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

ABSTRACT: 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.

SUBMITTER: Raikwar SP

PROVIDER: S-EPMC6344368 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Raikwar Sudhanshu P SP Thangavel Ramasamy R Dubova Iuliia I Selvakumar Govindhasamy Pushpavathi GP Ahmed Mohammad Ejaz ME Kempuraj Duraisamy D Zaheer Smita A SA Iyer Shankar S SS Zaheer Asgar A

Molecular neurobiology 20180427 1

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 ...[more]

PMID: 29704201

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Project description:Kidney renal clear cell carcinoma (KIRC) has the highest mortality rate and potential for invasion among renal cancers. The diagnosis and treatment of KIRC are becoming challenging because of its diverse pathogenic mechanisms. Glia (GMFB) is a highly conserved growth and differentiation factor for glia cells and neurons, and it is closely associated with neurodegenerative diseases. However, its role in KIRC remains unknown. The present study integrated bioinformatics approaches with suitable meta-analyses to determine the position of GMFB in KIRC. There was a significant decrease in Gmfb expression in KIRC kidneys compared with normal controls. Gmfb expression was negatively associated with pathologic stage, T and M stages, and histologic grade. Univariate and multivariate analyses showed that elevated Gmfb expression was an independent factor for a favorable prognosis. Furthermore, the nomogram verified that Gmfb is a low-risk factor for KIRC. Knockdown of Gmfb in Caki-2 cells increased viability and decreased p21 and p27 levels. Overexpression of Gmfb inhibited Caki-2 cell proliferation, migration, and invasion and decreased mitochondrial membrane potential. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses considering Gmfb co-expressed differentially expressed genes (DEGs) showed that collecting duct acid secretion and mineral absorption ranked were the most important upregulated and downregulated DEGs, respectively. The upregulated hub genes for DEGs were mainly involved in nucleosome assembly, nucleosome organization, and chromatin assembly, and the downregulated hub genes were primarily associated with keratinization. The ratio of tumor-infiltrating immune cells in KIRC tissues was evaluated using CIBERSORTx. The results showed that the Gmfb expression was significantly positively correlated with macrophage M2 cells and mast resting cell infiltration levels and negatively correlated with T follicular helper, T regulatory, and B plasma cell infiltration levels. The former cell types were associated with a beneficial outcome, while the latter had a worse outcome in patients with KIRC. In summary, this study identified GMFB as a novel independent biomarker and therapeutic target for KIRC, and it provides a helpful and distinct individualized treatment strategy for KIRC with a combination of molecular targets and tumor microenvironment.

Project description:The molecular mechanism mediating degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) is not yet fully understood. Previously, we have shown the contribution of glia maturation factor (GMF), a proinflammatory protein in dopaminergic neurodegeneration mediated by activation of mast cells (MCs). In this study, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal neurodegeneration and astro-glial activations were determined by western blot and immunofluorescence techniques in wild type (WT) mice, MC-deficient (MC-KO) mice and GMF-deficient (GMF-KO) mice, with or without MC reconstitution before MPTP administration. We show that GMF-KO in the MCs reduces the synergistic effects of MC and Calpain1 (calcium-activated cysteine protease enzyme)-dependent dopaminergic neuronal loss that reduces motor behavioral impairments in MPTP-treated mouse. Administration of MPTP increase in calpain-mediated proteolysis in nigral dopaminergic neurons further resulting in motor decline in mice. We found that MPTP administered WT mice exhibits oxidative stress due to significant increases in the levels of malondialdehyde, superoxide dismutase and reduction in the levels of reduced glutathione and glutathione peroxidase activity as compared with both MC-KO and GMF-KO mice. The number of TH-positive neurons in the ventral tegmental area, substantia nigra and the fibers in the striatum were significantly reduced while granulocyte macrophage colony-stimulating factor (GM-CSF), MC-Tryptase, GFAP, IBA1, Calpain1 and intracellular adhesion molecule 1 expression were significantly increased in WT mice. Similarly, tyrosine hydroxylase, dopamine transporters and vesicular monoamine transporters 2 proteins expression were significantly reduced in the SN of MPTP treated WT mice. The motor behavior as analyzed by rotarod and hang test was significantly reduced in WT mice as compared with both the MC-KO and GMF-KO mice. We conclude that GMF-dependent MC activation enhances the detrimental effect of astro-glial activation-mediated oxidative stress and neuroinflammation in the midbrain, and its inhibition may slowdown the progression of PD.

