Gap junction-mediated bidirectional signaling between human fetal hippocampal neurons and astrocytes

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.     

Functional expression of CCR2 by human fetal astrocytes

Astrocytes from different sources bind the chemokine monocyte chemoattractant factor (MCP-1), yet functional expression in these cells of CCR2, the major receptor for this ligand, has been a matter of controversy. Here we show that cultured human fetal astrocytes express CCR2 at the mRNA and protein levels, and display chemotaxis and calcium flux in response to MCP-1. Surface CCR2 protein expression and MCP-1 binding activity were observed to undergo near parallel downmodulation and recovery following MCP-1 exposure, supporting the argument that CCR2, and not another receptor, mediates MCP-1 ligation in these cells. Downmodulation was further determined to occur via receptor internalization, and to apparently proceed via both clathrin-coated vesicles and caveolae, the latter being a novel mode for the endocytosis of chemokine receptors. Insofar as MCP-1 is thought to mediate inflammatory and developmental processes within the central nervous system (CNS), such astrocyte responses to this chemokine are likely to significantly impact physiological and pathophysiological events at the blood-brain barrier and within the CNS parenchyma.     

水通道蛋白8在人胎盘胎膜中的表达

背景：胎盘和胎膜在母胎液体平衡过程中起着重要作用,而水穿过胎盘和胎膜运输的分子和细胞机制目前尚不十分清楚,推测母胎液体交换可能通过胎盘和胎膜上的水通道进行。 目的：观察人正常妊娠晚期胎盘与胎膜组织中水通道蛋白8蛋白质水平的表达及其分布。 设计、时间及地点：对比观察,于2005-07/12在广州医学院实验中心完成。 材料：收集正常足月妊娠剖宫分娩的胎盘与胎膜组织样本各5例,产妇年龄(27±5)岁,实验方法获医院伦理委员会批准。 方法：胎盘胎膜组织在分娩30 min内取样,用无菌生理盐水漂洗干净去除血液,立即放入液氮中,转运至-80 ℃冰箱保存备用。 主要观察指标：运用反转录-聚合酶链反应法、免疫组织化学法及Western印迹法检测水通道蛋白8在胎盘与胎膜组织中的表达与分布。 结果：反转录-聚合酶链反应显示,水通道蛋白8 mRNA在胎盘和胎膜组织中均有表达。水通道蛋白8 Western印迹检测胎盘和胎膜组织显示,一特异性条带在Mr45000左右。免疫组织化学结果显示,水通道蛋白8表达于胎盘合体滋养细胞、羊膜上皮细胞及平滑绒毛膜细胞滋养细胞。 结论：在蛋白质水平显示,水通道蛋白8表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜细胞滋养细胞。     

人脑胶质瘤中Fas及FasL的表达

目的　探讨Fas及FasL基因在人脑胶质瘤中的表达 ,与肿瘤的病理类型及恶性程度的关系。方法　应用Fas及FasL多抗和Ki 6 7单抗免疫组化染色检测 6例正常脑组织及 6 4例人脑胶质瘤。结果　Fas和FasL在胶质瘤中的总表达率分别为 83%和 75 % ,并随WHO分级升高而升高 ,与Ki 6 7LI呈正相关。Fas、FasL共同阳性率 (共表达率 )为 71% ,与肿瘤级别呈正相关。结论　脑胶质瘤中Fas及FasL表达与肿瘤病理类型及恶性程度密切相关     

HIV-1 and IL-1 beta regulate Fas ligand expression in human astrocytes through the NF-kappa B pathway

Reactive astrogliosis is a prominent pathological feature of HIV-1-associated dementia (HAD). We hypothesized that in HAD, astrocytes activated with proinflammatory stimuli such as IL-1beta express Fas ligand (FasL), a death protein. IL-1beta and HIV-1-activated astrocytes expressed FasL mRNA and protein. Luciferase reporter constructs showed that IL-1beta and HIV-1 upregulated FasL promoter activity (p<0.001). The NF-kappaB pathway was involved as shown by inhibition with SN50 and dominant negative IkappaBalpha mutants. Brain extracts from HAD patients had significantly elevated FasL levels compared to HIV-seropositive (p<0.001) and seronegative individuals (p<0.01). We propose that astrocyte expression of FasL may participate in neuronal injury in HAD.     

