The ameliorative effects of topical gemifloxacin alone or in combination with clobetasol propionate on imiquimod-induced model of psoriasis in mice.

Psoriasis is a lifelong immune-driven skin condition characterized by excessive epidermal overgrowth and inflammatory cell infiltration. Gemifloxacin is a fourth-generation fluoroquinolone with improved immunomodulatory and anti-inflammatory properties that are believed to possess an attractive role in psoriasis via suppressing the production of cytokines, chemokines, and eosinophil and neutrophil chemotaxis. The aim of this research is to investigate the ameliorative effects of prolonged topical gemifloxacin (GMF) alone and combined with clobetasol propionate (CLO) on an imiquimod (IMQ)-induced mouse model of psoriasis. Forty-eight Swiss albino mice were divided into six groups of eight. All groups except the negative controls got 62.5 mg of IMQ 5% topically for 8 days. Mice in the control group (controls) got Vaseline instead. Following the induction in the IMQ 5% group, mice in treatment groups CLO 0.05, GMF 1%, GMF 3%, and CLO + GMF obtained clobetasol propionate 0.05%, GMF 1% and 3%, and a combination of both, respectively, for an additional 8 days, rendering the experiment 16 days long. Our results revealed that gemifloxacin alleviated erythematous, thickened, and scaly psoriatic lesions and inhibited the tissue level of inflammatory cytokines, including interleukin (IL)-8, IL-17A, IL-23, tumor necrosis factor-α (TNF-α), and transforming growth factor-ß1 (TGF-ß1). The anti-inflammatory effect also occurred by hindering nuclear factor-kappa B (NF-κB) signaling and reversing histopathological problems. Gemifloxacin acts effectively in mitigating psoriasis-associated lesions and restricting NF-κB-mediated inflammation, recommending gemifloxacin as a promising adjuvant candidate for additional studies on the long-term treatment of autoimmune and autoinflammatory dermatoses like psoriasis.

Glia maturation factor beta deficiency protects against diabetic osteoporosis by suppressing osteoclast hyperactivity.

Excessive osteoclast activation, which depends on dramatic changes in actin dynamics, causes osteoporosis (OP). The molecular mechanism of osteoclast activation in OP related to type 1 diabetes (T1D) remains unclear. Glia maturation factor beta (GMFB) is considered a growth and differentiation factor for both glia and neurons. Here, we demonstrated that Gmfb deficiency effectively ameliorated the phenotype of T1D-OP in rats by inhibiting osteoclast hyperactivity. In vitro assays showed that GMFB participated in osteoclast activation rather than proliferation. Gmfb deficiency did not affect osteoclast sealing zone (SZ) formation but effectively decreased the SZ area by decreasing actin depolymerization. When GMFB was overexpressed in Gmfb-deficient osteoclasts, the size of the SZ area was enlarged in a dose-dependent manner. Moreover, decreased actin depolymerization led to a decrease in nuclear G-actin, which activated MKL1/SRF-dependent gene transcription. We found that pro-osteoclastogenic factors (Mmp9 and Mmp14) were downregulated, while anti-osteoclastogenic factors (Cftr and Fhl2) were upregulated in Gmfb KO osteoclasts. A GMFB inhibitor, DS-30, targeting the binding site of GMFB and Arp2/3, was obtained. Biocore analysis revealed a high affinity between DS-30 and GMFB in a dose-dependent manner. As expected, DS-30 strongly suppressed osteoclast hyperactivity in vivo and in vitro. In conclusion, our work identified a new therapeutic strategy for T1D-OP treatment. The discovery of GMFB inhibitors will contribute to translational research on T1D-OP.

Exploring the effect of glatiramer acetate on cerebral gray matter atrophy in multiple sclerosis.

BACKGROUND AND PURPOSE: Cerebral gray matter (GM) atrophy is a proposed measure of neuroprotection in multiple sclerosis (MS). Glatiramer acetate (GA) limits clinical relapses, MRI lesions, and whole brain atrophy in relapsing-remitting MS (RRMS). The effect of GA on GM atrophy remains unclear. We assessed GM atrophy in patients with RRMS starting GA therapy in comparison to a cohort of patients with clinically benign RRMS (BMS). DESIGN/METHODS: We studied 14 patients at GA start [age (mean ± SD) 44.2 ± 7.0 years, disease duration (DD) 7.2 ± 6.4 years, Expanded Disability Status Scale score (EDSS) (median,IQR) 1.0,2.0] and 6 patients with BMS [age 43.0 ± 6.1 years, DD 18.1 ± 8.4 years, EDSS 0.5,1.0]. Brain MRI was obtained at baseline and one year later (both groups) and two years later in all patients in the GA group except one who was lost to follow-up. Semi-automated algorithms assessed cerebral T2 hyperintense lesion volume (T2LV), white matter fraction (WMF), GM fraction (GMF), and brain parenchymal fraction (BPF). The exact Wilcoxon-Mann-Whitney test compared the groups. The Wilcoxon signed rank test assessed longitudinal changes within groups. RESULTS: During the first year, MRI changes did not differ significantly between groups (p > 0.15). Within the BMS group, WMF and BPF decreased during the first year (p = 0.03). Within the GA group, there was no significant change in MRI measures during each annual period (p > 0.05). Over two years, the GA group had a significant increase in T2LV and decrease in WMF (p < 0.05), while GMF and BPF remained stable (p > 0.05). MRI changes in brain volumes (GMF or WMF) in the first year in the GA group were not significantly different from those in the BMS group (p > 0.5). CONCLUSIONS: In this pilot study with a small sample size, patients with RRMS started on GA did not show significant GM or whole brain atrophy over 2 years, resembling MS patients with a clinically benign disease course.

