Smita Zaheer

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.

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.

Enhanced Expression of Glia Maturation Factor Correlates with Glial Activation in the Brain of Triple Transgenic Alzheimer’s Disease Mice

We previously demonstrated that glia maturation factor (GMF), a brain specific protein, isolated, sequenced and cloned in our laboratory, induce expression of proinflammatory cytokines and chemokines in the central nervous system. We also reported that the up-regulation of GMF in astrocytes leads to the destruction of neurons suggesting a novel pathway of GMF-mediated cytotoxicity of brain cells, and implicated its involvement in the pathogenesis of inflammatory neurodegenerative diseases. In the present study, we examined the expressions of GMF in triple-transgenic Alzheimer’s disease (3xTg-AD) mice. Our results show a 13-fold up-regulation of GMF and 8–12-fold up-regulation of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-1β, interferon gamma (IFN-γ), and chemokine (C–C motif) ligand 2 (CCL2) and C–X–C motif chemokine 10 (CXCL10/IP-10) mRNA as determined by quantitative real-time RT-PCR in the brain of 3xTg-AD mice as compared to non-transgenic (Non-Tg) mice. In conclusion, the increase in GMF and cytokine/chemokine expression was correlated with reactive glial fibrillary acidic protein positive astrocytes and ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglia in 3xTg-AD mice.

Transfection of C6 glioma cells with glia maturation factor upregulates brain-derived neurotrophic factor and nerve growth factor: trophic effects and protection against ethanol toxicity in cerebellar granule cells

Glial cells play active roles in neuronal survival, as well as neuroprotection against toxic insult. Recent studies suggest that the brain protein glia maturation factor (GMF) is involved in intracellular signaling in glia. This study investigated whether or not GMF plays a role in the survival-promoting and neuroprotective functions of glia. C6 glioma cells were transfected in vitro with GMF utilizing an adenovirus vector. The transfected cells overexpressed GMF intracellularly, but did not secrete the protein. The conditioned medium (CM) was obtained from the GMF-transfected cells (CM-GMF) and tested on primary neuronal cultures, consisting of cerebellar granule cells (CGC). The CGC cultures were utilized because these cultures have a background level of cell death, and the survival-promoting, i.e. neurotrophic effect, of the CM could be tested. In addition, since CGC cultures are ethanol-sensitive (ethanol enhances neuronal death), the neuroprotective effect of the CM against ethanol-induced cell death was tested also. We demonstrated that the CM-GMF had an enhanced neurotrophic effect as well as an increased neuroprotective effect against ethanol-induced cell death compared to control CM obtained from untransfected C6 cells (CM-Mock) or CM obtained from cells transfected with an unrelated gene (CM-LacZ). Because neurotrophins have trophic and protective effects, we investigated whether GMF-transfection upregulated the expression of neurotrophins in C6 cells. RT-PCR verified that GMF-transfected C6 cells had increased mRNA levels for BDNF and NGF. Immunoblotting corroborated the RT-PCR results and indicated that CM-GMF contained greater concentrations of BDNF and NGF protein compared to CM-Mock and CM-LacZ. A soluble TrkB-IgG fusion protein, which selectively binds BDNF and prevents its binding to the neuronal TrkB receptor, eliminated the neurotrophic effect of CM-GMF; whereas anti-NGF antibody was ineffective in preventing this effect, suggesting that the neurotrophic effect was due to BDNF. On the other hand, both the TrkB-IgG fusion protein and anti-NGF reduced neuroprotection, suggesting that BDNF and NGF both contribute to the neuroprotective effect of CM-GMF. In conclusion, GMF upregulates the expression of BDNF and NGF in C6 cells, and these factors exert neurotrophic and neuroprotective functions on primary neurons.

Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration

Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson’s disease (PD), Alzheimer’s disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1–42 (Aβ1–42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can also enter into the peripheral system through defective BBB, recruit immune cells into the brain, and exacerbate neuroinflammation. We suggest that mast cell-associated inflammatory mediators from systemic inflammation and brain could augment neuroinflammation and neurodegeneration in the brain. This review article addresses the role of some atypical inflammatory mediators that are associated with mast cell inflammation and their activation of glial cells to induce neurodegeneration.

Alzheimer's disease: evidence for the expression of interleukin-33 and its receptor ST2 in the brain.

Inflammatory responses are increasingly implicated in the pathogenesis of neurodegenerative diseases such as in Alzheimers disease (AD). Interleukin-33 (IL-33), a member of IL-1 family, is constitutively expressed in the central nervous system and thought to be an important mediator of glial cell response to neuropathological lesions. Proinflammatory molecules are highly expressed at the vicinity of amyloid plaques (APs) and neurofibrillary tangles (NFTs), the hallmarks of AD pathology. We have investigated the expression of IL-33 and ST2 in relation to APs and NFTs in human AD and non-AD control brains by immunohistochemistry. Sections from the entorhinal cortex, where APs and NFTs appear in early stages of AD, were used for immunohistochemistry. Mouse primary astrocytes were cultured and incubated with amyloid-β1-42 (Aβ1-42), component of plaque for 72 h and analyzed for the expression of IL-33 by flow cytometry. We found strong expression of IL-33 and ST2 in the vicinity of Aβ and AT8 labelled APs and NFTs respectively, and in the glial cells in AD brains when compared to non-AD control brains. IL-33 and ST2 positive cells were also significantly increased in AD brains when compared to non-AD brains. Flow cytometric analysis revealed incubation of mouse astrocytes with Aβ1-42 increased astrocytic IL-33 expression in vitro. These results suggest that IL-33, an alamin cytokine, may induce inflammatory molecule release from the glial cells and may play an important role in the pathogenesis of AD.