Growth factors for fetal and adult human astrocytes in culture

Using a recently developed method for the determination of cell proliferation in culture, we assessed the effects of several growth factors on their capability to promote proliferation of astrocytes derived from human fetuses or adults. Factors tested included fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, glial growth factor from bovine pituitary, nerve growth factor, interleukin-2, dibutyryl cyclic adenosine monophosphate, and 4 beta-phorbol 12,13-dibutyrate. The results show that astrocytes from human adults do not undergo proliferation in vitro even in the presence of these agents and that in the case of fetal astrocytes, only glial growth factor from bovine pituitary, platelet-derived growth factor and the phorbol ester were mitogenic. In addition, the capability of fetal astrocytes to proliferate appears to decrease with the increasing length of time they have been maintained in culture.     

Role of mitogen-activated protein kinases in inducible nitric oxide synthase and TNFalpha expression in human fetal astrocytes

Astrocytes are important sources of proinflammatory mediators such as iNOS and TNFalpha in the diseased central nervous system. In previous studies, we showed that the cytokine IL-1 plays a critical role in the activation of human astrocytes to express TNFalpha and the inducible form of nitric oxide synthase (iNOS). In the present study, we have addressed the role of the MAP-kinase pathway in the signaling events leading to the induction of these genes. Treatment with SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), potently inhibited IL-1-mediated induction of iNOS and TNFalpha in cultures of human fetal astrocytes. In contrast, PD98059, an upstream inhibitor of the extracellular regulated kinase (ERK)1/2 pathway, had little or no effect. Interestingly, SB203580 reduced the mRNA expression for iNOS, TNFalpha, and IL-6, indicating inhibition prior to translation. Transfection of astrocytes with a dominant-negative Jun-NH(2)-terminal kinase (JNK) construct also reduced iNOS expression. Western blot analysis showed phosphorylated p38 and JNK in IL-1-activated astrocytes, and phosphorylated ERK in both resting and activated cells. Electrophoretic mobility shift assay (EMSA) showed that IL-1 induced NF-kappaB and AP-1 DNA complex formation in astrocytes, and that SB203580 inhibited AP-1 complex formation. Taken together, these results demonstrate the differential roles played by the three MAP kinases in human astrocyte inflammatory gene activation and point to a crucial function of p38 and JNK MAP kinases in IL-1-mediated astrocyte activation.     

Immunocompetent cell markers in human fetal astrocytes and neurons in culture.

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregulatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex-class II antigen HLA-DR was observed in the cytoplasm and on the cell surface of GFA-P+ astrocytes and increased with time in culture and cell passages. Among the T-lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4-6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B-lymphocyte marker CD21, the cellular receptor for Epstein-Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B-cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events.     

Endocannabinoid signaling in astrocytes

The study of the astrocytic contribution to brain functions has been growing in popularity in the neuroscience field. In the last years, and especially since the demonstration of the involvement of astrocytes in synaptic functions, the astrocyte field has revealed multiple functions of these cells that seemed inconceivable not long ago. In parallel, cannabinoid investigation has also identified different ways by which cannabinoids are able to interact with these cells, modify their functions, alter their communication with neurons and impact behavior. In this review, we will describe the expression of different endocannabinoid system members in astrocytes. Moreover, we will relate the latest findings regarding cannabinoid modulation of some of the most relevant astroglial functions, namely calcium (Ca²⁺) dynamics, gliotransmission, metabolism, and inflammation.     

