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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 29, 2005 as doi:10.1096/fj.05-4873fje. |
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,,1
* Department of Psychiatry & Behavioral Neurosciences, and
Center for Molecular Medicine and Genetics, Wayne State University School of Medicine; and
John D. Dingell VA Medical Center, Detroit, Michigan, USA
Correspondence: John D. Dingell VA Medical Center, R&D Service (11R), 4646 John R, Detroit, MI 48201, USA. E-mail: donald.kuhn{at}wayne.edu
SPECIFIC AIMS
Microglia are the primary resident antigen-presenting cells within the central nervous system (CNS) and are thought to serve immune-like functions in protecting the brain against injury and invading pathogens. By mechanisms not yet fully understood, microglia can become activated in the early phases of neurodegenerative conditions and in response to certain neurotoxic drugs of abuse. Once activated, microglia produce and secrete reactive species, cytokines, and chemokines known to cause or enhance damage to neurons. A better understanding of the gene expression response of microglia to activation would provide important mechanistic information on how these cells participate in the process of neuronal damage. The specific aims of this study were to 1) elaborate the transcriptome of resting microglia; 2) activate cultured microglial cells with lipopolysaccharide (LPS), the neurotoxic HIV protein TAT, or dopamine (DA) quinone (DAQ), each of which has been linked causally to damage of the DA neuronal system via mechanisms that involve microglia; 3) characterize the gene expression response of microglia to activation by LPS, TAT, and DAQ using microarray analysis, and delineate genes whose expression is significantly increased or decreased in common by all three activators; and 4) define a functionally related network of microglial genes that participate in activation caused by LPS, TAT, and DAQ.
PRINCIPAL FINDINGS
1. Microglial transcriptome
Untreated mouse microglial BV-2 cells express 9882 annotated genes. These genes were stratified according to gene ontology (GO) analysis in GeneSpring®. As expected, all major cellular biological processes (BP) and Molecular Functions (MF) were populated by numerous microglial genes. Prominent BP classes included cellular communication (2227 genes), cell growth and/or maintenance (5532 genes), cellular metabolism (4066 genes) and development (2565 genes). It was interesting that BPs relating to host-pathogen interaction and responses to endogenous/exogenous stimuli included 1126 genes, an index of the immune-like functions subserved by microglia. Among MF classes in GO, binding (calcium, nucleotide, lipid and transcription factors) was predominant with 4241 genes, followed closely by catalytic activity (e.g., hydrolases, peptidases, phosphatases, and kinases) with 3774 genes. Defense immunity protein activity (552 genes) was also a highly represented MF class.
2. Gene expression profile of activated microglia
Microglial cells were activated by treatment with LPS, DAQ, or TAT and microarray analysis revealed that a total of 3127 genes were differentially regulated (increased or decreased in expression by 
2-fold) across all three treatments. The effects of LPS and TAT on microglial gene expression were similar, sharing 400 genes with increased expression and 220 genes that decreased in expression. DAQ was most distinct in its effects, increasing the expression of 354 genes and decreasing expression of 1557 genes. DAQ also caused expression changes in far more genes overall (564 increased, 1798 decreased) than either LPS (733 increased, 554 decreased) or TAT (413 increased, 255 decreased).
3. Microglial genes increased in expression by all activators
Gene expression profiles showed numerous similarities across all treatment conditions. A total of 116 genes were up-regulated by a factor of at least 2-fold by DAQ, TAT, and LPS. The changes in expression were statistically significant for 75 of these genes (P<0.05, ANOVA). Of the genes up-regulated by activation, 55 were not scored as present in the resting microglial transcriptome, but were significantly induced upon activation by all three activators (i.e., now scored present). Gene expression results from microarray analysis were used as input to the software program PathwayAssist, which uses KEGG, DIP, and BIND databases as well as natural language scans of PubMed to define connectivity among genes in order to delineate a functionally related network. A total of 42 of 88 nodes (representing 116 up-regulated genes) could be linked together in a direct manner, whereas 47 did not show apparent connectivity to other microglial genes. The up-regulated genes with highest connectivity included colony-stimulating factors 2/3, Icam1, interleukins 1
and 1ß, interleukin 6, Nos2, Ptgs2, and cyclin-dependent kinase inhibitor 1A. In addition, NF
B, Serpine 1 (serine or cysteine proteinase inhibitor), TNF receptors 1b and 5, Socs3, and chemokines Ccl4, Ccl5, Cxcl2, and Cxcl10 also showed substantial connectivity. It was also interesting that Mmp13, growth arrest, and DNA damage (Gadd) factors 45a and 45b, and GTP cyclohydrolase (Gch1; initial enzyme in the biosynthesis of tetrahydrobiopterin, the essential cofactor for Nos2) were significantly up-regulated under all activating conditions, with each sharing extensive connectivity with other microglial genes. Ptgs2 (i.e., Cox-2) was also linked broadly to numerous other members of this network including the nuclear transcription factor NFB, Il1, Il1ß, and Nos2. Taken together, these data reveal the highly coordinated gene expression response of microglia to activation that focuses on cytokines, chemokines, immune modulation, and inflammatory processes. Some novel genes that have not been linked heretofore to microglial activation were also significantly increased in expression by all activators. These genes (n=46, representing 55 gene transcripts) fall into five general functional categories including cell growth, differentiation, and survival (e.g., Axud1, Trim13, Fzd5, and Idb3), innate immunity/inflammatory responses (e.g., Myd116, Treml4, Irg1, and Sdc4), membrane trafficking (e.g., Rab11, Rab20, and Stx11), solute carriers (e.g., Slc7a11, Slc15a3, and Slc11a2), and chemokines (e.g., Ccrl2 and Cxcl10).
