Glia Maturation Factor Research Articles

Glia Maturation Factor Dependent Mast Cell Activation and Calpain 1 Synergize Dopaminergic Neuronal Loss and Behavioral Deficits in an MPTP Mouse Model of Parkinson's Disease

The molecular mechanism mediating nigrostriatal dopaminergic neurons degeneration in Parkinson's disease (PD) is not yet fully understood. We have shown that glia maturation factor (GMF), a proinflammatory protein mediated mast cells activation in the dopaminergic neurodegenerations. Here, we show that deficiency of GMF (GMF‐KO) and mast cell reduces the synergistic effects of mast cell proteases and calpain 1 dependent dopaminergic neuronal loss in the SN, and STR of the midbrain leads to improves motor behavioral impairments in an MPTP mouse model of PD. The MPTP induced nigrostriatal neurodegeneration and astro‐glial activations were determined by western blotting and immunocytochemistry. We found that MPTP administrated wild type (Wt) mice exhibits oxidative stress by significantly increased level of MDA and activity of SOD and reduced the level of GSH and the activity of GPx when compared with both mast cell deficient (MC‐KO) and GMF‐KO mice. The number of TH‐positive neurons in the ventral tegmental area (VTA), SN and the fibers in STR were significantly reduced and GFAP, IBA1, calpain 1 and ICAM 1 expressions were significantly increased in Wt mice. Similarly, we found that TH, DAT and VMAT2 proteins expression significantly reduced in the SN of Wt mice when compared with both the MC‐KO and GMF‐KO mice. The motor behavior in rotarod and hang test performance significantly reduced Wt mice as compared with both the MC‐KO and GMF‐KO mice. Our study shows that deficiency of GMF and mast cells protects nigrostriatal degeneration via inhibition of astro‐glial activation‐mediated oxidative stress and neuroinflammation in the MPTP mouse model of PD. These results suggest that GMF dependent mast cells activations enhances the detrimental effect of dopaminergic degeneration and its inhibition may be a beneficial therapeutic target of PD and other neurodegenerative disorders that are associated with neuroinflammation, mast cell‐astro‐glial activation‐derived cell death.Support or Funding InformationThis work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 and NS073670.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glia Maturation Factor‐Antibody Injection Reduces Behavioral Impairment, Neuro Inflammation and Amyloid Pathology in 5XFAD Mice Brains

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of beta‐amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. Emerging evidence suggested that in neurodegenerative disease, aggregation of these proteins can promotes neuroinflammation and neurodegeneration. Glia maturation factor (GMF), a neuroinflammatory protein isolated and cloned in our laboratory play an important role in the pathogenesis of AD. To study the relationship between overexpression of GMF, glial activation and neuroinflammation, we used 9‐month‐old 5XFAD mice, a transgenic model of AD. We intravenously (iv) injected single dose of GMF antibody and found that antibody injection leads to: improvements in behavioral deficits, decreased activation of glial cells and reduced expression of pro‐inflammatory cytokines and amyloid pathology. We report that aberrant expression of GMF, GFAP, IBA1, and co‐localization of GMF with GFAP, IBA1 and Aβ significantly more in 5XFAD mice as compared with wild type mice. However, following GMF antibody injection, we found significant reduced expression of GMF, GFAP, IBA1 and Aβ in the cortex region of 5XFAD mice. We conclude that GMF‐antibody may be used to reduce neuroinflammation in neurodegenerative diseases including Alzheimer's disease.Support or Funding InformationNational Institutes of Health Grants NS073670, AG048205 and Veterans Affairs Merit Award I01BX002477 to AZ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Co‐localization of Glia Maturation Factor and Progranulin in the Human Alzheimer's Disease Brains

