SHP-1 expression in avian mixed neural/glial cultures

The central nervous system response to injury includes astrocyte proliferation and hypertrophy as well as microglial activation and proliferation. However, not all glial cells enter the cell cycle following damage, and the mechanism that determines which glial cells will proliferate and which will remain quiescent has yet to be elucidated. Protein tyrosine phosphorylation has been shown to play an important role in the regulation of the cell cycle in a number of different systems and has been implicated in both astrocyte proliferation and differentiation. Of particular interest is the protein tyrosine phosphatase SHP-1 (Src homology 2-containing protein tyrosine phosphatase 1), which: (1) modulates cellular proliferation in the hematopoietic system, (2) is involved in various growth factor second messenger signaling cascades, and (3) has been demonstrated by our laboratory to increase in immunoreactivity within a subpopulation of astrocytes following deafferentation of the chicken auditory brainstem. These SHP-1+ cells appear to be those which fail to enter the cell cycle following deafferentation. The present study examines whether manipulation of cellular proliferation in vitro modifies the expression of SHP-1 immunoreactivity in mixed neural/glial cultures of the avian auditory brainstem. In addition, the effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate on cellular proliferation was assessed in these cultures. Our results demonstrate that SHP-1 expression can be modulated by changes in proliferation and that inhibiting tyrosine phosphatase activity results in increased proliferation. Taken together, these results indicate that SHP-1 may play central role in negatively regulating glial proliferation following injury.     

Integrin beta1 is involved in the signaling of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for the substantia nigra (SN) dopamine (DA) neurons. The transmembrane signaling of GDNF is mediated by a unique receptor system, including the ligand binding receptor GDNF family receptor alpha (GFRalpha) and the transmembrane signaling receptor Ret or neural cell adhesion molecule-140 (NCAM-140). Here, we found that another transmembrane cell adhesion molecule, integrin, a heterodimer consisting of alpha and beta subunits, also mediates the transmembrane signaling of GDNF. The results showed that the level of phosphorylated Src homology 2 domain containing (Shc), which was associated with the cytoplasmic domain of integrin beta1, increased after GDNF administration. Coimmunoprecipitation analysis demonstrated that integrin beta1 could form a complex with GFRalphal. The simulation of molecular modeling showed that four H-bonds were formed between integrin beta1 and GFRalpha. These data indicate that integrin beta1 is involved in the transmembrane signaling of GDNF and suggest that integrin beta1 may be an alternative signaling receptor for GDNF.     

Absence of the cell cycle inhibitor p21Cip1 reduces LPS-induced NO release and activation of the transcription factor NF-kappaB in mixed glial cultures

We have studied possible differences in glial activation between cells from wild-type and p21Cip1-/- mice. We compared the effect of serum mitogenic stimulation on proliferation rate and on the total number of glial cells after 7 days of culture. No differences between wild-type and p21Cip1-/- glial cells were observed. We also compared the effect of lipopolysaccharide (LPS) from Escherichia coli, an agent widely used to induce glial activation. Nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) release, and nuclear factor kappa-B (NF-kappaB) activation were evaluated as indicators of glial activation. We observed an attenuation of NO release and NF-kappaB activation in p21Cip1-/- glial cells when compared with glial cells from wild-type mice. In contrast, TNF-alpha release was enhanced in p21Cip1-/- glia. These results suggest that the cell cycle inhibitor p21Cip1 plays a role in the inflammatory response induced by LPS.     

Oxidative stress regulated heme-oxygenase-1 and glutathione S-transferase-m1 gene expression changes in cell lines exposed to melanins

To investigate the effects of oxidative stress on substantia nigra neuronal degeneration and death in patients with Parkinson’s disease, we treated neuroblastoma cells (SK-N-SH) and glioma cells with Fenton’s reagent, iron chelating agent, neuromelanin and dopamine melanin. We investigated the changes in expression of nine oxidative stress-related genes and proteins. The levels of mRNAs for heme-oxygenase-1 and glutathione S-transferase-m1 were significantly reduced in SK-N-SH cells exposed to oxidative stress, and increased in glial cells treated with deferoxamine. These results revealed that SK-N-SH neurons react sensitively to oxidative stress, which implies different outcomes between these two types of cells in the substantia nigra. Moreover, the influences of neuromelanin and dopamine melanin on cell function are varied, and dopamine melanin is not a good model for neuromelanin.     

