Asgar Zaheer

Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor-β

Glia maturation factor‐β, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor‐β, we screened a human brainstem cDNA library in δll. A 0.7‐kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor‐β, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.

Expression of Glia Maturation Factor β mRNA and Protein in Rat Organs and Cells

Abstract: Rat glia maturation factor β (GMF‐β) cDNA was obtained by reverse transcription of rat brain mRNA followed by polymerase chain reaction amplification, using primers from the human sequence. The deduced amino acid sequence of rat GMF‐β differed from the human counterpart in only three places: His27 in place of Asn, Val51 in place of Ile, and Leu93 in place of Val. The high degree of evolutionary conservation suggests that GMF‐β plays an essential role in animal cell physiology. The expression of GMF‐β mRNA in the rat was studied by the northern blot technique, using a rat cRNA probe corresponding to the entire coding region. GMF‐β mRNA was predominantly expressed in the brain and spinal cord, although trace levels were found in other organs, including testis and ovary. In the brain GMF‐β mRNA was detectable at as early as embryonic day 10, and persisted through as late as postnatal month 14, with minor variations in between. On the other hand, GMF‐β protein exhibited more obvious developmental changes, with its level increasing slowly prenatally and plateauing at 1 week after birth. GMF‐β mRNA and protein were also observed in several cultured cells. Some cells of neural origin contained higher levels of GMF‐β protein compared with cells derived from other sources. Through demonstration of mRNA and confirmation by immunoblotting, we conclude that GMF‐β is synthesized by rat organs and that GMF‐β is predominantly a brain protein.

Effects of glia maturation factor overexpression in primary astrocytes on MAP kinase activation, transcription factor activation, and neurotrophin secretion.

Using the replication-defective adenovirus vector, we overexpressed rat glia maturation factor (GMF) in primary astrocyte cultures derived from embryonic rat brains. Among the three isoforms of MAP kinase, there was a big increase in the phosphorylation of p38, as detected with Western blotting using the phosphospecific antibody. Likewise, there was a substantial increase in the phosphorylation of the transcription factor CREB. Using the electrophoretic mobility shift assay (EMSA), we found a stimulation in the transcription factor NF-κB. The activations of CREB and NF-κB were blocked by inhibitors of either p38 (SB-203580) or MEK (PD-098059), suggesting that they were events downstream of MAK kinase. There was an increased secretion of BDNF and NGF into the conditioned medium, along with an increase in their messenger RNA. The inductions of BDNF and NGF were also blocked by inhibitors of p38 and MEK, as well as by the inhibition of NF-κB with a decoy DNA sequence. Taken together, the results suggest that GMF functions intracellularly in astrocytes as a modulator of MAP kinase signal transduction, leading to a series of downstream events including CREB and NF-κB activation, resulting in the induction and secretion of the neurotrophins.

A novel role of glia maturation factor : induction of granulocyte-macrophage colony-stimulating factor and pro-inflammatory cytokines

The glia maturation factor (GMF), which was discovered in our laboratory, is a highly conserved protein predominantly localized in astrocytes. GMF is an intracellular regulator of stress‐related signal transduction. We now report that the overexpression of GMF in astrocytes leads to the destruction of primary oligodendrocytes by interactions between highly purified cultures of astrocytes, microglia, and oligodendrocytes. We infected astrocytes with a replication‐defective adenovirus carrying the GMF cDNA. The overexpression of GMF caused the activation of p38 MAP kinase and transcription factor NF‐κB, as well as the induction of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) mRNA and protein in astrocytes. Small interfering RNA‐mediated GMF knockdown completely blocked the GMF‐dependent activation of p38 mitogen‐activated protein kinase (MAPK), NF‐κB, and enhanced expression of GM‐CSF by astrocytes. Inhibition of p38 MAPK or NF‐κB by specific inhibitors prevented GM‐CSF production. The cell‐free conditioned medium from overexpressing GMF astrocytes contained 320 ± 33 pg/mL of GM‐CSF, which was responsible for enhanced production and secretion of TNF‐α, IL‐1β, IL‐6, and IP‐10 by microglia. Presence of these inflammatory cytokines in the conditioned medium from microglia efficiently destroyed oligodendrocytes in culture. These results suggest that GMF‐induced production of GM‐CSF in astrocytes is depending on p38 MAPK and NF‐κB activation. The GM‐CSF‐dependent expression and secretion of inflammatory cytokine/chemokine, TNF‐α, IL‐1β, IL‐6, and IP‐10, is cytotoxic to oligodendrocytes, the myelin‐forming cells in the central nervous system, and as well as neurons. Our results suggest a novel pathway of GMF‐initiated cytotoxicity of brain cells, and implicate its involvement in inflammatory diseases such as multiple sclerosis.

