DOCSLIB.ORG

TECHNISCHE UNIVERSITÄT MÜNCHEN Institut Für Experimentelle Genetik Helmholtz Zentrum München, Neuherberg Regulatory Mechanis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
TECHNISCHE UNIVERSITÄT MÜNCHEN Institut Für Experimentelle Genetik Helmholtz Zentrum München, Neuherberg Regulatory Mechanis TECHNISCHE UNIVERSITÄT MÜNCHEN Institut für Experimentelle Genetik Helmholtz Zentrum München, Neuherberg Regulatory mechanisms underlying atopic dermatitis: Functional characterization of the C11orf30/LRRC32 locus and analysis of genome-wide expression profiles in patients Judith Manz Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. M. Hrabe de Angelis Prüfer der Dissertation: 1. apl. Prof. Dr. J. Adamski 2. Prof. Dr. H. Witt 3. Prof. Dr. St. Weidinger Die Dissertation wurde am 14.06.2017 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 27.10.2017 angenommen. Am Ende wird alles gut. Wenn es nicht gut wird, ist es noch nicht das Ende. (Oscar Wilde) I TABLE OF CONTENTS TABLE OF CONTENTS ....................................................................................................................................... I SUMMARY ......................................................................................................................................................... VI ZUSAMMENFASSUNG .................................................................................................................................. VIII LIST OF ABBREVIATIONS .............................................................................................................................. X INTRODUCTION ........................................................................................................................................ 1 1.1 Atopic dermatitis ............................................................................................................................................ 1 1.1.1 Clinical features ..................................................................................................................................... 1 1.1.2 Epidemiology of atopic dermatitis ................................................................................................. 3 1.1.3 Pathophysiology of atopic dermatitis ........................................................................................... 3 1.1.3.1 T-helper subpopulations and the role of regulatory T cells in AD ............................... 7 1.1.4 Therapeutic strategies of atopic dermatitis ............................................................................ 10 1.1.5 Genetics of atopic dermatitis ......................................................................................................... 12 1.1.5.1 Genetic studies in atopic dermatitis ...................................................................................... 13 1.1.5.2 AD associated locus 11q13.5 – an pleiotropic locus for atopic diseases ................ 14 1.1.5.3 Follow-up of genome-wide association studies by functional studies ................... 17 1.2 Aim of the study ........................................................................................................................................... 18 RESULTS ................................................................................................................................................... 19 2.1 The GWA lead SNP rs7927894 and its proxy SNPs within the AD-associated ....................... 11q13.5 locus ................................................................................................................................................ 19 2.1.1 Allelic expression analysis of LRRC32 and C11orf30 transcripts ....................................... depending on rs7927894................................................................................................................ 20 2.1.2 Characterization of rs7927894 proxy SNPs within the 11q13.5 locus ........................ 22 2.2 Functional impact of SNP rs2155219 in the 11q13.5 locus ....................................................... 26 2.2.1 Allele-specific effect of rs2155219 on promoter regulation ............................................ 26 2.2.2 Regulatory effect on native LRRC32 and C11orf30 promotors ........................................ 33 2.2.3 Candidate gene expression in skin in dependence of rs2155219 genotype ............. 35 2.2.4 Analysis of differential protein-DNA binding patterns ...................................................... 36 2.3 Functional impact of LRRC32 coding variant rs79525962 ......................................................... 38 2.3.1 Targeted resequencing of 11q13.5 locus and identification of ........................................... low frequency missense variants ................................................................................................ 38 2.3.2 Protein modelling and bioinformatics prediction of missense variants ..................... 39 II 2.3.3 Impact of rs79525962/A407T on RNA level .......................................................................... 40 2.3.4 Impact of rs79525962/A407T on protein level .................................................................... 41 2.3.4.1 GARP protein expression determined by Western Blot analysis .............................. 41 2.3.4.2 Subcellular localization of GARP in HeLa and Cos-7 cells ............................................ 