Neuronal differentiation elicited by glial cell line-derived neurotrophic factor and ciliary neurotrophic factor in adrenal chromaffin cell line tsAM5D immortalized with temperature-sensitive SV40 T-antigen

To understand the characteristics of tsAM5D cells immortalized with the temperature-sensitive simian virus 40 large T-antigen, we first examined the responsiveness of the cells to ligands of the glial cell line-derived neurotrophic factor (GDNF) family. tsAM5D cells proliferated at the permissive temperature of 33 degrees C in response to either GDNF or neurturin, but not persephin or artemin. At the nonpermissive temperature of 39 degrees C, GDNF or neurturin caused tsAM5D cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, ciliary neurotrophic factor (CNTF) did not affect the GDNF-mediated cell proliferation at 33 degrees C but promoted the survival and differentiation of GDNF-treated cells at 39 degrees C. In the presence of GDNF plus CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of various neuronal marker genes, indicating that the cells had undergone neuronal differentiation. In addition, tsAM5D cells caused to differentiate by GDNF plus CNTF at 39 degrees C became dependent solely on nerve growth factor (NGF) for their survival and neurite outgrowth. Moreover, upon treatment with GDNF plus CNTF, the dopaminergic phenotype was suppressed by the temperature shift. Thus, we demonstrated that tsAM5D cells had the capacity to differentiate terminally into neuron-like cells in response to GDNF plus CNTF when the oncogene was inactivated by the temperature shift. This cell line provides a useful model system for studying the role of a variety of signaling molecules for GDNF/CNTF-induced neuronal differentiation.     

Establishment of an astrocyte progenitor cell line: Induction of glial fibrillary acidic protein and fibronectin by transforming growth factor-β1

An immortalized clonal cell line (AP-16) has been established from glial cultures obtained from neonatal mouse cerebra by multipassages under serum-free conditions. Immunofluorescent experiments showed that AP-16 cells expressed a marker for glial progenitors (A2B5) and did not express markers for oligodendrocytes (galactocerebroside) or mature astrocytes (glial fibrillary acidic protein: GFAP). Treatment with transforming growth factor-β1 (TGF-β1) or fetal calf serum (FCS) for 2 days induced AP-16 cells to differentiate into A2B5-negative, GFAP-positive, phenotypically mature astrocytes. AP-16 cells depended on epidermal growth factor for survival, and their growth was inhibited by FCS. These results indicate that AP-16 cells retained the properties of astrocyte progenitors. An enzyme-linked immunosorbent assay showed that AP-16 cells synthesized fibronectin and laminin, and that the expression of fibronectin was increased by TGF-β1. AP-16 cells should be useful for studying the roles of TGF-β1 in the differentiation of astrocyte progenitors. © 1993 Wiley-Liss, Inc.     

P2Y1受体对缺血状态下星形胶质细胞产生胶质原纤维酸性蛋白与胶质细胞源性神经营养因子的影响及相关信号通路

目的研究P2Y1受体对缺血时星形胶质细胞产生胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）及胶质细胞源性神经营养因子（glial cell line—derived neurotrophic factor,GDNF）的影响及其相关信号通路。方法分别利用右侧大腑中动脉线拴阻塞及培养细胞缺氧无营养后恢复正常培养,造成体内、外缺血再灌注模型。用免疫荧光标记、实时定量RT—PCR、Western blotting、酶联免疫吸附试验观察P2Y1受体、GDNF定位,检测GFAP、GDNF及信号分子的表达变化。结果与单纯性缺血组比较,用选择性拈抗剂MRS2179阻断P2Y1受体后,可使体内、外星形胶质细胞产生的GFAP减少,同时使其产生GDNF增加。体外缺氧无营养并阻断P2Y1受体后：可使磷酸化蛋白激酶B（Akt）及cAMP反应元件结合蛋（cAMP response element binding protein,CREB）升高,而使磷酸化JAK2及STAT3（Ser727）降低;JAK2的抑制剂AG490在降低磷酸化STAT3（Ser727）的同时也降低GFAP表达水平;PI3-K的抑制剂LY294002可降低磷酸化的Akt及CREB;MEK1／2抑制剂U0126可同时降低磷酸化的JAK2、STAT3 （Ser727）、Akt及CREB。结论P2Y1受体参与短时性缺血时星形胶质细胞GFAP及GDNF的产生过程,相关信号途径分别为JAK2／STAT3和P13-K／AKT／CREB,并且两条途径存在串话。     

Nitric oxide donor up-regulation of SDF1/CXCR4 and Ang1/Tie2 promotes neuroblast cell migration after stroke

