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-kappaB, 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-kappaB, and enhanced expression of GM-CSF by astrocytes. Inhibition of p38 MAPK or NF-kappaB 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-alpha, IL-1beta, 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-kappaB activation. The GM-CSF-dependent expression and secretion of inflammatory cytokine/chemokine, TNF-alpha, IL-1beta, 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.

Reduced severity of experimental autoimmune encephalomyelitis in GMF-deficient mice.

Glia maturation factor (GMF), a highly conserved brain-specific protein, isolated, sequenced and cloned in our laboratory. Overexpression of GMF in astrocytes induces the production and secretion of granulocyte-macrophage-colony stimulating factor (GM-CSF), and subsequent immune activation of microglia, expression of several proinflammatory genes including major histocompatibility complex proteins, IL-1beta, and MIP-1beta, all associated with the development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. Based on GMF's ability to activate microglia and induce well-established proinflammatory mediators, including GM-CSF, we hypothesize that GMF is involved in the pathogenesis of inflammatory disease EAE. In this present investigation, using GMF-deficient mice, we study the role of GMF and how the lack of GMF affects the EAE disease. Our results show a significant decrease in incidence, delay in onset, and reduced severity of EAE in GMF-deficient mice, and support the hypothesis that GMF plays a major role in the pathogenesis of disease.

GMF-knockout mice are unable to induce brain-derived neurotrophic factor after exercise.

We earlier reported that overexpression of glia maturation factor (GMF) in cultured astrocytes enhances the production of brain-derived neurotrophic factor (BDNF). The current study was conducted to find out whether BDNF production is impaired in animals devoid of GMF. To this end GMF-knockout (KO) mice were subjected to exercise and the neurotrophin mRNAs were determined by real-time RT-PCR. Compared to wild-type (WT) mice, there is a decrease in exercise-induced BDNF in the KO mice. The observation was correlated with the finding that, in WT mice, exercise increases GMF expression. The results are consistent with the hypothesis that GMF is necessary for exercise-induction of BDNF, and that GMF may promote neuroprotection through BDNF production.

Impaired motor performance and learning in glia maturation factor-knockout mice.

Glia maturation factor (GMF) is a unique brain protein localized in astrocytes and some neuronal populations. Studies with overexpression of GMF using adenovirus vector have uncovered its regulatory role in intracellular signal transduction and downstream induction of biologically active molecules, including the neurotrophins and cytokines. The current paper deals with the behavior of mice devoid of GMF protein (knockout). GMF-null mice developed normally without gross abnormality. When tested for simple position discrimination using a T-maze and for spatial memory using a Morris water maze, the knockout mice performed as well as the wild-type, showing no defect in maze learning. However, with beam walking, GMF-knockout mice performed poorly and failed to learn. Knockout mice were also defective in learning the eyeblink classical conditioning. Histologically, the knockout mice showed a loss of neurons in the inferior olive, which is a component of the circuitry of eyeblink conditioning, and is also essential for motor performance. The structural abnormality in GMF-null mice explained their impaired ability for both motor performance and motor learning.

Decreased copper-zinc superoxide dismutase activity and increased resistance to oxidative stress in glia maturation factor-null astrocytes.

Glia maturation factor (GMF) is a highly conserved protein found mainly in the nervous system. The current work was undertaken to investigate the effect of GMF expression in astrocytes on CuZn superoxide dismutase (CuZnSOD or SOD I) and on the vulnerability of the cells to H2O2 toxicity. Primary astrocyte cultures were derived from mice in which the GMF gene was completely deleted by homologous recombination (knockout). Astocytes derived from knockout animals displayed a lower level of CuZnSOD activity and protein. The reduction in CuZnSOD was restored by transfection with a GMF/adenovirus construct, and the resulting increase was blocked by the p38 MAP kinase inhibitor SB203580. There was no change in the other isoform of SOD (MnSOD or SOD II). Endogenous H2O2 was lower in the knockout cells, and the cells became more resistant to H2O2 toxicity compared to the wild type. In the GMF-null cells, concurrent with a decrease in CuZnSOD, the function of which is to convert superoxide to H2O2, there was an increase in the activity of the two enzymes that degrade H2O2: catalase and glutathione peroxidase. By regulating the redox state of the cell, GMF may be involved in a wide spectrum of cellular events ranging from survival, proliferation, differentiation, to death.

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.