DOCSLIB.ORG

Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms Erin A. Greguske ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intelꞏlectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. En la utilització o cita de parts de la tesi és obligat indicar el nom de la persona autora. ADVERTENCIA. La consulta de esta tesis queda condicionada a la aceptación de las siguientes condiciones de uso: La difusión de esta tesis por medio del servicio TDR (www.tdx.cat) y a través del Repositorio Digital de la UB (diposit.ub.edu) ha sido autorizada por los titulares de los derechos de propiedad intelectual únicamente para usos privados enmarcados en actividades de investigación y docencia. No se autoriza su reproducción con finalidades de lucro ni su difusión y puesta a disposición desde un sitio ajeno al servicio TDR o al Repositorio Digital de la UB. No se autoriza la presentación de su contenido en una ventana o marco ajeno a TDR o al Repositorio Digital de la UB (framing). Esta reserva de derechos afecta tanto al resumen de presentación de la tesis como a sus contenidos. En la utilización o cita de partes de la tesis es obligado indicar el nombre de la persona autora. WARNING. On having consulted this thesis you’re accepting the following use conditions: Spreading this thesis by the TDX (www.tdx.cat) service and by the UB Digital Repository (diposit.ub.edu) has been authorized by the titular of the intellectual property rights only for private uses placed in investigation and teaching activities. Reproduction with lucrative aims is not authorized nor its spreading and availability from a site foreign to the TDX service or to the UB Digital Repository. Introducing its content in a window or frame foreign to the TDX service or to the UB Digital Repository is not authorized (framing). Those rights affect to the presentation summary of the thesis as well as to its contents. In the using or citation of parts of the thesis it’s obliged to indicate the name of the author. Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms Thesis by Erin A Greguske Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms Doctoral Thesis presented by Erin A Greguske L’Hospitalet de Llobregat, 2019 Doctoral Program in Biomedicine Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms Department of Physiological Sciences Faculty of Medicine and Health Sciences, Campus Bellvitge Universitat de Barcelona Doctoral Candidate Thesis Director Erin A Greguske Dr. Jordi Llorens Baucells The work of this Thesis was financed by: - Grant FI-DGR 2015 (9015-355847/2014) “Damage and Repair in the Vestibular Sensory Epithelia during Chronic Ototoxicity and Recovery”, from the Agency for Management of University and Research Grants (AGAUR), Generalitat de Catalunya - Grant FPU14-05426 “Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms”, from the Ministry of Education, Culture and Sport of Spain - Project BFU2012-31164 “Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms”, from the Ministry of Economy and Competitiveness of Spain - Project BFU2015-66109-R “Damage and Repair of the Vestibular Synapses during Chronic Ototoxicity”, from the Ministry of Economy and Competitiveness of Spain - Minor Grant “Synaptic Damage in the Vestibular Epithelium in a Chronic Ototoxicity Model”, from the Ménière’s Society in the United Kingdom Acknowledgements Keeping it sweet and simple: Thank you to all my family and loved ones for believing in me every step of the way on this long academic journey. Your support and love were everything in helping me cultivate the passion I have for science and to always finish strong. Thank you to my friends, old and new, for the endless words of encouragement and hype, which kept me happy and sane. Thank you to my colleagues for your teachings that helped me persevere throughout my pursuit of knowledge and a special thanks to Benja and Josep of the CCiTUB and Klaas of the UMIC for all of your help. And thank you Jordi for your endless patience and guidance throughout the years; for believing in my academic strength and helping me achieve this goal. Index 1. Abbreviations………………………………………………………………..1 2. Abstract……………………………………………………………………...7 3. Introduction…………………………………………………………............11 