Amyloid plaques (APs) and neurofibrillary tangles (NFTs) are two major neuropathological hallmarks observed in the brains of Alzheimer's disease (AD) patients. Inflammatory mediators released from the activated glial cells and neurons induce neuroinflammation and neurodegeneration thereby exacerbating AD pathogenesis. We have previously shown that glia maturation factor (GMF), a proinflammatory brain protein is upregulated in the brains of AD patients. Additionally, we also found that the upregulated GMF is associated with APs and NFTs in the brains of AD patients. Progranulin a pleiotropic protein that serves as a precursor of active granulin peptides was also shown to be upregulated in the brains of AD patients. However, the association between GMF and progranulin has not yet been studied in the context of neurodegenerative diseases including AD. We analyzed frozen sections of temporal lobe from human AD patients in this study. Here, we investigated whether the upregulated GMF expression is associated with a corresponding increase in progranulin expression in the vicinity of APs in the AD brains using single and double immunohistochemistry and immunofluorescence staining. Our results show that progranulin immunoreactive neurons and activated microglia are present in the temporal cortex region of AD brains. We report increased progranulin and GMF expression in the vicinity of APs. Further, we also report significantly more progranulin expression in the neurons and glial cells in the brains of AD patients. Our data suggest that the increased expression of GMF and progranulin may synergize to play a critical role in neuroinflammation and neurodegeneration thereby exacerbating AD pathogenesis.Support or Funding InformationThis work was supported by the Veterans' Affairs Merit Award I01BX002477 and the National Institutes of Health Grants AG048205 and NS073670 to AZThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CRISPR/Cas9 Editing of Glia Maturation Factor Regulates Mitochondrial Dynamics by Attenuation of the NRF2/HO-1 Dependent Ferritin Activation in Glial Cells.

Microglial cells are brain specific professional phagocytic immune cells that play a crucial role in the inflammation- mediated neurodegeneration especially in Parkinson's disease (PD) and Alzheimer's disease. Glia maturation factor (GMF) is a neuroinflammatory protein abundantly expressed in the brain. We have previously shown that GMF expression is significantly upregulated in the substantia nigra (SN) of PD brains. However, its possible role in PD progression is still not fully understood. The Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) protein9 (CRISPR/Cas9) system is a simple, rapid and often extremely efficient gene editing tool at desired loci, enabling complete gene knockout or homology directed repair. In this study, we examined the effect of GMF editing by using the CRISPR/Cas9 technique in BV2 microglial cells (hereafter referred to as BV2-G) on oxidative stress and nuclear factor erythroid 2-related factor 2 (NRF2)/Hemeoxygenase1 (HO-1)-dependent ferritin activation after treatment with(1-methyl-4-phenylpyridinium) MPP+. Knockout of GMF in BV2-G cells significantly attenuated oxidative stress via reduced ROS production and calcium flux. Furthermore, deficiency of GMF significantly reduced nuclear translocation of NRF2, which modulates HO-1 and ferritin activation, cyclooxygenase 2 (COX2) and nitric oxide synthase 2 (NOS2) expression in BV2 microglial cells. Lack of GMF significantly improved CD11b and CD68 positive microglial cells as compared with untreated cells. Our results also suggest that pharmacological and genetic intervention targeting GMF may represent a promising and a novel therapeutic strategy in controlling Parkinsonism by regulating microglial functions. Targeted regulation of GMF possibly mediates protein aggregation in microglial homeostasis associated with PD progression through regulation of iron metabolism by modulating NRF2-HO1 and ferritin expression.

First description of enhanced expression of transforming growth factor-alpha (TGF-α) and glia maturation factor-beta (GMF-β) correlate with severity of neuropathology in border disease virus-infected small ruminants

