David von Schack

Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo

Neuropilin-1 is identified as a surface marker to distinguish different Foxp3+ T reg cell subsets under homeostatic conditions.

Dynamic Changes in the MicroRNA Expression Profile Reveal Multiple Regulatory Mechanisms in the Spinal Nerve Ligation Model of Neuropathic Pain

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3′-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca2+ channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca2+ channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca2+-induced cell death by modulating Ca2+ channels and promote neurite outgrowth via FKBP52 binding.

Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Tregs and CD4+FOXP3+Helios− T cells, followed by comparison with CD4+FOXP3−Helios− T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4+CD25+CD127lo/− T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226+TIGIT− population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.

Tofacitinib attenuates pathologic immune pathways in patients with psoriasis: A randomized phase 2 study

BACKGROUND Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. OBJECTIVE We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. METHODS Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. RESULTS In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/μm(2); day 1, median of 332 pSTAT1(+) cells/μm(2); and nonlesional, median of 155 pSTAT1(+) cells/μm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. CONCLUSIONS Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.

Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen-activated protein kinase and G-protein receptor kinase 2

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh‐regulated gene targets, Gli‐1 and Patched in a cyclopamine‐ and forskolin‐sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular‐signal‐regulated kinases or c‐jun N‐terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh‐dependent up‐regulation of Gli‐1, inter‐alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin‐like growth factor 2 or a novel Shh target, membrane‐associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively‐active mutant we show that Shh signaling to p38 MAPK and subsequent Gli‐1 transcription requires G‐protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G‐protein receptor kinase 2‐dependent p38 MAPK activity in regulating Shh‐mediated gene transcription in astrocytes.

Temporal proteomic profile of memory consolidation in the rat hippocampal dentate gyrus

Information storage in the brain depends on the ability of neurons to alter synaptic connectivity within key circuitries such as the hippocampus. Memory‐associated synaptic plasticity is mediated by a temporal cascade of de novo protein synthesis and altered protein processing. Here, we have used two‐dimensional difference in gel electrophoresis (2‐D DIGE) to investigate memory‐specific protein changes in the hippocampal dentate gyrus at increasing times following spatial learning. We identified 42 proteins that were significantly regulated in the first 24 h of spatial memory consolidation. Two distinct waves of protein expression regulation were evident, at 3 and 12 h post‐learning and this is in agreement with studies employing inhibitors of global translation. Functional classification of the memory‐associated proteins revealed that the majority of regulated proteins contributed either to cellular structure or cellular metabolism. For example, actins, tubulins and intermediate filament proteins, core proteins of the three major cytoskeletal components, were dynamically regulated at times that suggest a role in memory‐associated synaptic reorganization. Increased proteasome‐mediated protein degradation was evident in the early post‐training period including the down‐regulation of phosphoprotein enriched in astrocytes 15 kDa, a key inhibitor of extracellular signal‐regulated kinase signaling. Some of the most substantial protein expression changes were observed for secreted carrier proteins including transthyretin and serum albumin at 6–12 h post‐learning, regulations that could serve an important role in increasing the supply of retinoic acid and thyroid hormone, key synaptic plasticity‐promoting signals in the adult brain. Together these observations provide further insight into protein level regulations occurring in the hippocampus during spatial memory consolidation.

The molecular basis of the cooperation between EGF, FGF and eCB receptors in the regulation of neural stem cell function

Adult neurogenesis relies on EGF and FGF receptor (EGFR/FGFR) function and endocannabinoid (eCB) signalling. Here we have used a neural stem cell (NSC) line to determine how these systems cooperate to regulate neurogenesis. The results show the EGFR to be solely responsible for maintaining PI3K activation explaining its dominant role in promoting NSC survival. The EGFR and FGFR synergistically regulate the ERK/MAPK pathway, and this explains the requirement for both for optimal cell proliferation. The eCB receptors did not contribute to activation of the PI3K or ERK/MAPK pathways, highlighting the importance of another major proliferation pathway. The EGFR plays the dominant role in maintaining the transcriptome, with significant changes in the expression of over 3500 transcripts seen within hours of inhibition or activation of this receptor. The FGFR has a more modest effect on transcription with evidence for nodal integration with EGFR signalling at the level of the ERK/MAPK pathway. A common set of transcripts are regulated by the CB1 and CB2 receptors, with cooperation between these receptors and the EGFR apparent in the regulation of a pool of transcripts, most likely representing signal integration downstream from an as yet to be identified node. Finally, a first level molecular analysis of the transcriptional response shows regulation of a number of key growth factors, growth factor receptors and GPCRs to be under the control of the EGFR.

Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

Brown adipose tissue (BAT) plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1) that differentiates BAT from its energy storing white adipose tissue (WAT) counterpart. The clinical implication of “classical” BAT (originates from Myf5 positive myoblastic lineage) or the “beige” fat (originates through trans-differentiation of WAT) activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6) induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn) and Cyclooxygenase-2 (Cox2). Furthermore, pathway analyses using the Causal Reasoning Engine (CRE) identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R). Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

BackgroundRNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists.ResultsBy combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines.ConclusionThe high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.