Transcriptomics and Metabolomics of Reactive Oxygen Species Modulation in Near-Null Magnetic Field-Induced Arabidopsis thaliana.

The geomagnetic field (GMF) is a natural component of Earth's biosphere. GMF reduction to near-null values (NNMF) induces gene expression modulation that generates biomolecular, morphological, and developmental changes. Here, we evaluate the effect of NNMF on gene expression and reactive oxygen species (ROS) production in time-course experiments on Arabidopsis thaliana. Plants exposed to NNMF in a triaxial Helmholtz coils system were sampled from 10 min to 96 h to evaluate differentially expressed genes (DEGs) of oxidative stress responses by gene microarray. In 24-96 h developing stages, H2O2 and polyphenols were also analyzed from roots and shoots. A total of 194 DEGs involved in oxidative reactions were selected, many of which showed a fold change ≥±2 in at least one timing point. Heatmap clustering showed DEGs both between roots/shoots and among the different time points. NNMF induced a lower H2O2 than GMF, in agreement with the expression of ROS-related genes. Forty-four polyphenols were identified, the content of which progressively decreased during NNMF exposition time. The comparison between polyphenols content and DEGs showed overlapping patterns. These results indicate that GMF reduction induces metabolomic and transcriptomic modulation of ROS-scavenging enzymes and H2O2 production in A. thaliana, which is paralleled by the regulation of antioxidant polyphenols.

Glia maturation factor-ß induces ferroptosis by impairing chaperone-mediated autophagic degradation of ACSL4 in early diabetic retinopathy.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the world, and timely prevention and treatment are very important. Previously, we found that a neurodegenerative factor, Glia maturation factor-ß (GMFB), was upregulated in the vitreous at a very early stage of diabetes, which may play an important role in pathogenesis. Here, we found that in a high glucose environment, large amounts of GMFB protein can be secreted in the vitreous, which translocates the ATPase ATP6V1A from the lysosome, preventing its assembly and alkalinizing the lysosome in the retinal pigment epithelial (RPE) cells. ACSL4 protein can be recognized by HSC70, the receptor for chaperone-mediated autophagy, and finally digested in the lysosome. Abnormalities in the autophagy-lysosome degradation process lead to its accumulation, which catalyzes the production of lethal lipid species and finally induces ferroptosis in RPE cells. GMFB antibody, lysosome activator NKH477, CMA activator QX77, and ferroptosis inhibitor Liproxstatin-1 were all effective in preventing early diabetic retinopathy and maintaining normal visual function, which has powerful clinical application value. Our research broadens the understanding of the relationship between autophagy and ferroptosis and provides a new therapeutic target for the treatment of DR.

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 200 kD (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.

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-1ß), 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.

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.

Structural Basis of Arp2/3 Complex Inhibition by GMF, Coronin, and Arpin.

The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. To better understand Arp2/3 complex regulation, we used single-particle electron microscopy to compare the structures of Arp2/3 complex bound to three different inhibitory ligands: glia maturation factor (GMF), Coronin, and Arpin. Although the three inhibitors have distinct binding sites on Arp2/3 complex, they each induced an "open" nucleation-inactive conformation. Coronin promoted a standard (previously described) open conformation of Arp2/3 complex, with the N-terminal ß-propeller domain of Coronin positioned near the p35/ARPC2 subunit of Arp2/3 complex. GMF induced two distinct open conformations of Arp2/3 complex, which correlated with the two suggested binding sites for GMF. Furthermore, GMF synergized with Coronin in inhibiting actin nucleation by Arp2/3 complex. Arpin, which uses VCA-related acidic (A) motifs to interact with the Arp2/3 complex, induced the standard open conformation, and two new masses appeared at positions near Arp2 and Arp3. Furthermore, Arpin showed additive inhibitory effects on Arp2/3 complex with Coronin and GMF. Together, these data suggest that Arp2/3 complex conformation is highly polymorphic and that its activities can be controlled combinatorially by different inhibitory ligands.

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-1ß, ß-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.

Glia maturation factor modulates beta-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 Alzheimer's 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.

Identification and isolation of a full-length clone of mouse GMFB (Gmfb), a putative intracellular kinase regulator, differentially expressed in telencephalon.

We identified new transcribed sequences, using a differential display paradigm to select genes expressed in proliferating neuroblasts from mouse telencephalon at 10 days of embryonic development. In this systematic search, we isolated a 361-bp partial 3' untranslated region (3' UTR) homologous to the 3' UTR of the human gene encoding a putative intracellular kinase regulator, glia maturation factor beta (GMFB). We cloned a full-length, 4,311-bp mouse cDNA containing a 270-bp 5' UTR, a 3,615-bp 3' UTR, and an open reading frame of 426 nucleotides encoding a putative 142 amino-acid protein, identical to human GMFB, with the exception of two amino acids. This 4.3-kb transcript is present in a variety of adult tissues and is developmentally regulated as shown by Northern blot analysis. Differential expression in telencephalon was demonstrated by quantification of radioactive relative RT-PCR and confirmed by in situ hybridization. The isolation of this full-length clone of mouse Gmfb should facilitate investigation of the intracellular mechanisms involved in the development of telencephalon.

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-kappaB. The activations of CREB and NF-kappaB 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-kappaB 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-kappaB activation, resulting in the induction and secretion of the neurotrophins.