Dopamine increases glial cell line-derived neurotrophic factor in human fetal astrocytes

The use of fetal astrocytes for gene delivery into brains with neurodegenerative diseases has been suggested. Therefore, the effects of neurotransmitters in the brain on such cells are of interest. The presence of D1(D1A) receptors and the effect of dopamine on a fetal human astrocyte cell line (SVG cells) in vitro were examined. SVG cells expressed D1(D(1A)), but not D5(D1B) receptors, as shown by RT-PCR. Exposure to dopamine, apomorphine, and the specific D1 agonist, SKF-38393, increased glial-derived neurotrophic factor production of SVG cells, as well as intracellular free calcium. Exposure to the specific D1 antagonist, SCH 23390, blocked these effects. Thus, if implanted into a brain region rich in dopamine, or if transfected with the tyrosine hydroxylase gene, fetal astrocytes may serve as paracrine/autocrine cells capable of supplying critical growth factors to diseased brain tissue.     

Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes

Brain injury is often associated with proliferation and hypertrophic response of glial cells (reactive gliosis). We have previously reported immunosuppressant effects on survival of glioma cells and adult reactive astrocytes. In the present study, we demonstrate growth-inhibitory effect of FK506 on cortical astrocytes from newborn rats. FK506 inhibits Erk and PI-3K/Akt signaling, two crucial pro-survival pathways. The levels of phosphorylated Akt and p42/44 Erk decline in few hours after FK506 addition. Furthermore, in FK506-treated astrocyte cultures the levels of mRNA encoding PDGF, bFGF, and CNTF decreased. Downregulation of growth factor expression by FK506 may play a role in the inhibition of mitogenic/hypertrophic responses. FasL mRNA level was elevated and interaction of FasL with Fas receptor expressed in astrocytes may trigger cell death. Interestingly, expression of BDNF increased in a dose-dependent manner in FK506-treated astrocytes. Upregulation of BDNF mRNA and protein level in astrocytes exposed to FK506 may underlie neuroprotective action of FK506.     

Regulation of chemokine receptor expression in human microglia and astrocytes

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.     

Immunolabelling of spliceosomes in sections and cultured astrocytes of human fetal brain tissue

In the cell nucleus first large pre-mRNAs are synthesized which contain protein coding as well as non-coding sequences. The latter are removed in a process called splicing which takes place in nuclear spliceosomes. These spliceosomes consist among others of protein factors, such as the splicing factor SC35 being abundant in speckled regions of the cell nucleus. This study aims at determining immunostaining patterns using anti-SC35 in sections of the human fetal prosencephalon and cultured human astrocytes. Within the allocortical entorhinal region of the fifth gestational month the number, size and distribution of SC35-positive speckles varies considerably among the laminae which can, thus, clearly be delineated. The immature isocortical plate, however, does not display a laminar arrangement at this developmental stage. Differential immunostaining patterns can be seen in subcortical areas. Cultured human astrocytes reveal numerous speckles occupying a large portion of the nucleoplasm. On account of the SC35-immunostaining patterns no distinction of subpopulations of astrocytes is possible. The results demonstrate that SC35-immunoreactive speckles show lamina and area-specific characteristics of human fetal brain sections. Conspicuous differences in number, size and distribution of speckles are visible in different cytoarchitectonic structures; thus, architectonic borders stand out clearly in SC35-immunopreparations. The occurrence of area-specific immunolabelling of nuclear speckle domains reflects neuronal differentiation at the pre-translational level. It may be assumed that a distinct set of proteins, generated by a definite nerve cell type, can be correlated with a distinct morphology of spliceosomes. The in vitro finding indicates that anti-SC35 may well be used as a tool to study possible alterations of the speckles after, for instance, application of growth factors.     

