Insanity as a Defense to the Civil Fraud Penalty

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of proinflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-. signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression

Injury-induced perivascular niche supports alternative differentiation of adult rodent CNS progenitor cells.

Following CNS demyelination, oligodendrocyte progenitor cells (OPCs) are able to differentiate into either remyelinating oligodendrocytes (OLs) or remyelinating Schwann cells (SCs). However, the signals that determine which type of remyelinating cell is generated and the underlying mechanisms involved have not been identified. Here, we show that distinctive microenvironments created in discrete niches within demyelinated white matter determine fate decisions of adult OPCs. By comparative transcriptome profiling we demonstrate that an ectopic, injury-induced perivascular niche is enriched with secreted ligands of the BMP and Wnt signalling pathways, produced by activated OPCs and endothelium, whereas reactive astrocyte within non-vascular area express the dual BMP/Wnt antagonist Sostdc1. The balance of BMP/Wnt signalling network is instructive for OPCs to undertake fate decision shortly after their activation: disruption of the OPCs homeostasis during demyelination results in BMP4 upregulation, which, in the absence of Socstdc1, favours SCs differentiation.This work was funded by the National Science Centre grants, NN 401 584238 (MZ) and 2012/07/N/ NZ3/01943 (JU-P), a programme grant from the UK Multiple Sclerosis Society (RJMF, CZ) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute (RJMF)

The signal transducers Stat1 and Stat3 and their novel target Jmjd3 drive the expression of inflammatory genes in microglia

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of proinflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-. signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression

Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

<div><p>Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.</p></div

Deactivation of Rac1 activity rescues CD44 knockdown and hyaluronidase-induced morphological changes of astrocytes.

<p>A: Representative images of astrocytes transfected with CD44shRNA/pSuper or co-transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) constructs. The β-actin-RFP construct was used for cell visualization. Scale: 20 μm. B: Morphometric analysis of shape-describing parameters of cells treated as in A. One way ANOVA, area: F(3.112) = 2.456, p>0,05, solidity: F(3.114) = 30.173, p<0.001 Sidak post hoc test, circularity: F(3.114) = 13.834, p<0.001, branching: F(3.114) = 51, 825, p<0,001. Dunnett’s C post hoc test. C: Representative images of astrocytes transfected with pcDNA3-EGFP-Rac1-T17N (Rac1-DN) and β-actin-RFP constructs and treated with hyaluronidase. Scale: 20 μm. D: Morphometric analysis of shape-describing parameters of cells treated as in C. One way ANOVA, area: F(2.147) = 1.520, p>0.05, solidity: F(2.147) = 106.292, p<0.001, circularity: F(2.147) = 96.843, p<0.001, branching: F(2.147) = 135.932; p<0,001, Dunnett’s C post hoc tests.</p

Hyaluronidase treatment and CD44-knockdown leads to enhanced Rac1 activity.

<p>A: Cells were transfected with FRET based biosensor pRaichu-Rac1/1011X and then treated or not with hyaluronidase for 24h. YFP-CFP ratio was calculated as a readout of Rac1 activity. T-Student test, t(589) = 3.212; p<0,001. B: Cells were co-transfected with pRaichu-Rac1/1011X and pSuper/CD44shRNA/CD44-RFP constructs. YFP-CFP ratio was calculated as a readout of Rac1 activity. One way ANOVA, F(2.527) = 39.998; p<0.001, Sidak post hoc test.</p

Astrocytes treated with hyaluronidase acquire the stellate-like morphology.

<p>A. Hyaluronan digestion by hyaluronidase was evaluated by staining with hyaluronan binding protein (HABP) (red). Scale: 30 μm. B. Measurement of fluorescence intensity. One way ANOVA test was performed, F(2.57) = 53.169; p<0.001, Dunnett’s C post hoc. C. Representative images of astrocytes transfected with β-actin GFP and either untreated (control) or treated with hyaluronidase or heat inactivated hyaluronidase for 48h. Cell nuclei were visualized with DAPI staining. Scale: 30 μm.D. Morphometric analysis of shape-describing parameters of cells treated as described in C. One way ANOVA test was performed, area: F(2.57) = 2.658; p>0.05, solidity: F(2.57) = 16.814; p<0.001, circularity: F(2.57) = 13.799; p<0.001, branching: F(2.57) = 16.774; p<0,001 Dunnett’s C post hoc test.</p

CD44 regulates astrocyte morphology.

<p>A: Validation of CD44shRNA and CD44-GFP constructs. Astrocytes were transfected with pSuper, CD44shRNA or CD44-GFP constructs (together with β-actin-GFP plasmid) and then immunostained with anti-CD44 antibody (red). The level of CD44 expression was evaluated by measuring CD44 immunofluorescence (IF) signal intensity with the use of ImageJ program. One way ANOVA, F(2.71) = 71.187, p<0.001, Dunnett C post hoc tests. Scale: 30 μm. B: Morphological analysis of shape-describing parameters of astrocytes in 2D cultures co-transfected with pSuper, CD44shRNA or CD44shRNA/CD44Rescue constructs together with β-actin-GFP plasmid. One way ANOVA test was performed, area: F(3.150) = 8.169; p<0.001, solidity: F(3.153) = 21.454; p<0.001, circularity: F(3.153) = 18.873; p<0.001, Dunnett’s C post hoc tests, branching: F(3.151) = 33,478; p<0.001, Sidak post hoc test. Scale: 30 μm. C: The morphological analysis of shape-describing parameters of astrocytes in 3D cultures transfected with pSuper or CD44shRNA constructs (together with β-actin-GFP plasmid) or CD44-GFP. One way ANOVA test was performed, area: F(2.92) = 12.311; p<0.001, Sidak post hoc test; solidity: F(2.95) = 42.208; p<0.001, Dunnett’s C post hoc test, circularity: F(2.94) = 20.609; p<0.001, Dunnett’s C post hoc test, branching: F(2.95) = 17.703; p<0.001, Sidak post hoc test. Scale: 30 μm.</p

Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and \u3c10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology