Distribution of Secretory Pathway Ca 2+ ATPase (SPCA1) in Neuronal and Glial Cell Cultures

1. Secretory pathway Ca²⁺ ATPase type 1 (SPCA1) is a newly recognized Ca²⁺/Mn²⁺-transporting pump localized in membranes of the Golgi apparatus.2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.     

Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures

1. Secretory pathway Ca(2+) ATPase type 1 (SPCA1) is a newly recognized Ca(2+)/Mn(2+)-transporting pump localized in membranes of the Golgi apparatus. 2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues. 3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains. 4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells. 5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically. 6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, alpha-tubulin or MBP. 7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells. 8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.     

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.     

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.     

Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons,astrocytes, oligodendrocytes and microglial cells

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.     

Production of interleukin-1 receptor antagonist isoforms by microglia in mixed rat glial cells stimulated by lipopolysaccharide

Although the natural interleukin-1 receptor antagonist (IL-1Ra) has been shown to be produced by microglial cells in response to immune stimuli, nothing was known about the ability of these cells in primary culture to produce the different isoforms of IL-1Ra. Using RT-PCR, we first confirmed that mixed glial cell cultures from newborn rats respond to the cytokine inducer, lipopolysaccharide, by synthesizing IL-1Ra mRNA. Using double immunostaining, we showed that IL-1Ra was detected in microglia but not in astrocytes. Using Western blotting, we finally demonstrated that the IL-1Ra1 isoform was secreted in the supernatant of mixed glial cell cultures, and its production increased in response to lipopolysaccharide. The three different IL-1Ra isoforms were constitutively expressed in cell lysates and their levels increased after lipopolysaccharide treatment, except for IL-1Ra3. These results point to the ability of microglial cells in primary culture to produce the different isoforms of IL-1Ra.     

Histone Deacetylase 2 Inhibitor CAY10683 Alleviates Lipopolysaccharide Induced Neuroinflammation Through Attenuating TLR4/NF-κB Signaling Pathway

Neuroinflammation involves in the progression of many central nervous system diseases. Several studies have shown that histone deacetylase (HDAC) inhibitors modulated inflammatory responses in lipopolysaccharide (LPS) stimulated microglia. While, the mechanism is still unclear. The aim of present study was to investigate the effect of HDAC2 inhibitor CAY10683 on inflammatory responses and TLR4/NF-κB signaling pathways in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. The effect of CAY10683 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expressions of inflammatory cytokines were analyzed by western blotting and RT-PCR respectively. The TLR4 protein expression was measured by western blotting, immunofluorescence, immunohistochemistry respectively. The protein expressions of MYD88, phospho-NF-κB p65, NF-κB-p65, acetyl-H3 (AH3), H3, and HDAC2 were analyzed by western blotting. We found that CAY10683 could inhibit expression levels of inflammatory cytokine TNF-α and IL-1β in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. It could induce TLR4, MYD88, phospho-NF-κB p65, and HDAC2 expressions. Moreover, CAY10683 increased the acetylation of histones H3 in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. Taken together, our findings suggested that HDAC2 inhibitor CAY10683 could suppress neuroinflammatory responses and TLR4/NF-κB signaling pathways by acetylation after LPS stimulation.     

The Neurotoxic Effect of Cuprizone on Oligodendrocytes Depends on the Presence of Pro-inflammatory Cytokines Secreted by Microglia

In order to further characterize the still unknown mechanism of cuprizone-induced demyelination, we investigated its effect on rat primary oligodendroglial cell cultures. Cell viability was not significantly affected by this treatment. However, when concentrations of IFNγ and/or TNFα having no deleterious effects per se on cell viability were added together with cuprizone, cell viability decreased significantly. In mitochondria isolated from cuprizone-treated glial cells, we observed a marked decrease in the activities of the various complexes of the respiratory chain, indicating a disruption of mitochondrial function. An enhancement in oxidant production was also observed in cuprizone and/or TNFα-treated oligodendroglial cells. In in vivo experiments, inhibition of microglial activation with minocycline prevented cuprizone-induced demyelination. Based on the above-mentioned results we suggest that these microglial cells appear to have a very active role in cuprizone-induced oligodendroglial cell death and demyelination, through the production and secretion of pro-inflammatory cytokines. This work is dedicated with sincere friendship to Celia and Tony Campagnoni.     