Border disease (BD) is caused by Pestivirus and characterized by severe neuropathology, and histopathologically observed severe hypomyelination. We have previously shown that small ruminants infected with border disease virus (BDV) play an important role for neuropathology and pathogenesis of severe oxidative damage in brain tissue, neuronal mtDNA; in the production of high pathologic levels of nitric oxide; in glial cell activation and stimulation of intrinsic apoptosis pathway. This study aimed to investigate the relationship between glia maturation factor beta (GMF-β) and transforming growth factor alpha (TGF-α) expressions and the causes of BDV-induced neuropathology and to investigate their role in neuropathogenesis in a way that was not presented before. Expression levels of GMF-β and TGF-α were investigated. Results of the study revealed that the levels of GMF-β (P < 0.005) and TGF-α (P < 0.005) expression in the brain tissue markedly increased in the BDV-infected animals compared to the non-infected healthy control group. While TGF-α expressions were predominantly observed in neurons, GMF-β expressions were found in astrocytes, glial cells and neurons. These results were reasonable to suggest that BDV-mediated increased GMF-β might play a pivotal role neuropathogenesis and a different type of role in the mechanism of neurodegeneration/neuropathology in the process of BD. The results also indicated that increased levels of GMF up-regulation in glial cells and neurons causes neuronal destruction, suggesting pathological pathway involving GMF-mediated brain cell cytotoxicity. It is clearly indicated that the cause of astrogliosis is due to severe TGF-a expression. This is the first study to demonstrate the expression of GMF-β and TGF-α in neurons and reactive glial cells and its association with neuropathology in BD.

Glia Maturation Factor and Mast Cell-Dependent Expression of Inflammatory Mediators and Proteinase Activated Receptor-2 in Neuroinflammation.

Parkinson's disease (PD) is characterized by the presence of inflammation-mediated dopaminergic neurodegeneration in the substantia nigra. Inflammatory mediators from activated microglia, astrocytes, neurons, T-cells and mast cells mediate neuroinflammation and neurodegeneration. Administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induces PD like motor deficits in rodents. 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of MPTP activates glial cells, neurons and mast cells to release neuroinflammatory mediators. Glia maturation factor (GMF), mast cells and proteinase activated receptor-2 (PAR-2) are implicated in neuroinflammation. Alpha-synuclein which induces neurodegeneration increases PAR-2 expression in the brain. However, the exact mechanisms are not yet understood. In this study, we quantified inflammatory mediators in the brains of MPTP-administered wild type (Wt), GMF-knockout (GMF-KO), and mast cell knockout (MC-KO) mice. Additionally, we analyzed the effect of MPP+, GMF, and mast cell proteases on PAR-2 expression in astrocytes and neurons in vitro. Results show that the levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and the chemokine (C-C motif) ligand 2 (CCL2) were lesser in the brains of GMF-KO mice and MC-KO mice when compared to Wt mice brain after MPTP administration. Incubation of astrocytes and neurons with MPP+, GMF, and mouse mast cell protease-6 (MMCP-6) and MMCP-7 increased the expression of PAR-2. Our studies show that the absence of mast cells and GMF reduce the expression of neuroinflammatory mediators in the brain. We conclude that GMF along with mast cell interactions with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD and other neuroinflammatory disorders.

Molecular Association of Glia Maturation Factor with the Autophagic Machinery in Rat Dopaminergic Neurons: a Role for Endoplasmic Reticulum Stress and MAPK Activation.

Parkinson's disease (PD) is one of the several neurodegenerative diseases where accumulation of aggregated proteins like α-synuclein occurs. Dysfunction in autophagy leading to this protein build-up and subsequent dopaminergic neurodegeneration may be one of the causes of PD. The mechanisms that impair autophagy remain poorly understood. 1-Methyl-4-phenylpiridium ion (MPP+) is a neurotoxin that induces experimental PD in vitro. Our studies have shown that glia maturation factor (GMF), a brain-localized inflammatory protein, induces dopaminergic neurodegeneration in PD and that suppression of GMF prevents MPP+-induced loss of dopaminergic neurons. In the present study, we demonstrate a molecular action of GMF on the autophagic machinery resulting in dopaminergic neuronal loss and propose GMF-mediated autophagic dysfunction as one of the contributing factors in PD progression. Using dopaminergic N27 neurons, primary neurons from wild type (WT), and GMF-deficient (GMF-KO) mice, we show that GMF and MPP+ enhanced expression of MAPKs increased the mammalian target of rapamycin (mTOR) activation and endoplasmic reticulum stress markers such as phospho-eukaryotic translation initiation factor 2 alpha kinase 3 (p-PERK) and inositol-requiring enzyme 1α (IRE1α). Further, GMF and MPP+ reduced Beclin 1, focal adhesion kinase (FAK) family-interacting protein of 200kD (FIP200), and autophagy-related proteins (ATGs) 3, 5, 7, 16L, and 12. The combined results demonstrate that GMF affects autophagy through autophagosome formation with significantly reduced lysosomal-associated membrane protein 1/2, and the number of autophagic acidic vesicles. Using primary neurons, we show that MPP+ treatment leads to differential expression and localization of p62/sequestosome and in GMF-KO neurons, there was a marked increase in p62 staining implying autophagy deficiency with very little co-localization of α-synuclein and p62 as compared with WT neurons. Collectively, this study provides a bidirectional role for GMF in executing dopaminergic neuronal death mediated by autophagy that is relevant to PD.