Fas and Fas ligand expression in Alzheimer's disease

The Fas/Fas ligand (L) signaling system has been implicated in the control of cell death and cell survival of T and B lymphocytes and in a variety of cell types under particular pathological conditions. In the present study we examined the expression of Fas and Fas-L, by Western blotting and immunohistochemistry, in the human frontal cortex and hippocampus of individuals with advanced Alzheimer's disease (AD) and age-matched controls. Expression levels of Fas and Fas-L, as seen in Western blots, are preserved in the frontal cortex but decreased in the hippocampus in AD when compared with age-matched controls. Yet Fas and Fas-L immunoreactivity is found in remaining AD neurons in the frontal cortex and hippocampus. Moreover, Fas and Fas-L are expressed equally in tangle-bearing and non-tangle-bearing neurons, as revealed with double-labeling immunohistochemistry to Fas or Fas-L and tau or phosphorylated neurofilament epitopes. Dystrophic neurites of senile plaques are not stained with Fas and Fas-L antibodies. A moderate increase in Fas and a strong increase in Fas-L immunoreactivity occur in reactive astrocytes in AD. Yet there is no relationship between Fas or Fas-L expression and increased nuclear DNA vulnerability as revealed with double-labeling immunohistochemstry and in situ end-labeling of nuclear DNA fragmentation. Although the Fas/Fas-L system may have some effect in the control of reactive astrocytosis in AD, the present results show no evidence that Fas/Fas-L signals participate in specific processes of the disease, including neurofibrillary degeneration, dystrophic neurite formation, and cell death.     

Distinct patterns of stimulus-inducible chemokine mRNA accumulation in human fetal astrocytes and microglia

Interferon-gamma-inducible 10 kd protein (IP-10) is an ELR (Glu-Leu-Arg)(-) alpha chemokine with known chemotactic effects on T cells and monocytes, as well as anti-viral, anti-angiogenic, and anti-tumor effects. Previous studies have demonstrated that in cultured rat astrocytes and microglia, stimulation with LPS or virus can induce the expression of IP-10. In this study, we determined the pattern of IP-10 gene induction in primary human microglia and astrocytes by cytokines and LPS using ribonuclease protection assay. The expression of IP-10 mRNA was compared with that of other alpha (IL-8) and beta chemokines. The results showed that in human microglia, IP-10 expression was induced equally potently by LPS, IFNbeta or IFNgamma. "Proinflammatory" cytokines IL-1beta or TNFalpha also induced small amounts of IP-10 mRNA. "Anti-inflammatory" cytokines IL-4, IL-10 and TGFbeta were ineffective in inducing IP-10 in microglia. In human astrocytes, induction of IP-10 mRNA by cytokines was similar to that in microglia. LPS, however, was ineffective in inducing IP-10 in human astrocytes. The monocyte chemoattractant beta-chemokine I-309 mRNA was induced in human astrocytes and microglia by IFNbeta or IFNgamma, or by LPS in microglia, showing a tight co-regulation with IP-10 mRNA expression. In contrast to the potent induction of IP-10 and I-309 by IFNs in human glia, the ELR(+) alpha chemokine IL-8 mRNA was induced by IL-1beta and TNFalpha, and to a lesser extent by IFNbeta in microglia. IFNbeta but not IFNgamma was effective in inducing the expression of beta chemokines MIP-1alpha and MIP-1beta in human microglia, with the levels of mRNA similar to those induced by IL-1beta or TNFalpha. Neither MIP-1alpha nor MIP-1beta mRNAs were induced by any stimulation in human astrocytes. The induction of RANTES mRNA in microglia by IFNbeta, IL-1beta or TNFalpha was variable, showing no to low level expression depending on the case, whereas LPS provided a consistent inducing signal. In astrocytes, only cytokine combinations (IFN + IL-1beta) effectively induced the RANTES mRNA. These results demonstrate that distinct sets of chemokine genes are induced in human glial cells by cytokines and interferons. These results may have wide implications for inflammatory, vascular and neoplastic diseases of the CNS.     