Striatal dopamine-NMDA receptor interactions in the modulation of glutamate release in the substantia nigra pars reticulata in vivo: opposite role for D1 and D2 receptors

To investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 micro m), the peroxide disappeared from the incubation medium following first order kinetics with a half-time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 +/- 30.4 nmol x min(-1) x mg protein(-1) and 27.5 +/- 5.4 nmol x min(-1) x mg protein(-1) of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 +/- 0.54 min(-1) x mg protein(-1). Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.     

Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells

The expression of connexin36 (Cx36) was studied in primary cultures of rat brain glial cells: mature astrocytes, ameboid and ramified microglia and immature oligodendrocytes (at middle period of myelinogenesis). The data from these cells were compared with those obtained from cultures of neocortical and hypothalamic neurons. mRNA encoding Cx36 was investigated by RT-PCR, the Cx36 protein by immunocytochemistry using a polyclonal antibody against Cx36 in cells characterized by antibodies specific for the single cell types. The Cx36 was found in oligodendrocytes, both ameboid and ramified microglial cells and in neurons. Astrocytes showed no detectable expression of the Cx36. The expression of Cx36 in oligodendrocytes and microglial cells suggests an involvement of the direct cell-cell communication channels formed by Cx36 in myelin formation and in brain development, damage and repair processes.     

Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes,oligodendrocytes, microglial cells and neurones

Multidrug resistance proteins (Mrps) are ATP-driven export pumps that mediate the export of organic anions from cells. So far only little information is available on expression and physiological functions of Mrps in brain. The expression of mRNAs of six Mrp paralogs in rat brain, as well as in rat cultures enriched for neurones, astrocytes, oligodendrocytes and microglial cells, was studied by qualitative and semiquantitative RT-PCR analysis. In adult rat brain as well as in neural cell cultures the mRNAs coding for Mrp1, Mrp3, Mrp4 and Mrp5 were detected. Semiquantitative analysis revealed that the mRNAs coding for Mrp1 and Mrp5 were more abundant in the four cell culture types than mRNAs of the other Mrps. mRNAs coding for Mrp3 and Mrp4 were found at significant levels in cultured astrocytes and microglial cells, whereas cultures of neurones and oligodendrocytes contained only marginal quantities of these mRNAs. Putative physiological functions of Mrps in brain cells are discussed.     

Generation of oligodendroglial progenitors from neural stem cells

To understand how the differentiation of stem cells to oligodendroglial progenitors is regulated, we established cultures of neural stem cells from neonatal rat striatum in the presence of epidermal growth factor (EGF) as free-floating neurospheres that were then exposed to an increasing amount of B104 cell-conditioned medium (B104CM). The resultant cells proliferated in response to B104CM but no longer to EGF. In vitro analysis and transplantation studies indicated that these cells were committed to the oligodendroglial lineage, and they were thus referred to as oligospheres. Further characterization of their expression of early markers, cell cycle, migration, and self-renewal suggests that they were pre-O2A progenitors. RT-PCR analysis indicated that the oligosphere cells expressed mRNAs of platelet-derived growth factor α receptor in addition to fibroblast growth factor receptor but not EGF receptor; the latter two receptor mRNAs were expressed by neurosphere cells. Thus, the progression of stem cells to oligodendroglial progenitors is likely induced by factors in B104CM.     