Brain-Derived Glia Maturation Factor \u03b2 Participates in Lung Injury Induced by Acute Cerebral Ischemia by Increasing ROS in Endothelial Cells

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.

Glia maturation factor gamma, is a novel diagnostic marker of leukemia, has TAL1 binding sites in its promoter

Glia maturation factor gamma (GMFG) overexpression increases T-cell migration and adhesion. Its high expression in colorectal and ovarian cancer is associated with chemoresistance and adverse prognosis. However, it remains elusive if GMFG plays a role in the development and progression of leukemia. Tissue invasion in leukemia has long been known as the reason for disease progression as leukemic cells accumulates in bone marrow, spleen and lymph nodes. This tissue invasion requires cell motility and migration. The aim of the present study was to find any association of GMFG expression with leukemia and its potential to be a diagnostic marker. For this purpose, we have carried out expression profiling of GMFG among a cohort of leukemia patients. Liquid biopsies were collected and subjected to GMFG expression analysis. We found that GMFG expression was significantly elevated among leukemia patients of all age groups relative to their age match controls. Further promoter analysis of GMFG reveals binding sites for T-cell Acute Lymphocytic Leukemia 1 (TAL1) and associated transcription factors. TAL1 is master transcription factor which, in coordination with other factors like GATA-binding protein 1 (GATA-1) and LIM domain only 2 (LMO2), regulates gene expression in T-cell leukemia. The induced TAL1 expression coincides with upregulated GMFG expression. Together these data suggest that TAL1 may bind to GMFG promoter to increase its expression. GMFG may play a crucial role in the motility of leukemic cells and facilitates their tissue invasion which leads to therapy resistance.

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans

BackgroundThe GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length. MethodsWe have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. ResultsThe solution structure of CeGMF shows canonical ADF-H fold with two additional β-strands in the β4-β5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the β4-β5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of β4-β5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. ConclusionWe have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin. General significanceThis study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.

Mast Cell Proteases Activate Astrocytes and Glia-Neurons and Release Interleukin-33 by Activating p38 and ERK1/2 MAPKs and NF-κB

Inflammatory mediators released from activated microglia, astrocytes, neurons, and mast cells mediate neuroinflammation. Parkinson's disease (PD) is characterized by inflammation-dependent dopaminergic neurodegeneration in substantia nigra. 1-Methyl-4-phenylpyridinium (MPP+), a metabolite of parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), induces inflammatory mediators' release from brain cells and mast cells. Brain cells' interaction with mast cells is implicated in neuroinflammation. However, the exact mechanisms involved are not yet clearly understood. Mouse fetal brain-derived cultured primary astrocytes and glia-neurons were incubated with mouse mast cell protease-6 (MMCP-6) and MMCP-7, and mouse bone marrow-derived mast cells (BMMCs) were incubated with MPP+ and brain protein glia maturation factor (GMF). Interleukin-33 (IL-33) released from these cells was quantitated by enzyme-linked immunosorbent assay. Both MMCP-6 and MMCP-7 induced IL-33 release from astrocytes and glia-neurons. MPP+ and GMF were used as a positive control-induced IL-33 and reactive oxygen species expression in mast cells. Mast cell proteases and MPP+ activate p38 and extracellular signal-regulated kinases 1/2 (ERK1/2), mitogen-activated protein kinases (MAPKs), and transcription factor nuclear factor-kappa B (NF-κB) in astrocytes, glia-neurons, or mast cells. Addition of BMMCs from wt mice and transduction with adeno-GMF show higher chemokine (C-C motif) ligand 2 (CCL2) release. MPP+ activated glial cells and reduced microtubule-associated protein 2 (MAP-2) expression indicating neurodegeneration. IL-33 expression increased in the midbrain and striatum of PD brains as compared with age- and sex-matched control subjects. Glial cells and neurons interact with mast cells and accelerate neuroinflammation and these interactions can be explored as a new therapeutic target to treat PD.

