction medium. Optical density of tissue connexin43-immunoreactivity in each of the seven areas plotted against the density of connexin43 bands on Western blots gave a correlation coefficient of r = 0.90. Connexin43-immunoreactivity had a similar appearance in sections processed by PAP or immunofluorescence procedures and consisted of isolated or aggregates of puncta.(ABSTRACT TRUNCATED AT 250 WORDS)     

缺血后处理对大鼠脑缺血再灌注后缝隙连接蛋白43的影响

目的 观察缺血后处理对脑缺血再灌注后缝隙连接蛋白43(Cx43)的影响.方法 30只Wistar雄性大鼠随机分为假手术(Sham)组、缺血再灌注(I/R)组、缺血后处理(IP)组.采用线栓法建立大鼠大脑中动脉缺血模型,脑缺血2h后,I/R组予再灌注,IP组给予大脑中动脉血流再通30s,之后阻断血流30s,如此进行3个循环之后予永久再灌注.于脑缺血再灌注后24h行TTC染色观察脑梗死体积、神经功能缺损评分,应用激光共聚焦显微镜观察Cx43蛋白表达量及分布的变化,行各组间比较.结果 IP组脑梗死体积明显小于I/R组,IP组神经缺损评分低于I/R组,与Sham组相比,I/R组Cx43数量明显减低、分布紊乱,IP组Cx43表达数量及分布明显好于I/R组.结论 缺血后处理可减小脑梗死体积、减轻神经功能缺损,缺血后处理能抑制Cx43表达减少、改善Cx43分布情况.     

脑血管痉挛中缝隙连接蛋白Cx43磷酸化位点分析及S368位点与其关系的研究

目的 观察脑血管痉挛中缝隙连接蛋白Cx43的磷酸化位点及其S368位点磷酸化水平的变化,探讨其与脑血管痉挛的关系. 方法 新西兰大白兔78只按随机数字表法分为4组:正常对照组(n=6)、单纯脑池注血组(n=24)、甘珀酸(CBX)脑池处理组(n=24)和溶媒脑池处理组(n=24),后3组应用枕大池二次注血法建立兔蛛网膜下腔出血后脑血管痉挛模型及相应给药,并按1d、3d、7d、14d分成4亚组,每亚组6只.采用磷酸化蛋白富集试剂盒富集各组基底动脉中Cx43磷酸化总蛋白,再利用质谱技术鉴定出其磷酸化位点;应用Western blotting方法分析各组Cx43S368位点磷酸化水平的变化;通过数字减影血管造影技术(DSA)观察各组基底动脉直径变化情况. 结果 (1)质谱技术成功鉴定出Cx43的4个磷酸化位点,分别为Y265、S364、S365、S368.(2)Western blotting结果显示:正常对照组基底动脉Cx43 S368位点磷酸化水平较低(17.0％±2.3％);单纯脑池注血组及溶媒脑池处理组与正常对照组相比,Cx43 S368位点磷酸化水平在1d、3d、7d、14d各时间点均显著升高,差异有统计学意义(P＜0.05),且以7d表达最高,14d开始下降;CBX脑池处理组各时间点基底动脉Cx43 S368位点磷酸化水平显著低于单纯脑池注血组及溶媒脑池处理组,差异有统计学意义(P＜0.05).(3)DSA显示正常对照组第二次与首次造影基底动脉直径的百分比值平均为99.1％±1.3％,单纯脑池注血组、CBX脑池处理组、溶媒脑池处理组分别为66.1％±7.2％、91.3％±5.3％、63.7％±6.6％,CBX脑池处理组基底动脉直径显著狭窄于单纯脑池注血组,差异有统计学意义(P＜0.05). 结论 缝隙连接蛋白Cx43 S368位点磷酸化可能与脑血管痉挛密切相关,且其可能是CBX缓解脑血管痉挛的机制之一.     

