Oncostatin M promotes excitotoxicity by inhibiting glutamate uptake in astrocytes: implications in HIV-associated neurotoxicity.

BACKGROUND: Elevated levels of oncostatin M (OSM), an interleukin-6 cytokine family member, have been observed in HIV-1-associated neurocognitive disorders (HAND) and Alzheimer's disease. However, the function of OSM in these disease conditions is unclear. Since deficient glutamate uptake by astrocytes is instrumental in HAND-associated neurotoxicity, we hypothesized that OSM impairs glutamate uptake in astrocytes and thereby promotes neuronal excitotoxicity. METHODS: Primary cultures of mouse cortical astrocytes, neurons, microglia, and BV2 cell line were used. The expression of glutamate transporters (GLAST/EAAT1 and GLT-1/EAAT2) was investigated using real-time PCR and Western blot, and their activity was assessed by measuring (3)H-D-aspartate uptake. Neuronal toxicity was measured using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay and immunocytochemistry. A chimeric HIV-1 that infects murine cells (EcoHIV/NL4-3-GFP virus (EcoHIV)) was used to investigate whether the virus induces OSM, OSM receptor (OSMR)-ß, glycoprotein 130 (gp130), GLT-1, GLAST (mRNA and protein), and OSM release (ELISA) in cultured BV2 cells, primary microglia, or astrocytes. Statistical analyses of the data were performed using one-way ANOVA (to allow multiple comparisons) and two-tailed Student's t test. RESULTS: OSM treatment (10 ng/mL) time-dependently reduced GLAST and GLT-1 expression and inhibited (3)H-D-aspartate uptake in cultured astrocytes in a concentration-dependent manner, an effect prevented by the Janus kinase (JAK)/signal transducers and activators of transcription (STAT)3 inhibitor AG490. Down-regulation of astrocytic glutamate transport by OSM resulted in NMDA receptor-dependent excitotoxicity in cortical neurons. Infection with EcoHIV induced OSM gene expression and protein release in BV2 cells and microglia, but not in astrocytes. Conversely, EcoHIV caused a fivefold increase in OSMR-ß mRNA (but not gp130) and protein in astrocytes, but not in microglia, which did not express OSMR-ß protein. Finally, astrocytic expression of GLAST gene was unaffected by EcoHIV, whereas GLT-1 mRNA was increased by twofold. CONCLUSIONS: We provide first evidence that activation of JAK/STAT3 signaling by OSM inhibits glutamate uptake in astrocytes, which results in neuronal excitotoxicity. Our findings with EcoHIV suggest that targeting OSMR-ß signaling in astrocytes might alleviate HIV-1-associated excitotoxicity.

FeLV envelope protein (gp70) variable region 5 causes alterations in calcium homeostasis and toxicity of neurons.

In humans and animals, retroviruses have been implicated in nervous system disease. Our objective was to characterize the neurotoxicity of a peptide sequence derived from an animal retrovirus, the feline leukemia virus (FeLV). Using a peptide sequence from the subtype FeLV-C envelope protein variable region 5 (VR5), cytotoxicity was demonstrated in studies that evaluated neuronal survival, neurite outgrowth, and alterations in intracellular calcium ion concentration. The FeLV subtype isolate FeLV-CSarma possesses an envelope protein VR5 amino acid sequence that varies by four amino acids from the VR5 amino acid sequence of subtype FeLV-AGlasgow. The polypeptide representing the VR5 of FeLV-CSarma (FeLV-CVR5) is significantly more neurotoxic than the polypeptide sequence representing the VR5 of FeLV-AGlasgow (FeLV-AVR5). FeLV-CVR5 (> or = 3 microM) exposure resulted in significant dose-dependent neurotoxicity. Antibodies to FeLV-CVR5 blocked this effect. Neurite outgrowth was significantly reduced at all tested concentrations (3-12 microM) of FeLV-CVR5, with a 92% reduction in neurite length at 12 microM. FeLV-AVR5 was significantly less neurotoxic with respect to neurite outgrowth than was FeLV-CVR5. The significant reduction in neurotoxicity for FeLV-AVR5 illustrates the importance of the 4-amino-acid difference between it and FeLV-CVR5. Alterations in intracellular calcium ion concentration were associated with this neurotoxicity.

Alanine mutagenesis of conserved residues in the platelet-derived growth factor family: identification of residues necessary for dimerization and transformation.

Platelet-derived growth factor (PDGF) and vascular endothelial growth factor define a family of dimeric proteins characterized by eight conserved cysteine residues involved in disulfide bonds. Thirteen non-cysteine residues conserved among the platelet-derived/vascular endothelial growth factors were individually mutated to alanine in v-sis/PDGF-B. In addition, five other residues flanking F148 were also mutated to alanine. The resulting mutants were assayed for transformation of NIH3T3 cells, and the mutant proteins were assayed for their ability to dimerize. Four residues were found to be crucial for disulfide-linked dimer formation: P152 and G162 were mandatory, while R159 and H205 also contributed to efficient dimerization. Four of the mutant proteins (at residues N147, F148, L149 and K185) dimerized efficiently yet exhibited less than 50% transforming activity compared with wild-type v-sis. Two mutants (at residues D142 and F148) were located in a region important for PDGF receptor interaction and were further studied with regard to secretion and PDGF receptor autophosphorylation. A series of substitutions at residue F148 revealed a strong preference for aromatic amino acids. One mutant from this series (F148G) dimerized but was completely inactive for transformation. This study thus identifies four residues in v-sis/PDGF-B important for dimerization and also identifies additional residues critical for full activation of PDGF receptors. The E5 oncoprotein encoded by bovine papillomavirus type I exhibits two short regions of amino acid similarity when compared with the minimal transforming region of v-sis/PDGF-B. Several of the v-sis mutants discussed in this work affect residues that are also present in the E5 oncoprotein, including F148, L149 and H205.

Evidence for interaction between v-Mos and a p34cdc2 isoform, p35cdk.

The c-mos gene product (c-Mos) encodes a serine/threonine kinase required for activation of pre-MPF (maturation-promoting factor) to MPF in oocytes undergoing meiosis and also for stabilization of MPF leading to metaphase arrest in unfertilized eggs. In order to determine whether the v-mos gene product (v-Mos) causes neoplastic transformation via interaction with cell cycle control elements, we have searched for proteins that interact with v-Mos. Extracts of NIH3T3 cells transformed by v-Mos encoded by Moloney murine sarcoma virus (Mo-MuSV) were examined by gel filtration, by immunoprecipitation with antibodies to a conserved region of p34cdc2, and by binding to beads that contain cross-linked p13suc1, a protein known to bind p34cdc2. Gel filtration detected a 500-kDa complex that contained v-Mos and a p34cdc2 isoform, termed p35cdk. The 500-kDa macromolecular complex also exhibited histone H1 phosphorylation activity, consistent with the presence of a cdc2 isoform. The identity of p35cdk is based on its recognition by anti-cdc2 PSTAIR but not by anti-cdc2 C-terminal antibodies, which detect authentic p34cdc2. Structures containing v-Mos and p35cdk were also detected by experiments involving co-immunoprecipitation of v-Mos with anti-cdc2 PSTAIR antibodies. Furthermore, both v-Mos and the p35cdk co-precipitated with p13suc1-Sepharose beads. Our findings raise the possibility of a v-Mos-p35cdk regulatory interaction in cells transformed by Mo-MuSV.