Project description:BackgroundGlia maturation factor-γ (GMFG) regulates actin cytoskeletal organization and promotes the invasion of cancer cells. However, its expression pattern and molecular function in gliomas have not been clearly defined.MethodsIn this study, public datasets comprising 2,518 gliomas samples were used to explore GMFG expression and its correlation with malignancy in gliomas. Immunohistochemistry (IHC) staining was performed to determine the expression of GMFG in gliomas using an in-house cohort that contained 120 gliomas samples. Gene ontology enrichment analysis was conducted using the DAVID tool. The correlation between GMFG expression and immune cell infiltration was evaluated using TIMER, Tumor Immune Single-Cell Hub (TISCH) database, and IHC staining assays. The Kaplan-Meier analysis was performed to determine the prognostic role of GMFG and its association with temozolomide (TMZ) response in gliomas.ResultsThe GMFG expression was higher in gliomas compared with non-tumor brain tissues both in public datasets and in-house cohort. High expression of GMFG was significantly associated with WHO grade IV, IDH 1/2 wild-type, and mesenchymal (ME) subtypes. Bioinformatic prediction and IHC analysis revealed that GMFG expression obviously correlated with the macrophage marker CD163 in gliomas. Moreover, both lower grade glioma (LGG) and glioblastoma multiforme (GBM) patients with high GMFG expression had shorter overall survival than those with low GMFG expression. These results indicate that GMFG may be a therapeutic target for the treatment of such patients. Patients with low GMFG expression who received chemotherapy had a longer survival time than those with high GMFG expression. For patients who received ion radiotherapy (IR) only, the GMFG expression level had no effect on the overall survival neither in CGGA and TCGA datasets.ConclusionThe GMFG is a novel prognostic biomarker for patients with both LGG and GBM. Increased GMFG expression is associated with tumor-associated macrophages (TAMs) infiltration and with a bad response to TMZ treatment.

Project description:BackgroundGlia maturation factor-γ (GMFG) is reported to inhibit the actin nucleation through binding to the actin-related protein-2/3 complex (Arp2/3). Considering the main function of GMFG in actin remodeling, which is vital for immune response, angiogenesis, cell division and motility, GMFG is supposed to have important roles in tumor development, while up to now, only two studies described the role of GMFG in cancers. By investigating the clinical values of GMFG using The Cancer Genome Atlas (TCGA) data and the functional mechanisms of GMFG through analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, this study was aimed to better understand the impact of GMFG in pan-cancers and to draw more attentions for the future research of GMFG.MethodsRNA-seq and clinical data of cancer patients were collected from TCGA and analyzed by the Kaplan-Meier methods. GO and KEGG analyses were conducted using the online tools from the Database for Annotation, Visualization and Integrated Discovery (DAVID).ResultsCompared to the corresponding normal samples, GMFG was significantly upregulated in glioblastoma (GBM), kidney clear cell carcinoma (KIRC), lower grade glioma (LGG), acute myeloid leukemia (LAML), and pancreatic cancer (PAAD), testicular cancer (TGCT), but was downregulated in kidney chromophobe (KICH), lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) (P < 0.05 for all). High expression of GMFG predicted worse OS in GBM (HR = 1.5, P = 0.017), LGG (HR = 2.2, P < 0.001), LUSC (HR = 1.4, P = 0.022) and ocular melanomas (UVM) (HR = 7, P < 0.001), as well as worse DFS in LGG (HR = 1.8, P < 0.001) and prostate cancer (PRAD) (HR = 1.9, P = 0.004). In contrast, high expression of GMFG was associated with better OS in skin cutaneous melanoma (SKCM) (HR = 0.59, P < 0.001) and thymoma (THYM) (HR = 0.098, P = 0.031), as well as better DFS in bile duct cancer (CHOL) (HR = 0.2, P = 0.003). GMFG was mainly involved in the immune response, protein binding and cytokine-cytokine receptor interaction pathways, and was positively associated with multiple immunomodulators in most cancers.ConclusionOur study preliminarily identified that GMFG may cause different survivals for different cancers through modulating tumor progression, immune response status and tissue-specific tumor microenvironment (TME).

Project description:Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane.

Project description:Medulloblastoma is a malignant pediatric brain tumor. Current treatment following patient stratification into standard and high-risk groups using clinical features has improved survival. However, a subset of patients with standard risk features have unanticipated aggressive disease, underscoring the need for a better understanding of tumor biology and the development of novel treatments. Poor differentiation, a hallmark of medulloblastomas is associated with elevated expression levels of the repressor of neuronal differentiation called repressor element 1-silencing transcription factor (REST). Here, we assessed whether elevated REST expression levels had prognostic significance and whether its pharmacologic manipulation would promote neurogenesis and block tumor cell growth. REST levels in patient tumors were measured by immunohistochemistry and stratified into negative, low/moderate- (+/++/+++), and high-REST (+++++) groups. Kaplan-Meier curves revealed that patients with high-REST tumors had worse overall and event-free survival compared with patients with REST-negative or REST-low tumors. Because histone deacetylases (HDAC) are required for REST-dependent repression of neurogenesis, we evaluated a panel of HDAC inhibitors (HDACI) for their effects on growth and differentiation of established and primary REST-positive cell lines. MS-275, trichostatin-A (TSA), valproic acid (VPA), and suberoylanilide hydroxamic acid (SAHA) upregulated expression of the REST-target neuronal differentiation gene, Syn1, suggesting a potential effect of these HDACIs on REST function. Interestingly, VPA and TSA substantially increased histone acetylation at the REST promoter and activated its transcription, whereas SAHA unexpectedly promoted its proteasomal degradation. A REST-dependent decrease in cell growth was also observed following SAHA treatment. Thus, our studies suggest that HDACIs may have therapeutic potential for patients with REST-positive tumors. This warrants further investigation.

Dataset Information

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

Publications

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

Similar Datasets

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