Induction of STAP-1 promotes neurotoxic activation of microglia.

Activated microglia contribute to neurodegenerative processes in the brain and the retina. Via DNA-microarray analysis, we have previously identified up-regulation of several immune-related genes in the dystrophic retina of retinoschisin-deficient (Rs1h(-/Y)) mice. Here we report a strong overexpression of transcripts for the signal-transducing adaptor protein-1 (STAP-1) in isolated Rs1h(-/Y) microglia. Furthermore, STAP-1 expression was induced in activated bone marrow-derived macrophages as well as LPS-, interferon-gamma-, and CpG-stimulated myeloid cell lines. Ectopic expression of STAP-1 in BV-2 microglia changed the morphology and cytoskeletal organization of the cells and transformed ramified cells to an activated state. STAP-1 overexpression also leads to an interaction with the M-CSF receptor/c-Fms diminishing its ligand-dependent phosphorylation. Finally, STAP-1 expressing cells showed strongly reduced migration with increased cytotoxicity against 661W photoreceptor like cells. Taken together, our study implicates a previously unknown role of STAP-1 in pro-inflammatory microglia activation potentially contributing to neuronal apoptosis and degeneration.

Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype.

A disaccharide degradation product of chondrotin sulfate proteoglycan-disaccharide (CSPG-DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome-wide microarray analysis was used to identify specific gene expression profiles of CSPG-DS-stimulated BV-2 microglia-like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN-gamma-stimulated primary microglia using real-time quantitative RT-PCR validation. Importantly, a unique transcriptional phenotype with anti-inflammatory and IFN-gamma counter-regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG-DS-stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG-DS-treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN-gamma-induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG-DS-activated microglia. These might be starting points to explore the in vivo role of CSPG-DS as a bioactive microglia regulator and its potential, therapeutic application in immune-related, neurodegenerative disorders.

Inhibition of fat cell differentiation in 3T3-L1 pre-adipocytes by all-trans retinoic acid: Integrative analysis of transcriptomic and phenotypic data.

The process of adipogenesis is controlled in a highly orchestrated manner, including transcriptional and post-transcriptional events. In developing 3T3-L1 pre-adipocytes, this program can be interrupted by all-trans retinoic acid (ATRA). To examine this inhibiting impact by ATRA, we generated large-scale transcriptomic data on the microRNA and mRNA level. Non-coding RNAs such as microRNAs represent a field in RNA turnover, which is very important for understanding the regulation of mRNA gene expression. High throughput mRNA and microRNA expression profiling was performed using mRNA hybridisation microarray technology and multiplexed expression assay for microRNA quantification. After quantitative measurements we merged expression data sets, integrated the results and analysed the molecular regulation of in vitro adipogenesis. For this purpose, we applied local enrichment analysis on the integrative microRNA-mRNA network determined by a linear regression approach. This approach includes the target predictions of TargetScan Mouse 5.2 and 23 pre-selected, significantly regulated microRNAs as well as Affymetrix microarray mRNA data. We found that the cellular lipid metabolism is negatively affected by ATRA. Furthermore, we were able to show that microRNA 27a and/or microRNA 96 are important regulators of gap junction signalling, the rearrangement of the actin cytoskeleton as well as the citric acid cycle, which represent the most affected pathways with regard to inhibitory effects of ATRA in 3T3-L1 preadipocytes. In conclusion, the experimental workflow and the integrative microRNA-mRNA data analysis shown in this study represent a possibility for illustrating interactions in highly orchestrated biological processes. Further the applied global microRNA-mRNA interaction network may also be used for the pre-selection of potential new biomarkers with regard to obesity or for the identification of new pharmaceutical targets.

Posttranscriptional regulatory networks: from expression profiling to integrative analysis of mRNA and microRNA data.

Protein coding RNAs are posttranscriptionally regulated by microRNAs, a class of small noncoding RNAs. Insights in messenger RNA (mRNA) and microRNA (miRNA) regulatory interactions facilitate the understanding of fine-tuning of gene expression and might allow better estimation of protein synthesis. However, in silico predictions of mRNA-microRNA interactions do not take into account the specific transcriptomic status of the biological system and are biased by false positives. One possible solution to predict rather reliable mRNA-miRNA relations in the specific biological context is to integrate real mRNA and miRNA transcriptomic data as well as in silico target predictions. This chapter addresses the workflow and methods one can apply for expression profiling and the integrative analysis of mRNA and miRNA data, as well as how to analyze and interpret results, and how to build up models of posttranscriptional regulatory networks.