Augmented expression of glia maturation factor in Alzheimer's disease.

We have previously demonstrated that glia maturation factor (GMF), a brain-specific protein, isolated, sequenced, and cloned in our laboratory, is a prominent mediator of inflammation in the CNS leading to the death of neurons. In the present study, we demonstrate, for the first time, a significant upregulation of the GMF protein in various regions of Alzheimers disease (AD) brains compared with age-matched non-demented (ND) control brains. We analyzed AD and ND brain samples by quantitative enzyme-linked immunosorbent assay (ELISA) using a combination of highly specific monoclonal and polyclonal anti-GMF antibodies developed and characterized in our laboratory. For the comparison between ND controls and AD cases, we examined brain tissue from 12 ad cases (ages ranging from 78-92 years) and eight age-matched ND controls (ages ranging from 76-88 years). We observed a significant increase in GMF concentration in entorhinal cortex, parietal cortex, frontal cortex, occipital cortex, perirhinal cortex, and temporal cortex of AD patients. Our results clearly demonstrate that the GMF protein levels are significantly higher in all AD-affected brain regions than in ND controls. The immunohistochemistry analysis revealed co-localization of GMF with amyloid plaques (AP) and neurofibrillary tangles (NFTs) in AD brains. Our results imply that under conditions of neurodegeneration the expression of GMF is significantly upregulated.

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-1β, and MIP-1β, 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.

Suppression of neuro inflammation in experimental autoimmune encephalomyelitis by glia maturation factor antibody

Glia maturation factor (GMF), a protein primarily localized in the central nervous system (CNS) was isolated, sequenced and cloned in our laboratory. We previously demonstrated that GMF mediates the experimental autoimmune encephalomyelitis (EAE)-induced production of pro-inflammatory cytokines and chemokines in the central nervous system of mice. In the present study we show that immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) caused an early onset (days 7-9 post immunization) and severe EAE with a mean peak score of 3.5 ± 0.5 in mice. Neutralization of GMF with four injections of anti-GMF antibody 5 to 11 days post immunization delayed the time of onset (days 12-14 post immunization) and significantly reduced the severity of EAE (mean peak score of 1.5 ± 0.4). Consistent with these clinical scores, histological examination of the CNS of these mice revealed profound differences between GMF-antibody treated mice and isotype matched control-antibody treated mice. Histological analysis of the spinal cord and brain showed severe inflammation and demyelination in the control antibody-treated mice whereas significantly reduced inflammation and demyelination was detected in GMF-antibody-treated mice at days 8, 16, and 24 post immunization. The decreased incidence and reduced severity of EAE in GMF-antibody-treated mice was consistent with the significantly reduced expressions of pro-inflammatory cytokines and chemokines. Our overall results demonstrate that neutralization of endogenous GMF with an affinity purified GMF antibody significantly decreased the inflammation, severity and progression of immunization induced active, passive and relapsing-remitting EAE. Treatment of mice with isotype-matched control antibody did not have any effect on EAE. Taken together, these results demonstrate the critical role of GMF in EAE, and GMF antibody as a potent anti-inflammatory therapeutic agent for effectively suppressing EAE in mouse models of major types of multiple sclerosis (MS).

Dopaminergic Toxin 1-Methyl-4-Phenylpyridinium, Proteins α-Synuclein and Glia Maturation Factor Activate Mast Cells and Release Inflammatory Mediators

Parkinson’s disease (PD) is characterized by the presence of Lewy bodies and degeneration of dopaminergic neurons. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Lewy body component α-synuclein activates glia in PD pathogenesis. Mast cells and glia maturation factor (GMF) are implicated in neuroinflammatory conditions including Multiple Sclerosis. However, the role of mast cells in PD is not yet known. We have analyzed the effect of recombinant GMF, MPP+, α-synuclein and interleukin-33 (IL-33) on mouse bone marrow-derived cultured mast cells (BMMCs), human umbilical cord blood-derived cultured mast cells (hCBMCs) and mouse brain-derived cultured astrocytes by quantifying cytokines/chemokines released using ELISA or by detecting the expression of co-stimulatory molecules CD40 and CD40L by flow cytometry. GMF significantly released chemokine (C-C motif) ligand 2 (CCL2) from BMMCs but its release was reduced in BMMCs from GMF knockout mice. GMF, α-synuclein and MPP+ released IL-1β, β-hexosaminidase from BMMCs, and IL-8 from hCBMCs. GMF released CCL5, and IL-33- induced the expression of GMF from hCBMCs. Novel GMF expression was detected in hCBMCs and BMMCs by immunocytochemistry. GMF released tumor necrosis factor-alpha (TNF-α) from mouse astrocytes, and this release was greater in BMMC- astrocyte coculture than in individual cultures. Flow cytometry results showed increased IL-33 expression by GMF and MPP+, and GMF-induced CD40 expression in astrocytes. Proinflammatory mediator release by GMF, MPP+ and α-synuclein, as well as GMF expression by mast cells indicate a potential therapeutic target for neurodegenerative diseases including PD.