3.1. The Inner Ear…………………………………………………………....13 3.2. The Vertebrate Ear……………………………………………………....14 3.3. The Vestibular System…………………………………………………...14 3.4. The Vestibular Sensory Hair Cells……………………………………….16 3.5. The Vestibular Sensory Afferents………………………………………..17 3.6. Vestibular Sensory Hair Cell Signal Transmission………………………..19 3.7. Vestibular Signaling Pathway to the CNS………………………................21 3.8. The Auditory System……………………………………………………22 3.9. The Organ of Corti, the Sensory Epithelium of the Cochlea…………….23 3.10. The Cochlear Sensory Hair Cells……………………………………...23 3.11. The Cochlear Sensory Afferents……………………………………....25 3.12. The Auditory Efferent Pathway…………………………………….....25 3.13. Cochlear Sensory Hair Cell Signal Transmission………………….…...25 3.14. Auditory Signaling Pathway to the CNS………………………….........26 3.15. Ototoxicity and Aminoglycoside Antibiotics………………………......27 3.16. Animal Studies of Ototoxicity……………………………………........28 3.17. The Introduction of Nitriles for Progressive Ototoxic Studies………..29 4. Objectives…………………………………………………………………..33 5. Methodology……………………………………………………………….37 5.1. Animals………………………………………………………………….39 5.2. Ototoxic IDPN Exposure……………………………………………….39 5.3. Vestibular Dysfunction Ratings (VDRs)…………………………………41 5.4. Auditory Brainstem Response (ABR) Measurements…………………….41 5.5. Tissue Dissection and Epithelia/Ganglia Extraction…………………….42 5.6. Immunofluorescent Studies……………………………………………...42 5.6.1. First Protocol/Study………………………………………………43 5.6.2. Second Protocol/Study……………………………………………43 5.6.3. Third Protocol/Study……………………………………………...44 5.6.4. Fourth Protocol/Study………………………………………….....44 5.6.5. Fifth Protocol/Study……………………………………………....44 5.7. Cochlear Place-Frequency Maps…………………………………………47 5.8. Confocal Microscopy and Image Analysis………………………...............47 5.9. Scanning Electron Microscopy (SEM) Ultrastructural Studies………........49 5.10. Transmission Electron Microscopy (TEM) Ultrastructural Studies…....50 5.11. qRT-PCR Studies……………………………………………................50 5.12. RNA-sequencing (RNAseq)…………………………………................52 5.13. Data Analysis-SPSS………………………………………….................52 6. Results……………………………………………………………….............55 6.1. Preliminary Total RNA Isolation Study………………………………......57 6.1.1. Testing various protocols………………………………………........57 6.1.2. Qiagen proves to be the most consistent, efficient, and effective protocol……………………………………………………...............58 6.2. Characterizing Vestibular Damage…………………………………..........59 6.2.1. Effects of sub-chronic IDPN on 129S1 body weight…………..........59 6.2.2. Effects of sub-chronic IDPN on 129S1 vestibular function………...60 6.2.3. SEM analysis of stereociliary damage and HC loss following sub- chronic IDPN exposure………………………………………..........61 6.2.4. Light Microscopy and TEM analysis of calyceal junction loss, stereociliary fusion, and HC extrusion following sub-chronic IDPN exposure………………………………………………………….....64 6.2.5. Molecular analysis of calyceal junction dismantlement following sub- chronic IDPN exposure…………………………………………….66 6.2.6. mRNA expression of tenascin-c in the vestibular sensory epithelia is altered following sub-chronic IDPN exposure……………………...68 6.2.7. Molecular analysis of synaptic uncoupling and post-synaptic density protein alterations following sub-chronic IDPN exposure………….69 6.2.8. mRNA expression of various synaptic, scaffolding, and signaling proteins in the vestibular sensory epithelia and ganglia are altered following sub-chronic IDPN exposure……………………………..75 6.2.9. Preliminary RNAseq data of the vestibular ganglia and vestibular sensory epithelia demonstrate a depression in signal transmission in the afferent and cell-cell adhesion in the epithelia following sub- chronic IDPN exposure…………………………………………….76 6.2.10. Corroborating RNAseq results with qRT-PCR analyses of selected neurotrophic factors, synaptic, receptor, and kinase proteins and proteins associated with molecule transport…........................................81 6.3. Characterizing Auditory Damage…………………………………............82 6.3.1. Effects of sub-chronic IDPN on 129S2 body weight………….......82 6.3.2. Effects of sub-chronic IDPN on 129S2 vestibular function………83 6.3.3. IDPN exposure strongly affects the hearing of treated mice…........83 6.3.4. IDPN exposure causes a robust loss of OHCs, but IHCs remain