GMF as an Actin Network Remodeling Factor.

Glia maturation factor (GMF) has recently been established as a regulator of the actin cytoskeleton with a unique role in remodeling actin network architecture. Conserved from yeast to mammals, GMF is one of five members of the ADF-H family of actin regulatory proteins, which includes ADF/cofilin, Abp1/Drebrin, Twinfilin, and Coactosin. GMF does not bind actin, but instead binds the Arp2/3 complex with high affinity. Through this association, GMF catalyzes the debranching of actin filament networks and inhibits actin nucleation by Arp2/3 complex. Here, we discuss GMF's emerging role in controlling actin filament spatial organization and dynamics underlying cell motility, endocytosis, and other biological processes. Further, we attempt to reconcile these functions with its earlier characterization as a cell differentiation factor.

Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer's Disease Therapeutic Target.

Alzheimer's disease (AD) is a devastating, progressive neurodegenerative disorder that leads to severe cognitive impairment in elderly patients. Chronic neuroinflammation plays an important role in the AD pathogenesis. Glia maturation factor (GMF), a proinflammatory molecule discovered in our laboratory, is significantly upregulated in various regions of AD brains. We have previously reported that GMF is predominantly expressed in the reactive glial cells surrounding the amyloid plaques (APs) in the mouse and human AD brain. Microglia are the major source of proinflammatory cytokines and chemokines including GMF. Recently clustered regularly interspaced short palindromic repeats (CRISPR) based genome editing has been recognized to study the functions of genes that are implicated in various diseases. Here, we investigated if CRISPR-Cas9-mediated GMF gene editing leads to inhibition of GMF expression and suppression of microglial activation. Confocal microscopy of murine BV2 microglial cell line transduced with an adeno-associated virus (AAV) coexpressing Staphylococcus aureus (Sa) Cas9 and a GMF-specific guide RNA (GMF-sgRNA) revealed few cells expressing SaCas9 while lacking GMF expression, thereby confirming successful GMF gene editing. To further improve GMF gene editing efficiency, we developed lentiviral vectors (LVs) expressing either Streptococcus pyogenes (Sp) Cas9 or GMF-sgRNAs. BV2 cells cotransduced with LVs expressing SpCas9 and GMF-sgRNAs revealed reduced GMF expression and the presence of indels in the exons 2 and 3 of the GMF coding sequence. Lipopolysaccharide (LPS) treatment of GMF-edited cells led to reduced microglial activation as shown by reduced p38 MAPK phosphorylation. We believe that targeted in vivo GMF gene editing has a significant potential for developing a unique and novel AD therapy.

Glia maturation factor beta is required for reactive gliosis after traumatic brain injury in zebrafish

Gliosis is a hallmark of neural pathology that occurs after most forms of central nervous system (CNS) injuries including traumatic brain injury (TBI). Identification of genes that control gliosis may provide novel treatment targets for patients with diverse CNS injuries. Glia maturation factor beta (GMFB) is crucial in brain development and stress response. In the present study, GMFB was found to be widely expressed in adult zebrafish telencephalon. A gmfb mutant zebrafish was created using CRISPR/cas9. In the uninjured zebrafish telencephalon, glial fibrillary acidic protein (GFAP) fibers in gmfb mutants were disorganized and shorter than wild type zebrafish. After TBI, transformation of quiescent type I radial glial cells (RGC) to proliferative type II RGCs was significantly suppressed in the gmfb mutant. RGC proliferation and hypertrophy post-TBI was reduced in gmfb mutants, indicating that reactive gliosis was attenuated. TBI-induced acute inflammation was also found to be alleviated in the gmfb mutant. Morphological changes also suggest attenuation of microglial reactive gliosis. In a mouse model of TBI, GMFB expression was increased around the injury site. These GMFB+ cells were identified as astrocytes and microglia. Taken together, the data suggests that GMFB is not only required for normal development of GFAP fibers in the zebrafish telencephalon, but also promotes reactive gliosis after TBI. Our findings provide novel information to help better understand the reactive gliosis process following TBI.