44 2.3.4.3 GARP protein expression determined by flow cytometric analysis (FACS) ......... 46 2.3.4.4 FACS analysis of CTLA-4, FOXP3 and Neuropilin as specific ........................................... regulatory T cell markers........................................................................................................... 50 2.3.5 Analysis of soluble GARP via enzyme-linked immunosorbent assay (ELISA) .......... 51 2.4 Cutaneous mRNA expression patterns in atopic dermatitis ...................................................... 52 2.4.1 RNA quality assessment of samples isolated from skin biopsies ................................... 52 2.4.2 Quality control of gene expression data ................................................................................... 54 2.4.3 Genome-wide expression differences between non-lesional (AN) and .......................... lesional (AL) AD skin and healthy controls (NN) .................................................................. 55 2.4.4 Functional enrichment analyses of gene sets ......................................................................... 56 DISCUSSION ............................................................................................................................................ 60 3.1 The AD-associated polymorphism rs7927894 is not associated with ...................................... allelic expression imbalance of C11orf30 and LLRC32 in skin tissue ..................................... 60 3.2 Rs7927894 versus rs2155219 – a proxy SNP as potential regulatory variant .................. 62 3.2.1 Rs2155219 alters the DNA binding ability to SP1 transcription factor .......................... in Jurkat cells........................................................................................................................................ 65 3.2.2 The rs2155219 risk allele shows regulatory activity at the C11orf30/LRRC32 ........... gene locus .............................................................................................................................................. 66 3.2.3 Limitations of reporter gene assays ........................................................................................... 69 3.3 Do low frequency functional variants underlie the 11q association signal? ...................... 70 3.3.1 rs79525962 does not influence LRRC32 mRNA expression but alters ............................ GARP protein levels ........................................................................................................................... 71 3.3.2 rs79525962 might impair immune mechanisms by regulating ......................................... GARP protein expression ................................................................................................................ 73 3.4 Genome-wide cutaneous mRNA expression analysis in atopic dermatitis ......................... 75 3.4.1 Signature genes of the AD phenotype are involved in keratinization and ..................... skin inflammation .............................................................................................................................. 76 3.4.2 Dysregulation of potential precursor genes in non lesional AD skin ........................... 77 3.5 Conclusion ...................................................................................................................................................... 80 3.6 Outlook ............................................................................................................................................................. 81 METHODS ................................................................................................................................................ 83 4.1 Working with organisms .......................................................................................................................... 83 4.1.1 Working with eukaryotic cell lines ............................................................................................. 83
Load more

Recommended publications
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • 13809 P2X7 Receptor (E1E8T) Rabbit Mab
    Revision 1 C 0 2 - t P2X7 Receptor (E1E8T) Rabbit mAb a e r o t S Orders: 877-616-CELL (2355) [email protected] 9 Support: 877-678-TECH (8324) 0 8 Web: [email protected] 3 www.cellsignal.com 1 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source/Isotype: UniProt ID: Entrez-Gene Id: WB, IP H Endogenous 78 Rabbit IgG Q99572 5027 Product Usage Information 2. Valera, S. et al. (1994) Nature 371, 516-9. 3. North, R.A. and Surprenant, A. (2000) Annu Rev Pharmacol Toxicol 40, 563-80. Application Dilution 4. Skaper, S.D. et al. (2010) FASEB J 24, 337-45. 5. Bernardino, L. et al. (2008) J Neurochem 106, 271-80. Western Blotting 1:1000 6. Volonté, C. et al. (2003) Curr Drug Targets CNS Neurol Disord 2, 403-12. Immunoprecipitation 1:50 7. Chessell, I.P. et al. (2005) Pain 114, 386-96. 8. Dell'Antonio, G. et al. (2002) Neurosci Lett 327, 87-90. 9. Barden, N. et al. (2006) Am J Med Genet B Neuropsychiatr Genet 141B, 374-82. Storage 10. Lucae, S. et al. (2006) Hum Mol Genet 15, 2438-45. Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody. Specificity / Sensitivity P2X7 Receptor (E1E8T) Rabbit mAb recognizes endogenous levels of total P2X7 receptor protein. Species Reactivity: Human Source / Purification Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu462 of human P2X7 receptor protein.