Glutamate-induced calcium signaling in astrocytes

Astrocytes respond to the excitatory neurotransmitter glutamate with dynamic spatio-temporal changes in intracellular calcium [Ca²⁺]_i. Although they share a common wave-like appearance, the different [Ca²⁺]_i changes--an initial spike, sustained elevation, oscillatory intracellular waves, and regenerative intercellular waves--are actually separate and distinct phenomena. These separate components of the astrocytic Ca²⁺ response appear to be generated by two different signal transduction pathways. The metabotropic response evokes an initial spatial Ca²⁺ spike that can propagate rapidly from cell to cell and appears to involve IP₃. The metabotropic response can also produce oscillatory intracellular waves of various amplitudes and frequencies that propagate within cells and are sustained only in the presence of external Ca²⁺. The ionotropic response, however, evokes a sustained elevation in [Ca²⁺]_i associated with receptor-mediated Na⁺ and Ca²⁺ influx, depolarization, and voltage-dependent Ca²⁺ influx. In addition, the ionotropic response can lead to regenerative intercellular waves that propagate smoothly and nondecrementally from cell to cell, possibly involving Na⁺/Ca²⁺ exchange. All these astrocytic [Ca²⁺]_i changes tend to appear wave-like, traveling from region to region as a transient rise in [Ca²⁺]_i. Nevertheless, as our understanding of the cellular events that underlie these [Ca²⁺]_i changes grows, it becomes increasingly clear that glutamate-induced Ca²⁺ signaling is a composite of separate and distinct phenomena, which may be distinguished not based on appearance alone, but rather on their underlying mechanisms. © 1994 Wiley-Liss, Inc.     

Kainate/AMPA receptors expressed on human fetal astrocytes in long-term culture

Long-term cultivation of primary human fetal brain cells has yielded a homogeneous population of glial progenitors of extended life span. These human astrocyte precursor (HAP-1) cells have been in culture for greater than 1 year, are diploid, and do not form colonies in soft agar. The culture was established in 10% fetal calf serum (FCS), although cells greatly increase their proliferative rate when both basic fibroblast growth factor and FCS are present in the culture media. HAP-1 cells express the cytoskeletal proteins glial fibrillary acidic protein, vimentin, and nestin. HAP-1 cells express the AMPA/kainate receptor subunit genes GluRs 1, 3, and 4 and the kainate receptor subunit genes GluR6, KA1, and KA2. Immunohistochemistry confirms the expression of GluR subunit proteins. HAP-1 cells demonstrate a kainate-responsive current found to be blockable by CNQX. HAP-1 cells will serve in the study of human glial cells and ligand-gated ion channels and in the identification of compounds which might act as agonists or antagonists at these receptor-ion channel complexes. J. Neurosci. Res. 47:311–321, 1997. © 1997 Wiley-Liss, Inc.     

H-Ras increases urokinase expression and cell invasion in genetically modified human astrocytes through Ras/Raf/MEK signaling pathway

Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas.     

Regulation of P-glycoprotein by human immunodeficiency virus-1 in primary cultures of human fetal astrocytes

P-glycoprotein (P-gp), a drug efflux pump, is known to alter the bioavailability of antiretroviral drugs at several sites, including the brain. We have previously shown that human immunodeficiency virus-1 (HIV-1) glycoprotein 120 (gp120) induces proinflammatory cytokine secretion and decreases P-gp functional expression in rat astrocytes, a cellular reservoir of HIV-1. However, whether P-gp is regulated in a similar way in human astrocytes is unknown. This study investigates the regulation of P-gp in an in vitro model of gp120-triggered human fetal astrocytes (HFAs). In this system, elevated levels of interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α were detected in culture supernatants. Pretreatment with CCR5 neutralizing antibody attenuated cytokine secretion, suggesting that gp120-CCR5 interaction mediated cytokine production. Treatment with gp120 (R5-tropic) resulted in reduced P-gp expression (64%) and function as determined by increased (1.6-fold) cellular accumulation of [(3) H]digoxin, a P-gp substrate. Exposure to R5 or R5/X4-tropic viral isolates led to a downregulation in P-gp expression (75% or 90%, respectively), and treatment with IL-6 also showed lower P-gp expression (50%). Moreover, IL-6 neutralizing antibody blocked gp120-mediated P-gp downregulation, suggesting that IL-6 is a key modulator of P-gp. Gp120- or IL-6-mediated downregulation of P-gp was attenuated by SN50 (a nuclear factor-κB [NF-κB] inhibitor), suggesting involvement of NF-κB signaling in P-gp regulation. Our results suggest that, similarly to the case with rodent astrocytes, pathophysiological stressors associated with brain HIV-1 infection have a downregulatory effect on P-gp functional expression in human astrocytes, which may ultimately result in altered antiretroviral drug accumulation within brain parenchyma.