4. Microglial genes decreased in expression by all activators
A total of 94 genes were decreased in expression across all microglial activating conditions, and the changes in expression were statistically significant for 37 of these genes (P<0.05, ANOVA). Surprisingly, when this list of down-regulated genes was submitted to PathwayAssist to construct a potential gene network, only two could be linked (Hdac9 and Mef2c). Accordingly, the remaining 67 gene products have not been immediately linked to microglial activation and could be considered novel candidates for participation in the activation process. A closer manual examination of these down-regulated genes revealed interesting results. These genes could be classified into five general GO categories, including complement pathway/immune function (e.g., C1qr1, Il1rl1, and Nptxr), lipids, and membrane function (Crot, Depdc6, and Zdhhc14 domain), cell proliferation, migration, or adhesion (e.g., Dusp6/7, Fhod1, Itga6, and Sipa1), and membrane receptors and signaling (e.g., P2 purinergic receptor, mu opioid receptor, and Sestrin). Genes whose expression was most reduced by all activating conditions included Lyl1 (reduced 14-fold), Dusp6 (reduced 9-fold), Dusp7 (reduced 8-fold), P2ry1 receptor (reduced 5-fold), mu opioid receptor (Oprm1; reduced 43-fold), and Rgs2 (reduced 15-fold). Taken together, down-regulation of these genes would be consistent with conditions that are permissive of microglial migration and motility, lowered adhesion to matrix molecules, lessened phagocytosis, and reduction in the expression of receptors that would oppose chemotaxis and inflammation.
CONCLUSIONS AND SIGNIFICANCE
The present microarray analysis of the microglial gene expression response to activation reveals a broad panel of biomarker genes that could help define the signaling pathways used by microglia to mediate damage to DA neurons. In view of the role of microglia in mediating damage to the DA neuronal system in numerous clinically significant neurodegenerative conditions, a delineation of the gene expression profile of activated microglia could provide insight into the mechanisms by which these cells participate in the process of neuronal damage. A generalized scheme showing the microglial response to activation is presented in Fig. 1
. A panel of 116 common genes was increased in expression by all three activating conditions. When analyzed by PathwayAssist, 
50% of the up-regulated genes were found to be part of a dense network, with multiple connectivity among all genes. Taken together, these up-regulated genes could act in concert to support the neuronal-damaging phenotype of activated microglia. For example, increased production of cytokines and chemokines would drive inflammatory processes and could act in a feed-forward manner to enhance and perpetuate microglial activation. The increase in Gch1 would synthesize increased levels of tetrahydrobiopterin, an essential cofactor for Nos2, thereby allowing heightened or sustained production of nitric oxide. Similarly, matrix metalloproteinases degrade proteins of the extracellular matrix and enable migration of numerous cell types, including endothelial and microglial cells. Heightened production of nitric oxide is now known to disrupt the association of Mmp13 with caveolae, allowing increased extracellular Mmp13 abundance and increased collagen breakdown in the process of endothelial cell migration, a process that applies to microglia as well. Mining of various databases reveals that these genes, including Myd116, Treml4, Ccrl2, and Cxcl10 should perhaps be added to those listed above as participants in the neuronal-damaging phenotype of microglia because of their roles relating to mediation of CNS inflammation and innate immunity. The increase in expression of several genes for solute carriers is highly relevant because Slc7a11 encodes the cystine-glutamate antiporter through which microglia secrete the excitotoxic amino acid glutamate.
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Those genes whose expression was reduced in activated microglia but were not immediately linked by PathwayAssist can be assembled into a network that shifts the microglial phenotype toward neuronal destructive and away from neuronal protective. For example, C1qr1 is synthesized in microglia specifically within the CNS, and its heightened expression supports enhanced phagocytosis. Similarly, the neuronal pentraxin receptor (Nptxr) has been associated with clearance of debris from the synaptic space. Reductions in the expression of C1qr1 and Nptxr could diminish beneficial properties of microglia by lowering phagocytosis. Integrin 6 (Itga6) is an adhesion molecule that anchors cells to the extracellular matrix and contributes to tissue integrity; reductions in its expression could facilitate microglial transmigration and motility. Sestrin 1 (Sesn1) is a cysteine sulfinyl reductase that modulates peroxide signaling and antioxidant defenses, and reductions in its expression would lower the antioxidant firewall against over-oxidation. Microglia are known to express both purinergic (P2ry1) and µ-opioid receptors (Oprm1), both of which inhibit or limit microglial cell chemotaxis and are reduced in expression in activated microglia. Therefore, reductions in expression of "neuronally protective" receptors on microglia would limit chemotactic and anti-inflammatory capability, and act in concert with those up-regulated microglial genes that mediate inflammatory effects to amplify neuronal damage.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4873fje;
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