    [Show full text]
  • A Truncation Variant of the Cation Channel P2RX5 Is Upregulated During T Cell Activation
    A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation . Pierre Abramowski1,2", Christoph Ogrodowczyk3", Roland Martin1,4* , Olaf Pongs3,5 1 Institute for Neuroimmunology and Clinical Multiple Sclerosis Research (inims), ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2 Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3 Institute for Neural Signaltransduction, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4 Neuroimmunology and MS Research, Department of Neurology, University Hospital Zurich, Zurich, Switzerland, 5 Institute for Physiology, University Hospital Homburg, Homburg/Saar, Germany Abstract Members of the P2X family of ligand-gated cation channels (P2RX) are expressed by various cell types including neurons, smooth- and cardiac muscle cells, and leukocytes. The channels mediate signalling in response to extracellular ATP. Seven subunit isoforms (P2RX1-P2RX7) have been identified and these can assemble as homo- and heterotrimeric molecules. In humans, P2RX5 exists as a natural deletion mutant lacking amino acids 328–349 of exon 10, which are part of transmembrane (TM) 2 and pre-TM2 regions in other organisms like rat, chicken and zebrafish. We show that P2RX5 gene expression of human T lymphocytes is upregulated during activation. P2RX5 is recruited to the cell surface. P2RX5-siRNA- transfected CD4+ T cells produced twofold more IL-10 than controls. Surface and intracellular P2RX5 expression was upregulated in activated antigen-specific CD4+ T cell clones. These data indicate a functional role of the human P2RX5 splice variant in T cell activation and immunoregulation. Citation: Abramowski P, Ogrodowczyk C, Martin R, Pongs O (2014) A Truncation Variant of the Cation Channel P2RX5 Is Upregulated during T Cell Activation.
    [Show full text]
  • P2X1 (F-17): Sc-12208
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . P2X1 (F-17): sc-12208 BACKGROUND APPLICATIONS The P2X receptor family is comprised of ligand-gated ion channels that allow P2X1 (F-17) is recommended for detection of P2X1 of mouse, rat and for the increased permeability of calcium into the cell in response to extra - human origin by Western Blotting (starting dilution 1:200, dilution range cel lular ATP. The seven P2X receptors, P2X1-P2X7, form either homomeric or 1:100-1:1000), immunofluorescence (starting dilution 1:50, dilution range heteromeric channels or both. They are characterized by intracellular amino- 1:50-1:500) and solid phase ELISA (starting dilution 1:30, dilution range and carboxy-termini. P2X receptors are expressed in a wide variety of tissues, 1:30-1:3000). including neurons, prostate, bladder, pancreas, colon, testis and ovary. The P2X1 (F-17) is also recommended for detection of P2X1 in additional species, major function of the P2X receptors is to mediate synaptic transmissions be- including equine, canine, bovine and porcine. tween neurons and to other tissues via the binding of extracellular ATP, which acts as a neurotransmitter. The P2X receptors may be involved in the onset Suitable for use as control antibody for P2X1 siRNA (h): sc-42563, P2X1 of necrosis or apoptosis after prolonged exposure to high concentrations of siRNA (m): sc-42564, P2X1 shRNA Plasmid (h): sc-42563-SH, P2X1 shRNA extracellular ATP. Plasmid (m): sc-42564-SH, P2X1 shRNA (h) Lentiviral Particles: sc-42563-V and P2X1 shRNA (m) Lentiviral Particles: sc-42564-V.
    [Show full text]
  • Prdc Cdna Insert Product Line
    rP2X5/P2RX5 VersaClone cDNA Catalog Number: RDC2061 Specifications: Description This shuttle vector contains the complete ORF for the gene of interest, Gene: rP2rx5 along with a Kozak consensus sequence for optimal translation initiation. It is inserted NotI to AscI. The gene insert is flanked with Accession: NP_542958 convenient multiple cloning sites which can be used to easily cut and transfer the gene cassette into your desired expression vector. Insert size: 1381bp Preparation and Storage Concentration: 10µg at 0.2μg/μL Formulation cDNA is provided in 10 mM Tris-Cl, pH 8.5 Shipping Ships at ambient temperature Stability 1 year from date of receipt when stored at -20°C to -80°C Storage Use a manual defrost freezer and avoid repeated rP2X5/P2RX5 cDNA freeze-thaw cycles. Plasmid P2rx5 purinergic receptor P2X 5 BstAPI EcoO109I BbeI [ Rattus norvegicus (Norway ZraI KasI BmgBI AatII NarI rat) ] SspI HpaI SfoI EcoRV BmtI Also known as: P2x5 NheI ScaI NotI EagI BtgI Summary: NcoI StyI P2RX5 belongs to the family of purinoceptors for ATP. It functions AMP BlpI NmeAIII as ligand-gated ion channel. P2RX5 BsrFI is believed to form a trimer with BsaI AhdI RDC2061 EcoNI another member of this family, P2RX1. P2RX5 mRNA expression is 4115 bps rP2RX5 (1-455) detected in a significant portion of XcmI PshAI solid tumor cell lines, including renal BsaBI cell carcinoma (RCC), melanoma, BseRI colorectal carcinoma, brain cancer COLE1 and breast cancer. Expression was AlwNI also detected in a subset of primary SexAI solid tumor specimens derived from Bsu36I BbsI Van91I AscI RCC, brain cancer and breast cancer BsmI BssHII PciI PspXI Bst1107I patients.