Bipolar disorder in youth is associated with increased levels of vitamin D-binding protein

Genetic, dietary, and inflammatory factors contribute to the etiology of major mood disorders (MMD), thus impeding the identification of specific biomarkers to assist in diagnosis and treatment. We tested association of vitamin D and inflammatory markers in 36 adolescents with bipolar disorder (BD) and major depressive disorder (MDD) forms of MMD and without MMD (non-mood control). We also assessed the overall level of inflammation using a cell-based reporter assay for nuclear factor kappa-B (NFκB) activation and measuring antibodies to oxidized LDL. We found that these factors were similar between non-mood and MMD youth. To identify potential biomarkers, we developed a screening immunoprecipitation-sequencing approach based on inflammatory brain glia maturation factor beta (GMFβ). We discovered that a homolog of GMFβ in human plasma is vitamin D-binding protein (DBP) and validated this finding using immunoprecipitation with anti-DBP antibodies and mass spectrometry/sequencing analysis. We quantified DBP levels in participants by western blot. DBP levels in BD participants were significantly higher (136%) than in participants without MMD (100%). The increase in DBP levels in MDD participants (121.1%) was not statistically different from these groups. The DBP responds early to cellular damage by binding of structural proteins and activating inflammatory cells. A product of enzymatic cleavage of DBP has been described as macrophage-activating factor. Circulating DBP is comprised of heterogenous high and low molecular fractions that are only partially recognized by mono- and polyclonal ELISA and are not suitable for the quantitative comparison of DBP in non-mood and MDD participants. Our data suggest DBP as a marker candidate of BD warranting its validation in a larger cohort of adolescent and adult MMD patients.

Glia Maturation Factor Dependent Inhibition of Mitochondrial PGC-1α Triggers Oxidative Stress-Mediated Apoptosis in N27 Rat Dopaminergic Neuronal Cells.

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting over five million individuals worldwide. The exact molecular events underlying PD pathogenesis are still not clearly known. Glia maturation factor (GMF), a neuroinflammatory protein in the brain plays an important role in the pathogenesis of PD. Mitochondrial dysfunctions and oxidative stress trigger apoptosis leading to dopaminergic neuronal degeneration in PD. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α or PPARGC-α) acts as a transcriptional co-regulator of mitochondrial biogenesis and energy metabolism by controlling oxidative phosphorylation, antioxidant activity, and autophagy. In this study, we found that incubation of immortalized rat dopaminergic (N27) neurons with GMF influences the expression of peroxisome PGC-1α and increases oxidative stress, mitochondrial dysfunction, and apoptotic cell death. We show that incubation with GMF reduces the expression of PGC-1α with concomitant decreases in the mitochondrial complexes. Besides, there is increased oxidative stress and depolarization of mitochondrial membrane potential (MMP) in these cells. Further, GMF reduces tyrosine hydroxylase (TH) expression and shifts Bax/Bcl-2 expression resulting in release of cytochrome-c and increased activations of effector caspase expressions. Transmission electron microscopy analyses revealed alteration in the mitochondrial architecture. Our results show that GMF acts as an important upstream regulator of PGC-1α in promoting dopaminergic neuronal death through its effect on oxidative stress-mediated apoptosis. Our current data suggest that GMF is a critical risk factor for PD and suggest that it could be explored as a potential therapeutic target to inhibit PD progression.