Expression of mRNAs of multiple growth factors and receptors by astrocytes and glioma cells: Detection with reverse transcription-polymerase chain reaction

Summary1. Although glial cells in culture are known to secrete growth factors and are also known to be responsive to some of them, detailed comparisons are difficult because the bulk of information was based on various animals of origin, developmental stages, growth properties, culture age, and culture conditions.2. To present a unified picture of the growth factors and their receptors found in glial cells, we surveyed the expression of messenger RNAs of a panel of growth factors and receptors, using reverse transcription-polymerase chain reaction (RT-PCR), in three common glial cell types: rat astrocytes in primary culture, rat glioma line C6, and human glioma line A172.3. We observed that normal and neoplastic glial cells in culture express multiple growth factors and also possess most of the receptors to these factors, suggesting multiple autocrine functions. In addition, glia produce growth factors known to be capable of acting on neurons, implicating paracrine function involving glia-neuron interaction. Glial cells also produce growth factors and receptors that are capable of communicating with hematopoietic cells, suggesting neuroimmunologic interaction. What is most interesting is that glial cells express receptors for growth factors previously thought to be acting on neurons only.4. The current study demonstrates the feasibility of screening from a small sample a large number of growth factors and receptors. The method portends future clinical application to biopsy or necropsy samples from brain tumors or pathologic brains suffering from degenerative diseases such as Alzheimers or Parkinsons disease.

Overexpression of glia maturation factor in astrocytes leads to immune activation of microglia through secretion of granulocyte–macrophage-colony stimulating factor

We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective adenovirus carrying the rat glia maturation factor (GMF) cDNA. Affymetrix microarray analysis showed a big increase in the expression of several major histocompatibility complex (MHC) class II proteins along with interleukin-1beta (IL-1beta). Subsequent study using reverse transcription-polymerase chain reaction (RT-PCR) yielded the same results with the mixed culture, but not with pure astrocytes or pure microglia. We also noticed that the GMF/virus construct infected only astrocytes but not microglia. This led us to suspect that overexpression of GMF in astrocytes resulted in the secretion of an active substance that stimulated the microglia to express MHC II and IL-1beta. We identified this substance as granulocyte-macrophage-colony stimulating factor (GM-CSF). MHC II are unique to antigen-presenting cells such as microglia and monocytes. The results suggest that GMF in astrocytes can initiate a series of events, leading to immune activation in the nervous system, and implicates its involvement in autoimmune diseases such as multiple sclerosis.

Glia maturation factor modulates β-amyloid-induced glial activation, inflammatory cytokine/chemokine production and neuronal damage