    [Show full text]
  • Acutemicroglialrespons
    ACUTE MICROGLIAL RESPONSES TO SINGLE NON-EPILEPTOGENIC VS. EPILEPTOGENIC SEIZURES IN MOUSE HIPPOCAMPUS A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience By Alberto Sepulveda-Rodriguez, B.S. Washington, D.C. June 5, 2019 Copyright 2019 by Alberto Sepulveda-Rodriguez All Rights Reserved ii ACUTE MICROGLIAL RESPONSES TO SINGLE NON-EPILEPTOGENIC VS. EPILEPTOGENIC SEIZURES IN MOUSE HIPPOCAMPUS Alberto Sepulveda-Rodriguez, B.S. Thesis Advisor: Stefano Vicini, Ph.D. ABSTRACT As the resident macrophage of the central nervous system, microglia are in a uniquely privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. In one of the prototypical rodent models of seizure-induced epilepsy, hippocampal microglia become activated after prolonged, damaging seizures known as Status Epilepticus (SE). However, since SE comprises both neuronal hyperactivity and injury, the specific mechanisms triggering this microglial activation remain unclear, as does its relevance to the ensuing epileptogenic processes. The present studies employed another well-established seizure model, electroconvulsive shock (ECS), to study the effect of paroxysmal/ictal neuronal hyperactivity on mouse hippocampal microglia, in the absence of concomitant neuronal degeneration. Unlike SE, ECS did not cause neuronal injury and did not alter hippocampal CA1 microglial and astrocytic density, morphology nor baseline process motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices and similarly upregulated expression of the iii chemokine CCL2.
    [Show full text]
  • Identification of a Subset of Immunosuppressive P2RX1
    ARTICLE https://doi.org/10.1038/s41467-020-20447-y OPEN Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis Xu Wang1,2,4, Li-Peng Hu1,4, Wei-Ting Qin1,4, Qin Yang1,4, De-Yu Chen2,4, Qing Li1, Kai-Xia Zhou1, Pei-Qi Huang1, Chun-Jie Xu1, Jun Li1, Lin-Li Yao1, Ya-Hui Wang1, Guang-Ang Tian1, Jian-Yu Yang3, ✉ ✉ ✉ Min-Wei Yang3, De-Jun Liu3, Yong-Wei Sun 3 , Shu-Heng Jiang 1 , Xue-Li Zhang 1 & ✉ Zhi-Gang Zhang 1 1234567890():,; The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechan- isms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1- deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA- activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immuno- suppressive P2RX1-negative neutrophils. 1 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R.
