Thomas Stempfl

A new fate mapping system reveals context-dependent random or clonal expansion of microglia

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Validation of microarray‐based resequencing of 93 worldwide mitochondrial genomes

The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip® Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of ≥99.98% for the MitoChip. The good performance was achieved by using in‐house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of ≥4 C bases or a sequence variant within 12 bases up‐ or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing. Hum Mutat 0,1–8, 2008.

Differential gene expression involved in oxidative stress response caused by triethylene glycol dimethacrylate.

Triethylene glycol dimethacrylate (TEGDMA) is a comonomer that is released from dental resin-based materials into hydrophilic solvents. The compound reduces cell vitality, and causes genotoxicity in mammalian cells in vitro. Here, we used gene expression profiling, combined with pathway analysis tools, to identify the molecular events associated with TEGDMA cytotoxicity in human fibroblasts using Affymetrix HG-U133A 2.0 GeneChip arrays. Increased ROS production and a cell cycle delay caused by 3mm TEGDMA after a 6h exposure were related to a cell response at the transcriptional level. The predominant biological processes associated with the genes that were differentially expressed in untreated and treated cell cultures included oxidative stress, cellular growth, proliferation and morphology, cell death, gene expression as well as DNA replication and repair. The most significantly upregulated genes were GEM (17-fold), KLHL24, DDIT4, TGIF, DUSP5 and ATF3, which are all related to the regulation of the cell structure, stress response, and cell proliferation. TXNIP was the most downregulated transcript (five-fold), whose gene product regulates the cellular redox balance. The downregulation of NRG1, ASPM, FBXO5, and PLK2 is linked to the regulation of cell proliferation and cell structure. The underlying mechanisms of the up- and downregulation of genes seem to be activated by the production of ROS, and the related regulation of the cellular redox balance disturbed in the presence of TEGDMA appears to be of the utmost importance. The coordinated induction of genes coding for oxidative stress response and antioxidant proteins is a critical mechanism of protection against TEGDMA-induced cell damage.

RBP2-H1/JARID1B is a transcriptional regulator with a tumor suppressive potential in melanoma cells

The RBP2‐H1/JARID1B nuclear protein belongs to the ARID family of DNA‐binding proteins and is a potential tumor suppressor that is lost during melanoma development. As we have recently shown, one physiological function of RBP2‐H1/JARID1B is to exert cell cycle control via maintenance of active retinoblastoma protein. We now add new evidence that RBP2‐H1/JARID1B can also directly regulate gene transcription in a reporter assay system, either alone or as part of a multimolecular complex together with the developmental transcription factors FOXG1b and PAX9. In melanoma cells, chromatin immunoprecipitation combined with promoter chip analysis (ChIP‐on‐chip) suggests a direct binding of re‐expressed RBP2‐H1/JARID1B to a multitude of human regulatory chromosomal elements (promoters, enhancers and introns). Among those, a set of 23 genes, including the melanoma relevant genes CDK6 and JAG‐1 could be confirmed by cDNA microarray analyses to be differentially expressed after RBP2‐H1/JARID1B re‐expression. In contrast, in nonmelanoma HEK 293 cells, RBP2‐H1/JARID1B overexpression only evokes a minor transcriptional response in cDNA microarray analyses. Because the transcriptional regulation in melanoma cells is accompanied by an inhibition of proliferation, an increase in caspase activity and a partial cell cycle arrest in G1/0, our data support an anti‐tumorigenic role of RBP2‐H1/JARID1B in melanocytic cells.

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‐γ‐stimulated primary microglia using real‐time quantitative RT‐PCR validation. Importantly, a unique transcriptional phenotype with anti‐inflammatory and IFN‐γ 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‐γ‐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.

Identification of new genes associated with melanoma.

Purpose:  Repeated failures in melanoma therapy made clear that the molecular mechanisms leading to melanoma are still poorly understood. In this study, we aim to provide a more comprehensive understanding of the transcriptional profiles and signalling pathways associated with melanoma.

Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis

BackgroundExperimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female Dark Agouti (DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats.MethodsUntreated DA rats and DA rats immunised with MOG protein were sacrificed at defined time points. Total RNA was isolated from spinal cord tissue and used for hybridization of Affymetrix rat genome arrays RG U34 A-C. Selected expression values were confirmed by RealTime PCR.Adult neural stem cells were incubated with recombinant secretory leukocyte protease inhibitor (SLPI). Proliferation was assessed by BrdU incorporation, cyclin D1 and HES1 expression by RealTime PCR, cell differentiation by immunofluorescence analysis and IkappaBalpha degradation by Western blot.ResultsAmong approximately 26,000 transcripts studied more than 1,100 were differentially regulated. Focussing on functional themes, we noticed a sustained downregulation of most of the transcripts of the cholesterol biosynthesis pathway. Furthermore, we found new candidate genes possibly contributing to regenerative processes in the spinal cord. Twelve transcripts were solely upregulated in the recovery phase, including genes not previously associated with repair processes. Expression of SLPI was upregulated more than hundredfold during EAE attack. Using immunohistochemistry, SLPI was identified in macrophages, activated microglia, neuronal cells and astrocytes. Incubation of adult neural stem cells (NSC) with recombinant SLPI resulted in an increase of cell proliferation and of differentiation towards oligodendrocytes. These processes were paralleled by an upregulation of the cell-cycle promotor cyclin D1 and a suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of IkappaBalpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation.ConclusionWe identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

Bacteria-Triggered Systemic Immunity in Barley Is Associated with WRKY and ETHYLENE RESPONSIVE FACTORs But Not with Salicylic Acid

Infection of barley with bacteria induces systemic resistance against a secondary Xanthomonas translucens infection and does not appear to be associated with salicylic acid. Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.

FGFR3 mutation affects cell growth, apoptosis and attachment in keratinocytes

FGFR3 mutations have recently been identified in several benign epidermal skin lesions such as seborrheic keratosis, epidermal nevus and solar lentigo. The functional consequences of these mutations in human skin are as yet unknown. In this study we analyzed the functional effects of the most common FGFR3 mutation in benign skin tumors, the R248C FGFR3 hotspot mutation, in human HaCaT keratinocytes. The cells were stably transduced with either the R248C or wildtype FGFR3 IIIb cDNA using a retroviral vector system. FGFR3 mutant and wildtype cells showed similar growth rates at subconfluence. However, at confluence FGFR3 mutant keratinocytes revealed a significantly higher cell number than wildtype cells. Furthermore, FGFR3 mutant cells showed significantly lower levels of apoptosis and decreased attachment to fibronectin compared with FGFR3 wildtype cells. Expression of mutant FGFR3 did not alter migration and senescence. Microarray analysis revealed only a few differentially expressed genes between FGFR3 mutant and wildtype keratinocytes. Enhanced phosphorylation of ERK1/2 was observed in confluent R248C mutant HaCaT cells compared with wildtype keratinocytes. Our results suggest that an increased cell number at confluence along with a decreased apoptosis may contribute to the development of acanthotic tumors in FGFR3 mutant skin in vivo.

An integrated workflow for analysis of ChIP-chip data

Although ChIP-chip is a powerful tool for genome-wide discovery of transcription factor target genes, the steps involving raw data analysis, identification of promoters, and correlation with binding sites are still laborious processes. Therefore, we report an integrated workflow for the analysis of promoter tiling arrays with the Genomatix ChipInspector system. We compare this tool with open-source software packages to identify PU.1 regulated genes in mouse macrophages. Our results suggest that ChipInspector data analysis, comparative genomics for binding site prediction, and pathway/network modeling significantly facilitate and enhance whole-genome promoter profiling to reveal in vivo sites of transcription factor-DNA interactions.