Astrocytes promote TNF-mediated toxicity to oligodendrocyte precursors

Neuroinflammation and increased production of tumor necrosis factor (TNF) in the CNS have been implicated in many neurological diseases including white matter disorders periventricular leukomalacia and multiple sclerosis. However, the exact role of TNF in these diseases and how it mediates oligodendrocyte injury remain unclear. Previously, we demonstrated that lipopolysaccharide (LPS) selectively kills oligodendrocyte precursors (preOLs) in a non-cell autonomous fashion through the induction of TNF in mixed glial cultures. Here, we report that activation of oligodendroglial, but not astroglial and microglial, TNFR1 is required for LPS toxicity, and that astrocytes promote TNF-mediated preOL death through a cell contact-dependent mechanism. Microglia were the sole source for TNF production in LPS-treated mixed glial cultures. Ablation of TNFR1 in mixed glia completely prevented LPS-induced death of preOLs. TNFR1-expressing preOLs were similarly susceptible to LPS treatment when seeded into wildtype and TNFR1(-/-) mixed glial cultures, demonstrating a requirement for oligodendroglial TNFR1 in the cell death. Although exogenous TNF failed to cause significant cell death in enriched preOL cultures, it became cytotoxic when preOLs were in contact with astrocytes. Collectively, our results demonstrate oligodendroglial TNFR1 in mediating inflammatory destruction of preOLs and suggest a previously unrecognized role for astrocytes in promoting TNF toxicity to preOLs.     

Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

血清饥饿可诱导人血管平滑肌细胞再分化

体外培养的分化型血管平滑肌细胞 (vascularsmoothmusclecells ,VSMC)以特异性标志基因表达、长梭形外观及对兴奋剂刺激产生收缩反应为其表型特征 .以血清饥饿法培养处于超汇合 (overconfluence)状态的人VSMC ,观察其分化型标志基因表达活性及其与细胞形态特征和收缩反应性之间的关系 ,探讨细胞生存环境对VSMC基因表达及表型的影响 .研究显示 ,生长至超汇合的VSMC由含血清培养转为血清饥饿后 ,收缩蛋白如SMα肌动蛋白 (SMα actin)、SM2 2α、h1 calponin、肌球蛋白重链 (MHC)SM1和SM2亚型的表达活性明显上调 ,证实血清饥饿诱导的收缩蛋白基因表达和血清应答因子 (serumresponsefactor ,SRF)与CArG顺式元件结合活性的增强有关 .同时 ,血清饥饿还可激活参与VSMC分化调节的转录调控因子SmLIM、Gax和分化相关蛋白HRG 1基因的转录 .随着血清饥饿培养时间的延长 ,VSMC逐渐形成多层、束状、成极性排列的形式 ,对兴奋剂刺激产生的收缩反应明显增强 .结果表明 ,超汇合状态的去分化型VSMC脱离血清刺激后 ,可以再分化成熟并重新获得收缩能力     

Localization of MCC (mutated in colorectal cancer) in various tissues of mice and its involvement in cell differentiation.

Localization of the MCC (mutated in colorectal cancer) gene product, a cell cycle-regulating protein mutated in several colorectal tumors, in various mouse tissues was examined by immunohistochemistry and immunoelectron microscopy. MCC was localized on microvilli and in the apical cytoplasm in renal proximal tubule epithelial cells and pancreatic acinar cells. In hepatocytes, MCC was exclusively detected on microvilli. MCC was highly expressed in the cerebral cortex and the molecular layer of the cerebellar cortex and was partially associated with membrane organelles in neuronal elements. Adrenal chromaffin cells showed little expression of MCC. MCC was localized to the cell margins of ependymal cells, thyroid follicular cells, and anterior pituitary cells. In parotid acinar cells, only the apical surface was immunopositive. MCC was not expressed in skeletal and cardiac muscle. MCC was present at lateral cell borders in the duodenum and colon epithelium. In addition, the apical cytoplasm of colon epithelial cells exhibited intense immunoreactivity. The amount of MCC increased during differentiation of NGF-treated PC12 cells. In conclusion, MCC was expressed in differentiated cells and was associated with the plasma membrane and membrane organelles. In addition to the negative regulation of the cell cycle, MCC may be involved in cell differentiation.