Load more

Recommended publications
  • Anti-Rab11 Antibody (ARG41900)
    Product datasheet [email protected] ARG41900 Package: 100 μg anti-Rab11 antibody Store at: -20°C Summary Product Description Goat Polyclonal antibody recognizes Rab11 Tested Reactivity Hu, Ms, Rat, Dog, Mk Tested Application IHC-Fr, IHC-P, WB Host Goat Clonality Polyclonal Isotype IgG Target Name Rab11 Antigen Species Mouse Immunogen Purified recombinant peptides within aa. 110 to the C-terminus of Mouse Rab11a, Rab11b and Rab11c (Rab25). Conjugation Un-conjugated Alternate Names RAB11A: Rab-11; Ras-related protein Rab-11A; YL8 RAB11B: GTP-binding protein YPT3; H-YPT3; Ras-related protein Rab-11B RAB25: RAB11C; CATX-8; Ras-related protein Rab-25 Application Instructions Application table Application Dilution IHC-Fr 1:100 - 1:400 IHC-P 1:100 - 1:400 WB 1:250 - 1:2000 Application Note IHC-P: Antigen Retrieval: Heat mediation was recommended. * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control Hepa cell lysate Calculated Mw 24 kDa Observed Size ~ 26 kDa Properties Form Liquid Purification Affinity purification with immunogen. Buffer PBS, 0.05% Sodium azide and 20% Glycerol. Preservative 0.05% Sodium azide www.arigobio.com 1/3 Stabilizer 20% Glycerol Concentration 3 mg/ml Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. Note For laboratory research only, not for drug, diagnostic or other use.
    [Show full text]
  • Supplementary Table 1: Adhesion Genes Data Set
    Supplementary Table 1: Adhesion genes data set PROBE Entrez Gene ID Celera Gene ID Gene_Symbol Gene_Name 160832 1 hCG201364.3 A1BG alpha-1-B glycoprotein 223658 1 hCG201364.3 A1BG alpha-1-B glycoprotein 212988 102 hCG40040.3 ADAM10 ADAM metallopeptidase domain 10 133411 4185 hCG28232.2 ADAM11 ADAM metallopeptidase domain 11 110695 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 195222 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 165344 8751 hCG20021.3 ADAM15 ADAM metallopeptidase domain 15 (metargidin) 189065 6868 null ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme) 108119 8728 hCG15398.4 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 117763 8748 hCG20675.3 ADAM20 ADAM metallopeptidase domain 20 126448 8747 hCG1785634.2 ADAM21 ADAM metallopeptidase domain 21 208981 8747 hCG1785634.2|hCG2042897 ADAM21 ADAM metallopeptidase domain 21 180903 53616 hCG17212.4 ADAM22 ADAM metallopeptidase domain 22 177272 8745 hCG1811623.1 ADAM23 ADAM metallopeptidase domain 23 102384 10863 hCG1818505.1 ADAM28 ADAM metallopeptidase domain 28 119968 11086 hCG1786734.2 ADAM29 ADAM metallopeptidase domain 29 205542 11085 hCG1997196.1 ADAM30 ADAM metallopeptidase domain 30 148417 80332 hCG39255.4 ADAM33 ADAM metallopeptidase domain 33 140492 8756 hCG1789002.2 ADAM7 ADAM metallopeptidase domain 7 122603 101 hCG1816947.1 ADAM8 ADAM metallopeptidase domain 8 183965 8754 hCG1996391 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 129974 27299 hCG15447.3 ADAMDEC1 ADAM-like,
    [Show full text]
  • Rabbit Anti-CASPR/FITC Conjugated Antibody-SL11128R-FITC
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-CASPR/FITC Conjugated antibody SL11128R-FITC Product Name: Anti-CASPR/FITC Chinese Name: FITC标记的轴突蛋白4/少突胶质细胞抗体 Neurexin4; caspr 1; Caspr; Caspr1; Cntnap 1; Cntnap1; CNTP 1; CNTP1; CNTP1_HUMAN; Contactin associated protein 1; Contactin-associated protein 1; Alias: MHDNIV; NCP 1; NCP1; Neurexin 4; Neurexin IV; Neurexin-4; Nrxn 4; Nrxn4; p190; Paranodin. Organism Species: Rabbit Clonality: Polyclonal React Species: Human,Mouse,Rat,Dog,Pig,Cow,Horse, Flow-Cyt=1:50-200ICC=1:50-200IF=1:50-200 Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 154kDa Cellular localization: The cell membrane Form: Lyophilized or Liquid Concentration: 1mg/ml immunogen: KLH conjugated synthetic peptide derived from human CASPR/Neurexin4 Lsotype: IgGwww.sunlongbiotech.com Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. background: Neurexins comprise a family of neuronal cell surface proteins, which include neurexin I (NRXN1), neurexin II (NRXN2), neurexin III (NRXN3) and CASPR (neurexin IV). Product Detail: Neurexins I-III are expressed as ? and ∫ isoforms.