Analysis of the interactions between GMF and Arp2/3 complex in two binding sites by molecular dynamics simulation

The Arp2/3 complex plays a key role in nucleating actin filaments branching. The glia maturation factor (GMF) competes with activators for interacting with the Arp2/3 complex and initiates the debranching of actin filaments. In this study, we performed a comparative analysis of interactions between GMF and the Arp2/3 complex and identified new amino acid residues involved in GMF binding to the Arp2/3 complex at two separate sites, revealed by X-ray and single particle EM techniques. Using molecular dynamics simulations we demonstrated the quantitative and qualitative changes in hydrogen bonds upon binding with GMF. We identified the specific amino acid residues in GMF and Arp2/3 complex that stabilize the interactions and estimated the mean force profile for the GMF using umbrella sampling. Phylogenetic and structural analyses of the recently defined GMF binding site on the Arp3 subunit indicate a new mechanism for Arp2/3 complex inactivation that involves interactions between the Arp2/3 complex and GMF at two binding sites.

Co-Expression of Glia Maturation Factor and Apolipoprotein E4 in Alzheimer's Disease Brain.

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for Alzheimer's disease (AD). The E4 allele of ApoE plays a crucial role in the inflammatory and neurodegenerative processes associated with AD. This is evident from the multiple effects of the ApoE isoforms in amyloid-β (Aβ) aggregation. Glia maturation factor (GMF) is a brain-specific neuroinflammatory protein that we have previously demonstrated to be significantly upregulated in various regions of AD brains compared to non-AD control brains and that it induces neurodegeneration. We have previously reported that GMF is predominantly expressed in the reactive astrocytes surrounding amyloid plaques (APs) in AD brain. In the present study, using immunohistochemical and dual immunofluorescence staining, we show the expression and colocalization of GMF and ApoE4 in AD brains. Our results show that ApoE4 is present within the APs of AD brain. Further, we found that GMF and ApoE4 were strongly expressed and co-associated in APs and in the reactive astrocytes surrounding APs in AD. An increased expression of GMF in APs and neurofibrillary tangles in the AD brain, and the co-localization of GMF and ApoE4 in APs suggest that GMF and ApoE4 together should be contributing to the neuropathological changes associated with AD.

Glia Maturation Factor-Gamma Modulates M2 Activation of Macrophage Via Suppression of Mitochondria Function

