ATM Influences the Efficiency of TCRβ Rearrangement,Subsequent TCRβ-Dependent T Cell Development,and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4⁻CD8⁻ double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4⁺CD8⁺ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4⁺CD8⁺ thymocytes, resulting in reduced numbers of CD4⁺CD8⁺ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4⁺CD8⁺ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.     

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.     

Cell migration: GSK3β steers the cytoskeleton's tip

Directed cell migration polarizes the cytoskeleton, allowing the cell to move toward migratory cues. In this issue, Wu et?al. (2011) demonstrate that the glycogen synthase kinase 3β (GSK3β) controls microtubule architecture and polarized movement of skin stem cells during wound healing in mammals by regulating the microtubule crosslinking protein ACF7.     

GSK-3β and MMP-9 Cooperate in the Control of Dendritic Spine Morphology

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3β (GSK-3β) has been implicated. Despite much evidence of the involvement of GSK-3β in functional synaptic plasticity, it is unclear how GSK-3β controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3β in neurons to investigate how GSK-3β affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3β activity increased the number of thin spines, whereas lacking GSK-3β increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3β activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3β overexpressing mice. Administration of the nonspecific GSK-3β inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3β and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3β activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3β-induced synaptic alterations.     

Genetic Diversity of Human Enterovirus 68 Strains Isolated in Kenya Using the Hypervariable 3′- End of VP1 Gene

Reports of increasing worldwide circulation of human enterovirus-68 (EV68) are well documented. Despite health concerns posed by resurgence of these viruses, little is known about EV68 strains circulating in Kenya. In this study, we characterized 13 EV68 strains isolated in Kenya between 2008 and 2011 based on the Hypervariable 3′- end of the VP1 gene. Viral RNA was extracted from the isolates and partial VP1 gene amplified by RT-PCR, followed by nucleotide sequencing. Alignment of deduced amino acid sequences revealed substitutions in Kenyan EV68 isolates absent in the prototype reference strain (Fermon). The majority of these changes were present in the BC and DE-loop regions, which are associated with viral antigenicity and virulence. The Kenyan strains exhibited high sequence homology with respect to those from other countries. Natural selection analysis based on the VP1 region showed that the Kenyan EV68 isolates were under purifying selection. Phylogenetic analysis revealed that majority (84.6%) of the Kenyan strains belonged to clade A, while a minority belonged to clades B and C. Overall, our results illustrate that although EV68 strains isolated in Kenya were genetically and antigenically divergent from the prototype strain (Fermon), they were closely related to those circulating in other countries, suggesting worldwide transmissibility. Further, the presence of shared mutations by Kenyan EV68 strains and those isolated in other countries, indicates evolution in the VP1 region may be contributing to increased worldwide detection of the viruses. This is the first study to document circulation of EV68 in Kenya.     

Expression of the Clustered NeuAcα2–3Galβ O-Glycan Determines the Cell Differentiation State of the Cells

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2–3Galβ O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1–60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1–60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.     

SUMO1 promotes Aβ production via the modulation of autophagy

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-β (Aβ) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aβ levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aβ production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aβ was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aβ production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.     

SUMO1 promotes Aβ production via the modulation of autophagy

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-β (Aβ) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aβ levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aβ production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aβ was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aβ production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.     

Mechanosensitivity of the 2nd Kind: TGF-β Mechanism of Cell Sensing the Substrate Stiffness

Cells can sense forces applied to them, but also the stiffness of their environment. These are two different phenomena, and here we investigate the mechanosensitivity of the 2^nd kind: how the cell can measure an elastic modulus at a single point of adhesion—and how the cell can receive and interpret the chemical signal released from the sensor. Our model uses the example of large latent complex of TGF-β as a sensor. Stochastic theory gives the rate of breaking of latent complex, which initiates the signaling feedback loop after the active TGF-β release and leads to a change of cell phenotype driven by the α-smooth muscle actin. We investigate the dynamic and steady-state behaviors of the model, comparing them with experiments. In particular, we analyse the timescale of approach to the steady state, the stability of the non-linear dynamical system, and how the steady-state concentrations of the key markers vary depending on the elasticity of the substrate. We discover a crossover region for values of substrate elasticity closely corresponding to that of the fibroblast to myofibroblast transition. We suggest that the cell could actively vary the parameters of its dynamic feedback loop to ‘choose’ the position of the transition region and the range of substrate elasticity that it can detect. In this way, the theory offers the unifying mechanism for a variety of phenomena, such as the myofibroblast conversion in fibrosis of wounds and lungs and smooth muscle cell dysfunction in cardiac disease.     