    [Show full text]
  • Mouse Rab11fip2 Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Rab11fip2 Knockout Project (CRISPR/Cas9) Objective: To create a Rab11fip2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Rab11fip2 gene (NCBI Reference Sequence: NM_001164367 ; Ensembl: ENSMUSG00000040022 ) is located on Mouse chromosome 19. 5 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 5 (Transcript: ENSMUST00000171986). Exon 2 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from the coding region. Exon 2 covers 33.33% of the coding region. The size of effective KO region: ~443 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 5 Legends Exon of mouse Rab11fip2 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 913 bp section downstream of Exon 2 is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • Downloaded the “Top Edge” Version
    bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. 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. 1 Drosophila models of pathogenic copy-number variant genes show global and 2 non-neuronal defects during development 3 Short title: Non-neuronal defects of fly homologs of CNV genes 4 Tanzeen Yusuff1,4, Matthew Jensen1,4, Sneha Yennawar1,4, Lucilla Pizzo1, Siddharth 5 Karthikeyan1, Dagny J. Gould1, Avik Sarker1, Yurika Matsui1,2, Janani Iyer1, Zhi-Chun Lai1,2, 6 and Santhosh Girirajan1,3* 7 8 1. Department of Biochemistry and Molecular Biology, Pennsylvania State University, 9 University Park, PA 16802 10 2. Department of Biology, Pennsylvania State University, University Park, PA 16802 11 3. Department of Anthropology, Pennsylvania State University, University Park, PA 16802 12 4 contributed equally to work 13 14 *Correspondence: 15 Santhosh Girirajan, MBBS, PhD 16 205A Life Sciences Building 17 Pennsylvania State University 18 University Park, PA 16802 19 E-mail: [email protected] 20 Phone: 814-865-0674 21 1 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. 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. 22 ABSTRACT 23 While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non- 24 neuronal phenotypes, functional studies evaluating these regions have focused on the molecular 25 basis of neuronal defects.
    [Show full text]
  • (Caspr) and Contactin Form a Complex That Is Targeted to the Paranodal Junctions During Myelination
    The Journal of Neuroscience, November 15, 2000, 20(22):8354–8364 Contactin-Associated Protein (Caspr) and Contactin Form a Complex That Is Targeted to the Paranodal Junctions during Myelination Jose C. Rios,1 Carmen V. Melendez-Vasquez,1 Steven Einheber,1 Marc Lustig,2 Martin Grumet,2 John Hemperly,5 Elior Peles,6 and James L. Salzer1,3,4 Departments of 1Cell Biology, 2Pharmacology, 3Neurology, and the 4Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016, 5BD Technologies, Research Triangle Park, North Carolina 27709, and 6Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel Specialized paranodal junctions form between the axon and the associated specifically with Caspr in the paranodes, whereas a closely apposed paranodal loops of myelinating glia. They are higher-molecular-weight form of contactin, not associated with interposed between sodium channels at the nodes of Ranvier Caspr, is present in central nodes of Ranvier. These results and potassium channels in the juxtaparanodal regions; their suggest that the targeting of contactin to different axonal do- precise function and molecular composition have been elusive. mains may be determined, in part, via its association with Caspr. We previously reported that Caspr (contactin-associated protein) Treatment of myelinating cocultures of Schwann cells and neu- is a major axonal constituent of these junctions (Einheber et al., rons with RPTP␤–Fc, a soluble construct containing the carbonic 1997). We now report that contactin colocalizes and forms a cis anhydrase domain of the receptor protein tyrosine phosphatase complex with Caspr in the paranodes and juxtamesaxon.
    [Show full text]
  • Itraq‑Based Proteomics Analysis of the Therapeutic Effects of Combined Anticancer Bioactive Peptides and Oxaliplatin on Gastric Cancer Cells
    ONCOLOGY REPORTS 43: 201-217, 2020 iTRAQ‑based proteomics analysis of the therapeutic effects of combined anticancer bioactive peptides and oxaliplatin on gastric cancer cells YANAN XU1, XIAN LI2 and XIULAN SU1,2 1Department of Cell Biology, College of Basic Medicine, Capital Medical University, Beijing 100069; 2Clinical Medical Research Center, The Affiliated Hospital of Inner Mongolia Medical University, Inner Mongolia Autonomous Region 010050, P.R. China Received April 2, 2019; Accepted September 25, 2019 DOI: 10.3892/or.2019.7406 Abstract. The combination of chemotherapeutic modalities cells treated with ACBP, OXA and ACBP-OXA exhibited 17 may be more effective in treating gastric cancer compared (10 up- and 7 downregulated), 111 (27 up- and 84 downregu- with any modality alone. Previous studies have demonstrated lated) and 128 (53 up- and 75 downregulated) differentially that the combination of anticancer bioactive peptides (ACBP) expressed proteins, respectively. Of the 256 differentially and oxaliplatin (OXA) significantly inhibited the growth of the expressed proteins, 6 (TPX2, NUSAP1, TOP2A, YAP, MKi-67 gastric cancer cell line MKN-45, promoted the apoptosis of and GPC4) were verified by the parallel reaction monitoring MKN-45 cells, and caused an irreversible arrest of the MKN-45 method, which revealed that TPX2, NUSAP1, TOP2A, YAP, cell cycle in the G2/M phase. In the present study, an isobaric MKi-67 and GPC4 expression decreased with ACBP-OXA tag for relative and absolute quantitation (iTRAQ)-based treatment. The cellular localization, functional annotation and quantitative proteomics technique was used to determine the biological pathways of differentially expressed proteins were effect of ACBP-OXA treatment on the proteomics profile examined by Gene Ontology and Kyoto Encyclopedia of Genes of MKN-45 cells.
    [Show full text]
  • Mir-15A and Mir-16-1 Cluster Functions in Human Leukemia
    MiR-15a and miR-16-1 cluster functions in human leukemia George A. Calin*†‡, Amelia Cimmino*§, Muller Fabbri*, Manuela Ferracin¶, Sylwia E. Wojcik*, Masayoshi Shimizu*§, Cristian Taccioli*, Nicola Zanesi*, Ramiro Garzon*, Rami I. Aqeilan*, Hansjuerg Alder*, Stefano Volinia*ʈ, Laura Rassenti**, Xiuping Liu*, Chang-gong Liu*, Thomas J. Kipps**, Massimo Negrini¶, and Carlo M. Croce*†† *Human Cancer Genetics Program and †Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210; §Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, 80138 Naples, Italy; ¶Department of Experimental and Diagnostic Medicine, Interdepartment Center for Cancer Research and ʈMorphology and Embryology Department, University of Ferrara, 44100 Ferrara, Italy; and **Department of Medicine, University of California at San Diego, La Jolla, CA 92093 Contributed by Carlo M. Croce, January 16, 2008 (sent for review December 6, 2007) MicroRNAs (miRNAs) are short noncoding RNAs regulating gene megakaryocytic leukemia (22). These data support a role for expression that play roles in human diseases, including cancer. Each miR-15a and miR-16-1 as tumor-suppressor genes (TSGs) in CLLs miRNA is predicted to regulate hundreds of transcripts, but only few and perhaps in other malignancies in which these genes are lost or have experimental validation. In chronic lymphocytic leukemia (CLL), down-regulated. the most common adult human leukemia, miR-15a and miR-16-1 are Here, to investigate the mechanism of action of miR-15a and lost or down-regulated in the majority of cases. After our previous miR-16-1 as tumor suppressors in leukemias, we analyzed the work indicating a tumor suppressor function of miR-15a/16-1 by effects of miR-15a and miR-16-1 on transcriptome and proteome in targeting the BCL2 oncogene, here, we produced a high-throughput MEG-01 leukemic cells.