Macrophages are innate immune cells of dynamic phenotype as they can acquire distinct phenotypes and biological functions depending on the microenvironment and the metabolic state. In response to various signals, macrophages can undergo M1 activation (stimulated by TLR ligand and interferon-gamma) or M2 activation (stimulated by IL-4/IL-13, immunocomplexes, TGFbeta, and IL-10). M1 and M2 macrophages have distinct metabolic phenotypes that differ from those of resting macrophages. M1 macrophages rely on aerobic glycolysis, while M2 macrophages use fatty acid oxidation to fuel mitochondrial oxidative phosphorylation. IL-4/IL13 induced M2 macrophages polarization has an enhanced mitochondrial oxygen-consumption rate, whereas M1 has a significantly decreased oxygen-consumption rate. This indicates that macrophage phenotypes can be regulated by the different aspects of cellular metabolism. Thus, the identification of molecules and mechanisms associated with the phenotypic switch of them provides a molecular basis for macrophage-centered therapeutic strategies. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG-knockdown macrophages enhanced IL-4/IL-13-stimulated M2 markers and promoted iron metabolism change to iron recycling states, but its underlying molecular mechanism remains unclear. In this study, to explore the possible role of GMFG on mitochondrial function, we measured mitochondrial respiration by determining the oxygen consumption rate (OCR) and glycolytic flux in GMFG-knockdown M0 (unactivated), M1 and M2 activated bone marrow-derived macrophages using an extracellular flux analyzer. We observed that decreased basal oxygen consumption (ORC), diminished maximal respiratory capacity (MRC) and reduced cellular ATP levels in GMFG-knockdown M0 and M2 macrophages compared with control siRNA transfected macrophages. Whereas, there were no detectable changes in the rate of extracellular acidification (ECAR) in GMFG-knockdown cells. Importantly, oxygen consumption in GMFG-knockdown cells were reduced to the same level of TGFbeta-stimulated cells, but not to the level of M1 macrophages, reflecting the loss of mitochondria fitness similar to TGFbeta-stimulated M2 macrophages. Additionally, we observed a moderately increased mitochondrial ROS levels in GMFG-knockdown cells which is similar to increased levels in M2 macrophages, but significantly lower than the increased level in M1 macrophages. We did not observe decreased mitochondrial membrane potential, but found moderately increased mitochondria mass in the GMFG-knockdown cells, which may be due to the accumulation of dysfunctional mitochondria. Moreover, we found that suppressed OCR in GMFG-knockdown cells were accompanied by suppressed mitochondrial biogenesis as shown by decreased protein expression levels of mitochondrial complex I, ISCU, MnSOD2 and CuZnSOD1 by immunoblotting analysis. We also found that GMFG expression was downregulated by treatment with hydrogen peroxide at a concentration of 0.25 mM, which accompanied with increased protein levels of TfR1 and IRP1 in macrophages. Increased mROS, TfR1 and decreased MnSOD2, CuZnSOD1 in GMFG-knockdown cells can be attenuated by treatment of 5 mM NAC and 0.01 mM MitoTEMPO for 1 hour. Furthermore, we found that the remarkable decreased protein level of Dynamin-related protein 1(Drp1), which promoted mitochondria fission, in GMFG-knockdown cells. Consistent with this, confocal microscope analysis exhibited predominantly punctate mitochondria in GMFG-knockdown cells compared with the elongated tubules mitochondria in control cells. Finally, immunoblotting analysis showed that GMFG-knockdown macrophages showed an increase of phosphorylated-CREB, which might be contributed to enhanced M2 markers in GMFG-knockdown cells. Together, these results suggest that downregulation of GMFG skewing macrophages toward a M2 phenotype through suppression of mitochondrial function, which associated with impaired mitochondria fission/fusion balance. Our results indicate that GMFG plays an important role in maintaining mitochondrial function and dynamic, which is necessary for macrophage reprogramming. DisclosuresNo relevant conflicts of interest to declare.

Co-Localization of Glia Maturation Factor with NLRP3 Inflammasome and Autophagosome Markers in Human Alzheimer's Disease Brain.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the presence of intracellular neurofibrillary tangles (NFTs) containing hyperphosphorylated tau, and the extracellular deposition of amyloid plaques (APs) with misfolded amyloid-β (Aβ) peptide. Glia maturation factor (GMF), a highly conserved pro-inflammatory protein, isolated and cloned in our laboratory, has been shown to activate glial cells leading to neuroinflammation and neurodegeneration in AD. We hypothesized that inflammatory reactions promoted by NLRP3-Caspase-1inflammasome pathway trigger dysfunction in autophagy and accumulation of Aβ which is amplified and regulated by GMF in AD. In this study, using immunohistochemical techniques we analyzed components of the NLRP3 inflammasome and autophagy- lysosomal markers in relation to Aβ, p-tau and GMF in human postmortem AD and age-matched non-AD brains. Tissue sections were prepared from the temporal cortex of human postmortem brains. Here, we demonstrate an increased expression of the inflammasome components NLRP3 and Caspase-1 and the products of inflammasome activation IL-1β and IL-18 along with GMF in the temporal cortex of AD brains. These inflammasome components and the pro-inflammatory cytokines co-localized with GMF in the vicinity and periphery of the APs and NFTs. Moreover, using double immunofluorescence staining, AD brain displayed an increase in the autophagy SQSTM1/p62 and LC3 positive vesicles and the lysosomal marker LAMP1 that also co-localized with GMF, Aβ and hyperphosphorylated p-tau. Our results indicate that in AD, the neuroinflammation promoted by the NLRP3 inflammasome may be amplified and regulated by GMF, which further impairs clearance of protein aggregates mediated by the auto-phagosomal pathway.