Glia maturation factor (GMF), discovered and characterized in our laboratory, is a highly conserved protein primarily localized in mammalian central nervous system. Previously we demonstrated that GMF is required in the induced production of proinflammatory cytokines and chemokines in brain cells. We now report that ventricular infusion of human amyloid beta peptide1-42 (Abeta1-42) in mouse brain caused glial activation and large increases in the levels of GMF as well as induction of inflammatory cytokine/chemokine known for launching the neuro inflammatory cascade in Alzheimers disease (AD). To test the hypothesis that GMF is involved in the pathogenesis of AD, we infused Abeta1-42 in the brain of GMF-deficient (GMF-KO) mice, recently prepared in our laboratory. GMF-deficient mice showed reduced glial activation and significantly suppressed proinflammatory cytokine/chemokine production following Abeta infusion compared to wild type (Wt) mice. The decrease in glial activation in the GMF-KO mice is also associated with significant reduction in Abeta induced loss of pre-synaptic marker, synaptophysin, and post-synaptic density protein-95 (PSD 95). We also examined the potential relationship between GMF or lack of it with learning and memory using the T-maze, Y-maze, and water maze, hippocampal-dependent spatial memory tasks. Our results show that memory retention was improved in GMF-KO mice compared to Wt controls following Abeta infusion. Diminution of these Abeta1-42 effects in primary cultures of GMF-KO astrocyte and microglia were reversed by reconstituted expression of GMF. Taken together, our results indicate a novel mediatory role of GMF in the neuro-inflammatory pathway of Abeta and its pro-inflammatory functions.

Activation of nuclear factor-κB in C6 rat glioma cells after transfection with glia maturation factor

Abstract: The 17‐kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication‐defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor‐κB (NF‐κB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double‐stranded oligonucleotide probe containing the consensus binding sequence for NF‐κB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF‐κB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen‐activated protein kinase inhibitor SB‐203580 also suppressed the GMF‐activated NF‐κB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12‐myristate 13‐acetate activated NF‐κB whether or not GMF was overexpressed. Along with NF‐κB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF‐κB nuclear translocation was blocked by its specific decoy DNA, implicating NF‐κB as an upstream mediator of this anti‐oxidant enzyme. The p38 inhibitor SB‐203580 also blocked the GMF‐activated SOD. As NF‐κB and SOD are both pro‐survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.

Polyclonal antibody localizes glia maturation factor β-like immunoreactivity in neurons and glia

A rabbit polyclonal antibody (91-01) was raised against recombinant human glia maturation factor beta (r-hGMF-beta). The antibody did not cross-react with a number of other growth factors on ELISA test. When compared with the monoclonal antibody G2-09 previously obtained, 91-01 immunoblotted the same protein band in rat brain extract. However, unlike G2-09 which immunostained only astrocytes and Bergmann glia, 91-01 stained neurons as well. Many but not all neurons in the central and peripheral nervous system were positive for GMF-beta. The larger cell population stained by the polyclonal antibody was most likely due to its increased sensitivity, although other explanations are possible. The presence of GMF-beta-like immunoreactivity in both neurons and glia raises the possibility of a wider range of cell-cell interaction than was previously considered.

Inactivation of (Na-++K-+)-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells.

The mechanism of action of the cytotoxic protein P6 isolated from cobra venom (Naja naja) which shows preferential cytotoxicity particularly to Yoshida sarcoma cells has been studied by its effects on the membrane-bound enzyme (Na-++K-+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of a variety of cell systems. Evidence obtained with Yoshida sarcoma cells, dog and human erythrocytes and three tissue culture cell lines KB (human oral carcinoma), Hela (human cervix carcinoma) and L-132 (human lung embryonic) shows that inhibition of (Na-++K-+)-ATPase by the P6 protein can be correlated with its lytic activity. (Na-++k-+)-ATPase of Yoshida sarcoma membrane fragments inactivated by P6 protein could be reconstituted by the addition of phosphatidylserine and phosphatidic acid. It is conceivable that lysis of cells by the P6 protein may be due to an imbalance of K-+ and Na-+ in the cell which leads to swelling and disintegration of the membrane structure. Observations indicate that the P6 protein combines with membrane constituents of susceptible cells. The overall evidence suggests that both the specificity of its protein structure and the highly basic nature of the P6 protein are factors which enable it to compete with the lipid moiety maintaining the (Na-++k-+)-ATPase of the susceptible cells in proper conformation for activity.