    [Show full text]
  • Sensory Neuronal P2RX4 Receptors Controls BDNF Signaling In
    www.nature.com/scientificreports OPEN Sensory neuronal P2RX4 receptors controls BDNF signaling in infammatory pain Received: 12 April 2017 Sarah Lalisse1,2,3, Jennifer Hua1,2,3, Manon Lenoir1,2,3, Nathalie Linck1,2,3, Accepted: 27 December 2017 François Rassendren 1,2,3 & Lauriane Ulmann1,2,3 Published: xx xx xxxx Chronic infammatory and neuropathic pains are major public health concerns. Potential therapeutic targets include the ATP-gated purinergic receptors (P2RX) that contribute to these pathological types of pain in several diferent cell types. The purinergic receptors P2RX2 and P2RX3 are expressed by a specifc subset of dorsal root ganglion neurons and directly shape pain processing by primary aferents. In contrast the P2RX4 and P2RX7 are mostly expressed in myeloid cells, where activation of these receptors triggers the release of various pro-infammatory molecules. Here, we demonstrate that P2RX4 also controls calcium infux in mouse dorsal root ganglion neurons. P2RX4 is up-regulated in pain-processing neurons during long lasting peripheral infammation and it co-localizes with Brain- Derived Neurotrophic Factor (BDNF). In the dorsal horn of the spinal cord, BDNF-dependent signaling pathways, phosphorylation of Erk1/2 and of the GluN1 subunit as well as the down regulation of the co-transporter KCC2, which are triggered by peripheral infammation are impaired in P2RX4-defcient mice. Our results suggest that P2RX4, expressed by sensory neurons, controls neuronal BDNF release that contributes to hyper-excitability during chronic infammatory pain and establish P2RX4 in sensory neurons as a new potential therapeutic target to treat hyperexcitability during chronic infammatory pain. Pain is a sensory modality that is encoded by specialized nociceptive neurons in the dorsal root ganglion (DRG).
    [Show full text]
  • Electroconvulsive Shock Enhances Responsive Motility and Purinergic Currents in Microglia in the Mouse Hippocampus
    New Research Disorders of the Nervous System Electroconvulsive Shock Enhances Responsive Motility and Purinergic Currents in Microglia in the Mouse Hippocampus Alberto Sepulveda-Rodriguez,1,2 Pinggan Li,1,4 Tahiyana Khan,1,2 James D. Ma,1 Colby A. Carlone,1 P. Lorenzo Bozzelli,2,3 Katherine E. Conant,2,3 Patrick A. Forcelli,1,2 and Stefano Vicini1,2 https://doi.org/10.1523/ENEURO.0056-19.2019 1Department of Pharmacology and Physiology, Georgetown University, Washington, DC 20007, 2Interdisciplinary Program in Neuroscience, Georgetown University, Washington, DC 20007, 3Department of Neuroscience, Georgetown University, Washington, DC 20007, 4Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Abstract Microglia are in a privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. Hippocampal microglia become activated after prolonged, damaging seizures known as status epilepticus (SE). However, since SE causes both hyperactivity and injury of neurons, the mechanisms triggering this activation remain unclear, as does the relevance of the microglial activation to the ensuing epileptogenic processes. In this study, we use electroconvulsive shock (ECS) to study the effect of neuronal hyperactivity without neuronal degeneration on mouse hippocampal microglia. Unlike SE, ECS did not alter hippocampal CA1 microglial density, morphology, or baseline motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices, and similarly upregu- lated expression of the chemokine C-C motif chemokine ligand 2 (CCL2). Whole-cell patch-clamp recordings of hippocampal CA1sr microglia showed that ECS enhanced purinergic currents mediated by P2X7 receptors in the absence of changes in passive properties or voltage-gated currents, or changes in receptor expression.
    [Show full text]
  • Dl-3-N-Butylphthalide Attenuates Mouse Behavioral Deficits to Chronic
    Wang et al. Translational Psychiatry (2020) 10:49 https://doi.org/10.1038/s41398-020-0731-z Translational Psychiatry ARTICLE Open Access Dl-3-n-butylphthalide attenuates mouse behavioral deficits to chronic social defeat stress by regulating energy metabolism via AKT/CREB signaling pathway Wei Wang1,2,3,TingWang1,2,4, Shunjie Bai1,2,5, Zhi Chen1,2, Xunzhong Qi1,2 andPengXie1,2,3,4,6,7 Abstract Major depressive disorder (MDD) is a severe mental disorder associated with high rates of morbidity and mortality. Current first-line pharmacotherapies for MDD are based on enhancement of monoaminergic neurotransmission, but these antidepressants are still insufficient and produce significant side-effects. Consequently, the development of novel antidepressants and therapeutic targets is desired. Dl-3-n-butylphthalide (NBP) is a compound with proven efficacy in treating ischemic stroke, yet its therapeutic effects and mechanisms for depression remain unexplored. The aim of this study was to investigate the effect of NBP in a chronic social defeat stress model of depression and its underlying molecular mechanisms. Here, we examined depression-related behavior and performed a targeted metabolomics analysis. Real-time quantitative polymerase chain reaction and western blotting were used to examine key genes and proteins involved in energy metabolism and the AKT/cAMP response element-binding protein (CREB) signaling pathway. Our results reveal NBP attenuates stress-induced social deficits, anxiety-like behavior and despair 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; behavior, and alters metabolite levels of glycolysis and tricarboxylic acid (TCA) cycle components. NBP affected gene expression of key enzymes of the TCA cycle, as well as protein expression of p-AKT and p-CREB.
