GABAergic dysfunction is present in the hippocampus in schizophrenia (SZ) and bipolar disorder (BD). The trisynaptic pathway was ''deconstructed'' into various layers of sectors CA3/2 and CA1 and gene expression profiling performed. Network association analysis was used to uncover genes that may be related to regulation of glutamate decarboxylase 67 (GAD 67), a marker for this system that has been found by many studies to show decreased expression in SZs and BDs. The most striking change was a down-regulation of GAD 67 in the stratum oriens (SO) of CA2/3 in both groups; CA1 only showed changes in the SO of schizophrenics. The network generated for GAD 67 contained 25 genes involved in the regulation of kainate receptors, TGF- and Wnt signaling, as well as transcription factors involved in cell growth and differentiation. In SZs, IL-1, (GRIK2/3), TGF-2, TGF-R1, histone deacetylase 1 (HDAC1), death associated protein (DAXX), and cyclin D2 (CCND2) were all significantly up-regulated, whereas in BDs, PAX5, Runx2, LEF1, TLE1, and CCND2 were significantly down-regulated. In the SO of CA1 of BDs, where GAD67 showed no expression change, TGF- and Wnt signaling genes were all up-regulated, but other transcription factors showed no change in expression. In other layers/sectors, BDs showed no expression changes in these GAD 67 network genes. Overall, these results are consistent with the hypothesis that decreased expression of GAD 67 may be associated with an epigenetic mechanism in SZ. In BD, however, a suppression of transcription factors involved in cell differentiation may contribute to GABA dysfunction.epigenetics ͉ network association analysis ͉ PAX5 ͉ Runx2 ͉ HDAC1
Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1β release. In contrast, we report that although N-GSDMD is required for IL-1β secretion in NLRP3activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3 + autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1β via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.
The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injuryinduced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratchwounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent proteinelabeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. (Am J Pathol 2016, 186: 285e296; http://dx
Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of GABA. We found that PNECs were the only cellular source of GABA in airways. In addition, PNECs expressed NT4 as a target-derived mechanism underlying PNEC innervation during development. Early life allergen exposure elevated the level of NT4 and caused PNEC hyperinnervation and nodose neuron hyperactivity. Associated with aberrant PNEC innervation, the authors discovered that GABA hypersecretion was required for the induction of mucin Muc5ac expression. In contrast, mice were protected from allergen-induced mucus overproduction and changes along the nerve-PNEC axis without any defects in inflammation. Last, GABA installation restored mucus overproduction in mice after early life allergen exposure. Together, our findings provide the first evidence for NT4-dependent neural regulation of PNEC secretion of GABA in a neonatal disease model. Targeting the nerve-PNEC axis may be a valid treatment strategy for mucus overproduction in airway diseases, such as childhood asthma.-Barrios, J., Patel, K. R., Aven, L., Achey, R., Minns, M. S., Lee, Y., Trinkaus-Randall, V. E., Ai, X. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion.
Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death.
Lee A, Derricks K, Minns M, Ji S, Chi C, Nugent MA, Trinkaus-Randall V. Hypoxia-induced changes in Ca 2ϩ mobilization and protein phosphorylation implicated in impaired wound healing. Am J Physiol Cell Physiol 306: C972-C985, 2014. First published March 26, 2014; doi:10.1152/ajpcell.00110.2013.-The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca 2ϩ wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca 2ϩ signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca 2ϩ dynamic patterns were altered in response to decreased O 2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca 2ϩ mobilization and localization of N-methyl-D-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca 2ϩ -mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair. hypoxia; wound healing; imaging; cell communication DAMAGE TO TISSUE activates intricate underlying mechanisms that mediate the healing process. Communication between cells is generated immediately after injury and continues during the migration phase and the later phases of proliferation and extracellular matrix reassembly (13,14,23). This reepithelialization requires the precise control of glutamatergic and purinergic signaling pathways. Injured cells release nucleotides that serve as ligands for cell-surface purinergic receptors, and injured neurons release components, including ATP and glutamate, that bind their respective receptors. The release of nucleotides can modulate Ca 2ϩ homeostasis (4,11,43,52,54).Neuronal transmitters such as glutamate bind to metabotropic G protein-coupled receptors or to ionotropic receptors, including 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors. It has been reported that epithelial cells do not respond to AMPA or kainate, but they do respond to glutamate in neuronal wound media and to NM...
The aim of this study was to examine whether glutamatergic inputs onto GABA interneurons via the kainate receptor in the anterior cingulate cortex may be altered in schizophrenia and bipolar disorder. Hence, in a cohort of 60 post-mortem human brains from schizophrenia, bipolar disorder, and normal control subjects, we simultaneously labeled the mRNA for the GluR5 or GluR6 subunit of the kainate receptor with [(35)S] and the mRNA for the 67 kD isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD)(67) with digoxigenin using an immunoperoxidase method. The density of the GAD(67) mRNA-containing neurons that co-expressed GluR5 mRNA was decreased by 43% and 40% in layer 2 of the anterior cingulate cortex in schizophrenia and bipolar disorder, respectively. In contrast, the density of the GAD(67) mRNA-containing cells that expressed GluR6 mRNA was unaltered in either condition. Furthermore, the amount of GluR5 or GluR6 mRNA in the GAD(67) mRNA-expressing cells that contained a detectable level of these transcripts was also unchanged. Finally, the density of cells that did not contain GAD(67) mRNA, which presumably included all pyramidal neurons, but expressed the mRNA for the GluR5 or GluR6 subunit was not altered. Thus, glutamatergic modulation of inhibitory interneurons, but not pyramidal neurons, via kainate receptors containing the GluR5 subunit appears to be selectively altered in the anterior cingulate cortex in schizophrenia and bipolar disorder.
Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18−/− and Dectin-1−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4−/− corneas; however, we found no difference between C57BL/6 and PAD4−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.
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