Detection of a novel pattern of connexin 43 immunoreactivity responsive to dehydration in rat hypothalamic magnocellular nuclei

Immunocytochemical expression of Connexin 43 (Cx 43) in the rat Supraoptic Nucleus was analyzed following dehydration, using sequence-specific anti-Cx 43 antibodies (designated 13-8300, 71-0700, and sc-9059) that exhibit differential recognition of Cx 43. Punctate and longitudinally arranged immunostaining patterns of Cx 43 labeling, as evidenced by antibody sc-9059, was detected overlaying the nucleus of magnocellular neuroendocrine cells. This novel form of longitudinally arranged Cx 43 immunoreactivity was modified by dehydration and halothane exposure, but not lactation.     

Differential expression of connexin 43 in the chick tangential vestibular nucleus

The chick tangential nucleus is a major vestibular nucleus whose principal cells receive convergent inputs from primary vestibular and nonvestibular fibers and participate in the vestibular reflexes. During development, the principal cells gradually acquire the mature firing pattern in part by losing a specific potassium current around hatching (H). Here we focus on characterizing the expression of connexin 43 (Cx43), a gap junction protein found mainly between astrocytes in the mature brain. The astrocytic syncytium plays an important role in maintaining extracellular potassium ion balance in the brain. Accordingly, it is important to characterize the potential of this syncytium to communicate during the critical developmental age of hatching. Using fluorescence immunocytochemistry, we investigated whether Cx43 staining was concentrated in specific cellular compartments at H1 by applying well-known markers for astrocytes (glial fibrillary acidic protein; GFAP), oligodendrocytes (antimyelin), neurons (microtubule-associated protein 2), and synaptic terminals (synaptotagmin). GFAP-positive astrocytes and GFAP-negative nonneuronal cells around the principal cell bodies were labeled with Cx43, suggesting that Cx43 was expressed exclusively by nonneuronal cells near the neuronal elements. Next, the developmental pattern of expression of Cx43 was studied at embryonic day 16 (E16), H1, and H9. At E16, Cx43 was present weakly as random small clusters in the tangential nucleus, whereas, at H1, overall staining became localized, with increases in size, brightness, and number of immunostained clusters. Finally, at H9, Cx43 staining decreased, but cluster size and location remained unchanged. These results suggest that Cx43 is developmentally regulated with a peak at birth and is associated primarily with astrocytes and nonneuronal cells near the principal cell bodies.     

Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia

The prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2) is up-regulated in the brain of rodents during cerebral ischemia and contributes to ischemic brain injury. This study sought to determine whether COX-2 is also up-regulated in the human brain in the acute stages of cerebral ischemic infarction. Paraffin-embedded sections from patients who died 1–2 days following infarction in the middle cerebral artery territory were processed for COX-2 immunohistochemistry. COX-2 immunoreactivity was observed in infiltrating neutrophils, in vascular cells and in neurons located at the border of the infarct. The data suggest that COX-2 up-regulation is also relevant to cerebral ischemia in humans and raise the possibility that COX-2 reaction products participate in the mechanisms of ischemic injury also in the human brain. Received: 12 January 1999 / Revised, accepted: 8 March 1999     

Gap junction protein connexin43 (GJA1) in the human glaucomatous optic nerve head and retina

Primary open angle glaucoma is characterised by the progressive and irreversible death of retinal ganglion cells. Experimental evidence suggests that the initial site of injury to the retinal ganglion cell is at or near the lamina cribrosa or in the peripapillary retina. However, the mediators of axonal injury remain poorly understood. The purpose of this study was to investigate the expression of the gap junction protein connexin43 (GJA1) in the human glaucomatous optic nerve head and retina as a potential mediator of axonal injury. Using affinity isolated polyclonal antibodies to the C-terminal segment of human connexin43, the expression of connexin43 was determined in post-mortem human eyes with primary open angle glaucoma and age-matched controls. In normal eyes, connexin43 was present on glial fibrillary acidic protein (GFAP)-positive astrocytes in the retinal ganglion cell layer and optic nerve head. In glaucomatous eyes, increased connexin43 immunoreactivity was observed at the level of the lamina cribrosa and in the peripapillary and mid-peripheral retina in association with glial activation. This novel finding may suggest that gap junction communication is a potential mediator of retinal ganglion cell injury in glaucoma.     

Occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema

The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney＇s free falling method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve cell edema, degenera- tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradually de- creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradually increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.