Editorial Expression of Concern: Sox9 Protects against Human Lung Fibroblast Cell Apoptosis Induced by LPS through Activation of the AKT/GSK3β Pathway

Biochemistry (Moscow) -     

GRK5 influences the phosphorylation of tau via GSK3β and contributes to Alzheimer's disease

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase whose dysfunction results in cognitive impairment and Alzheimer-like pathology, including tau hyperphosphorylation. However, the mechanisms whereby GRK5 influences tau phosphorylation remain incompletely understood. In the current study, we showed that GRK5 influenced the phosphorylation of tau via glycogen synthase kinase 3β (GSK3β). The activity of both tau and GSK3β in the hippocampus was increased in aged GRK5-knockout mice, which is consistent with what occurs in APP/PS1 transgenic mice. Furthermore, GRK5 regulated the activity of GSK3β and phosphorylated tau in vitro. Regardless of changes of GRK5 protein levels, tau hyperphosphorylation remained reduced after GSK3β activity was inhibited, suggesting that GRK5 may specifically influence tau hyperphosphorylation by modulating GSK3β activity. Taken together, our findings suggest that GRK5 deficiency contributes to the pathogenesis of Alzheimer's disease by influencing the hyperphosphorylation of tau through the activation of GSK3β.     

Effect of the CALHM1 G330D and R154H Human Variants on the Control of Cytosolic Ca2+ and Aβ Levels

CALHM1 is a plasma membrane voltage-gated Ca²⁺-permeable ion channel that controls amyloid-β (Aβ) metabolism and is potentially involved in the onset of Alzheimer''s disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF)  = 0.015%), but that G330D is not (MAF  = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca²⁺ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca²⁺ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca²⁺ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.     

Effect of Chlorpromazine on the Binding of Oestradiol-17β by Human Uterine Cytosol

Abstract

Chlorpromazine increases the binding of oestradiol-17β by human uterine cytosol in vitro. This effect is due to an increase in the number of receptor sites, and the dissociation constant for oestradiol-17β is unaffected by chlorpromazine.     

Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-β Signaling Pathway

Post-operative adhesions are a critical problem in pelvic and abdominal surgery despite a multitude of studies dedicated to finding modalities to prevent their occurrence. Ghrelin administration promotes an anti-fibrotic response in a surgical mouse model of adhesion-induction, but the mechanisms mediating this effect have not been established. In the current study, the molecular mechanisms that underlie the anti-adhesion effect of ghrelin were investigated. Post-surgical adhesions were experimentally created in C57BL/6 wild-type mice via a combination of ischemic peritoneal buttons and cecal multiple abrasions. Ghrelin or saline intraperitoneal injections were given twice daily from two days before surgery to selected time points post-surgically to assess the phenotypic and molecular effects of treatment (1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) after surgery). Endpoints included the scoring of adhesions and gene and protein expression analysis of pro-fibrogenic factors conducted on peritoneal ischemic tissue by quantitative PCR and Western blot. Ghrelin administration significantly reduced post-surgical adhesions and down-regulated pro-inflammatory gene and protein expression, including Tgfb3 and Tgfbr2. The up-regulation of inhibitory proteins Smad6 and Smad7 confirmed the ghrelin-induced blockage of TGF-β signaling. Ghrelin is a candidate therapeutic drug for post-operative adhesion prevention, inhibiting inflammatory responses via blockage of the TGF-β signaling pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.