We tested the hypothesis that a nitric oxide donor, DETA-NONOate, up-regulates stromal cell-derived factor-1 (SDF1) and angiopoietin 1 (Ang1) in the ischemic brain and their respective receptors chemokine CXC motif receptor 4 (CXCR4) and Tie2 in the subventricular zone (SVZ) and thereby promote SVZ neuroblast cell migration after stroke. C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAo), and 24 hr later DETA-NONOate (0.4 mg/kg) or phosphate-buffered solution was intravenously administered. Mice were sacrificed at 14 days for histological assessment or sacrificed at 3 days for analysis by real-time polymerase chain reaction and migration after MCAo. To elucidate whether SDF1/CXCR4 and Ang1/Tie2 pathways mediate DETA-NONOate-induced SVZ migration after stroke, SDF1alpha, Ang1 peptide, a specific antagonist of CXCR4 (AMD3100), and a neutralizing antibody of Tie2 (anti-Tie2) were used in vitro. DETA-NONOate significantly increased the percentage area of doublecortin (DCX, a marker of migrating neuroblasts)-immunoreactive cells in the SVZ and ischemic boundary zone. DETA-NONOate significantly increased the expression of SDF1 and Ang1 in the ischemic border and up-regulated CXCR4 and Tie2 in the SVZ compared with MCAo control. DCX-positive cell migration from SVZ explants was significantly increased in the DETA-NONOate treatment group compared with MCAo-alone animals. In vitro, SDF1alpha and Ang1 significantly increased SVZ explants cell migration. In addition, inhibition of CXCR4 or Tie2 significantly attenuated DETA-NONOate-induced SVZ cell migration. Our data indicate that treatment of stroke with a nitric oxide donor up-regulates SDF1/CXCR4 and Ang1/Tie2 pathways and thereby likely increases SVZ neuroblast cell migration.     

Role of monocyte chemoattractant protein‐1 (MCP‐1/CCL2) in migration of neural progenitor cells toward glial tumors

Neural progenitor cells (NPCs) have been investigated as potential vehicles for brain tumor therapy because they have been shown to migrate toward central nervous system gliomas and can be genetically engineered to deliver cytotoxic agents to tumors. The mechanisms that regulate migration of NPCs to tumors are not fully understood. By means of microarray analysis, polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemistry, we found that monocyte chemoattractant protein‐1 (MCP‐1/CCL‐2) was expressed in experimental brain tumor cells in vivo and in vitro. CCR2, the receptor for MCP‐1, was expressed on C17.2 NPCs. We used a modified Boyden chamber assay and found increased migration of NPCs in vitro in response to MCP‐1. By means of an in vivo model for NPC migration, we found evidence of NPC migration toward areas of MCP‐1 infusion in rat brains. An understanding of NPC migration mechanisms may be used to enhance delivery of cytotoxic agents to brain tumor cells. © 2009 Wiley‐Liss, Inc.     

Effects of the HIV-1 Envelope Glycoprotein gp120 in Cerebellar Cultures. [Ca2+]i Increases in a Glial Cell Subpopulation

The various types of cells present in cultures prepared from the postnatal rat cerebellum, identified by their gross morphology and immunocytochemistry, were loaded with the specific dye fura-2 and analysed individually for [Ca²⁺]_i changes induced by the HIV-1 envelope glycoprotein gp120 and a variety of other treatments. In granule neurons [Ca²⁺]_i increases were induced by high KCl and glutamate (mainly through the NMDA receptor) while in type-1 astrocytes this effect was observed after serotonin, carbachol and also quisqualate. In contrast, administration of gp120 was always without effect in these cells. Type-2 astrocytes (an arborized cell type responsive to agonists targeted to the glutamatergic AMPA and cholinergic receptors) were also most often unresponsive to the viral glycoprotein. However, among the cells exhibiting the arborized phenotype, a subpopulation (-13%) responded to gp120 with conspicuous [Ca²⁺]_i increases sustained by both release from intracellular stores and influx across the plasma membrane. These responses to the viral protein did not involve activation of either voltage-gated Ca²⁺ channels or glutamatergic receptors. Although not yet conclusively identified by specific cytochemical markers, the gp120-responsive cells resemble type-2 astrocytes and differ from neurons and type-1 astrocytes both in gross phenotype and in a number of receptor/channel properties: positivity to AMPA and cholinergic agonists; negativity to NMDA, serotonin and high KCl. From these results it is concluded that a subpopulation of glial cells is affected by gp120. The role of these cells in HIV brain infection and damage requires further studies to be precisely established.     