    [Show full text]
  • A Focus on Microglia and P2X4R
    JPET Fast Forward. Published on February 29, 2020 as DOI: 10.1124/jpet.120.265017 This article has not been copyedited and formatted. The final version may differ from this version. JPET # 265017 Title: Sex-dependent mechanisms of chronic pain: A focus on microglia and P2X4R Authors and Affiliations: Katherine Halievski1, 2, Shahrzad Ghazisaedi1, 2, 3, Michael W. Salter1, 2, 3 1. Neurosciences and Mental Health Program, The Hospital for Sick Children, Toronto ON 2. The University of Toronto Centre for the Study of Pain, Toronto ON Downloaded from 3. The Department of Physiology, University of Toronto, Toronto ON jpet.aspetjournals.org at ASPET Journals on September 24, 2021 1 JPET Fast Forward. Published on February 29, 2020 as DOI: 10.1124/jpet.120.265017 This article has not been copyedited and formatted. The final version may differ from this version. JPET # 265017 Running title: Microglia-P2X4R sex differences in pain Corresponding author: Michael W. Salter The Hospital for Sick Children 686 Bay St. Toronto, ON M5G 0A4 Canada tel: (416) 813-6272 fax: (416) 813-7921 [email protected] Number of text pages: 15 Number of references: 72 Downloaded from Number of tables: 1 Number of words in the Abstract: 137 Number of words in the Body: 5013 jpet.aspetjournals.org Abbreviations: brain-derived neurotrophic factor (BDNF) chemokine (C-C motif) ligand (CCL) colony-stimulating factor 1 (CSF-1) interleukin-6 (IL-6) interferon regulatory factor (IRF) at ASPET Journals on September 24, 2021 lipopolysaccharide (LPS) N-methyl-D-aspartate receptor (NMDAR) periaqueductal gray (PAG) peripheral nerve injury (PNI) purinergic receptor, ionotropic (P2XR) prostaglandin E2 (PGE-2) vesicular nucleotide transporter (VNUT) Section assignment: Special Section: Sexual Dimorphism in Neuroimmune Cells 2 JPET Fast Forward.
    [Show full text]
  • Extracellular ATP Released from Candida Albicans Activates Non-Peptidergic Neurons to Augment Host Defense
    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.27.921049; this version posted January 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Extracellular ATP released from Candida albicans activates non-peptidergic neurons to augment host defense SHORT TITLE (70 characters): Candida albicans secretes ATP to augment host defense Tara N Edwards¶,1, Shiqun Zhang¶,1, Andrew Liu1, Jonathan A. Cohen1, Paul Yifan Zhou1, Selene Mogavero2, Bernhard Hube2, Judith Berman3, Marie-Elisabeth Bougnoux4,5, Alicia R. Mathers1, Sarah L. Gaffen6, Kathryn M. Albers7,8, H. Richard Koerber7,8, Brian M. Davis7,8, Christophe D'Enfert4, Daniel H Kaplan1,* 1 Departments of Dermatology and Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania 2 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute (HKI), Jena, Germany 3 School of Molecular Cell Biology & Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel 4 Unité Biologie et Pathogénicité Fongiques, Département de Mycologie, Institut Pasteur, USC2019 INRA, Paris, France 5 Unité de Parasitologie-Mycologie, Service de Microbiologie clinique, Hôpital Necker- Enfants-Malades, Assistance Publique des Hôpitaux de Paris (APHP), Paris, France 6 Division of Rheumatology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 7 Pittsburgh Center for Pain Research, University of Pittsburgh, Pittsburgh, PA 15261, USA 8 Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15261, USA * Corresponding author E-mail: [email protected] (DHK) ¶Authors provided equal contribution Keywords: Candida albicans, ATP, peripheral nervous system, sensory neuron, non- peptidergic neuron.
    [Show full text]