Activation of the extracellular signal-regulated kinases 1 and 2 by glial cell line-derived neurotrophic factor and its relation to neuroprotection in a mouse model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be neuroprotective in animal models of the dopamine deficiency in Parkinson's disease. To examine the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in this process, we infused a single dose of GDNF into the striatum of mice and analyzed the effect on ERK1/2 by immunohistochemistry and Western blot analysis. GDNF caused an increase in the phosphorylation of ERK1/2 both in the striatum and in tyrosine hydroxylase-positive neurons in the substantia nigra. In the striatum, the increase in ERK1/2 phosphorylation was evident by 3 hr and persisted for at least 7 days, whereas, in the substantia nigra, an increase in phosphorylated ERK1/2 was first evident at 24 hr and persisted for at least 7 days. The increase in phosphorylated ERK1/2 was maximal at 0.45 microg GDNF at the time points examined. GDNF also protected dopamine terminals against the loss of tyrosine hydroxylase immunoreactivity normally associated with the intrastriatal administration of 6-hydroxydopamine (0.5 microg/0.5 microl). However, this was observed only at a much higher dose of GDNF, 4.5 microg. Thus, our results suggest that the ability of GDNF to protect dopamine neurons cannot be explained solely in terms of its influence on ERK1/2 and that the role of other signaling pathways should be explored.     

Antiapoptotic mechanism of cannabinoid receptor 2 agonist on cisplatin-induced apoptosis in the HEI-OC1 auditory cell line

Cisplatin is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of cisplatin. The present study investigated the effects of the cannabinoid receptor 2 (CB2) ligand JWH-015 on cisplatin-induced apoptosis. CB2 mRNA was constitutively expressed in the auditory cell line HEI-OC1. By using MTT assay, DNA fragmentation, and FACS analysis, we demonstrated that apoptosis induced by cisplatin was inhibited by treatment with JWH-015 in a dose-dependent manner. Activation of caspase-3, caspase-8, and caspase-9 was detected after treatment with cisplatin, and the cleavage of poly-(ADP)-ribose polymerase (PARP) was observed within cisplatin-treated HEI-OC1 cells. JWH-015 inhibited the activation of caspase-3, caspase-8, and caspase-9; cleavage of PARP; and release of cytochrome c. JWH-015 also inhibited the apoptosis through activation of the extracellular signal-regulated kinase pathway. Finally, JWH-015 inhibited cisplatin-induced reactive oxygen species and tumor necrosis factor-alpha production. Collectively, these findings show that blocking a critical step in apoptosis by using JWH-015 may be a useful strategy to prevent harmful side effects of cisplatin ototoxicity in patients having to undergo chemotherapy.     

Altered expression of CHL1 by glial cells in response to optic nerve injury and intravitreal application of fibroblast growth factor-2

The close homologue of L1 (CHL1) is a member of the L1 family of cell recognition molecules. The protein is expressed by a variety of nerve cell types and subpopulations of glial cells in vivo and promotes elongation of neurites and survival of nerve cells in vitro. Here we demonstrate that glial cells up-regulate expression of CHL1 in response to an intraorbital crush of the adult mouse optic nerve. We also demonstrate that a single intravitreal application of fibroblast growth factor-2 (FGF-2) increases expression of CHL1 in retinal astrocytes and Müller cells. Elevated expression of CHL1 by glial cells in injured optic nerves and astrocytes and Müller cells in FGF-2-treated retinas suggests a role of the protein in the lesioned central nervous system. Results also suggest that trophic factors might exert part of their biological function by modifying expression of cell recognition molecules.     

Insulin-like growth factor binding proteins-1 and -2 differentially inhibit rat oligodendrocyte precursor cell survival and differentiation in vitro

Insulin-like growth factor-1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and induces myelination. Its actions are modulated by IGF binding proteins (IGFBPs) that are present in the extracellular fluids or on the cell surface. Additionally, IGFBPs are also known to exert actions that are independent of IGF-1. We studied whether IGF-binding proteins (IGFBPs)-1 and -2 modulate rat oligodendrocyte precursor (O2A) cell survival and differentiation in vitro both in the absence and presence of exogenously added IGF-1. The data reveal that IGFBP-1 and -2 reduced O2A cell survival in the absence and presence of exogenously added IGF-1. The effects of IGFBP-1 on cell survival in the presence of exogenously added IGF-1 were IGF-1-dependent, whereas IGFBP-2 displayed both IGF-1-dependent and IGF-1-independent effects. Furthermore, IGFBP-1 and -2 inhibited O2A cell differentiation in the presence of IGF-1 as reflected by decreased expression levels of two myelin proteins, CNPase (2',3'-cyclic nucleotide 3'-phosphohydrolase) and MAG (myelin associated glycoprotein). Analysis of medium samples revealed that O2A cells do not secrete proteases that degrade these IGFBPs. Taken together the data show that IGFBP-1 and -2 are negative effectors of oligodendrocyte survival and differentiation. Accordingly, the role of IGFBPs should be explicitly taken into account when investigating IGF-1 effects on oligodendrocytes, especially in the context of therapeutic purposes.