    [Show full text]
  • The Role of Proteoglycans in the Initiation of Neural Tube Closure
    The role of proteoglycans in the initiation of neural tube closure Oleksandr Nychyk Thesis submitted to UCL for the degree of Doctor of Philosophy 2017 Developmental Biology of Birth Defects Developmental Biology & Cancer Programme UCL Great Ormond Street Institute of Child Health Declaration of contribution I, Oleksandr Nychyk confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in my thesis. ___________________________________________ Oleksandr Nychyk 2 Abstract Neurulation is the embryonic process that gives rise to the neural tube (NT), the precursor of the brain and spinal cord. Recent work has emphasised the importance of proteoglycans in convergent extension movements and NT closure in lower vertebrates. The current study is focused on the role of proteoglycans in the initiation of NT closure in mammals, termed closure 1. In this project, the initial aim was to characterise the ‘matrisome’, or in vivo extracellular matrix (ECM) composition, during mammalian neurulation. Tissue site of mRNA expression and protein localisation of ECM components, including proteoglycans, were then investigated showing their distinct expression patterns prior to and after the onset of neural tube closure. The expression analysis raised various hypothesis that were subsequently tested, demonstrating that impaired sulfation of ECM proteoglycan chains worsens the phenotype of planar cell polarity (PCP) mutant loop tail (Vangl2Lp) predisposed to neural tube defects. Exposure of Vangl2Lp/+ embryos to chlorate, an inhibitor of glycosaminoglycan sulfation, during ex vivo whole embryo culture prevented NT closure, converting Vangl2Lp/+ to the mutant Vangl2Lp/Lp pathophenotype. The same result was obtained by exposure of Vangl2Lp/+ + embryos to chondroitinase or heparitinase.
    [Show full text]
  • LRP1 Regulates Peroxisome Biogenesis and Cholesterol
    RESEARCH ARTICLE LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair Jing-Ping Lin1, Yevgeniya A Mironova2, Peter Shrager3, Roman J Giger1,2,4,5* 1Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI, United States; 2Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, MI, United States; 3Department of Neuroscience, University of Rochester Medical Center, Rochester, NY, United States ; 4Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, United States; 5Interdepartmental Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, MI, United States Abstract Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling molecule broadly expressed by neurons and glia. In adult mice, global inducible (Lrp1flox/ flox;CAG-CreER) or oligodendrocyte (OL)-lineage specific ablation (Lrp1flox/flox;Pdgfra-CreER) of Lrp1 attenuates repair of damaged white matter. In oligodendrocyte progenitor cells (OPCs), Lrp1 is required for cholesterol homeostasis and differentiation into mature OLs. Lrp1-deficient OPC/ OLs show a strong increase in the sterol-regulatory element-binding protein-2 yet are unable to maintain normal cholesterol levels, suggesting more global metabolic deficits. Mechanistic studies revealed a decrease in peroxisomal biogenesis factor-2 and fewer peroxisomes in OL processes. / *For correspondence: Treatment of Lrp1À À OPCs with cholesterol or activation of peroxisome proliferator-activated [email protected] receptor-g with pioglitazone alone is not sufficient to promote differentiation; however, when combined, cholesterol and pioglitazone enhance OPC differentiation into mature OLs. Collectively, Competing interests: The our studies reveal a novel role for Lrp1 in peroxisome biogenesis, lipid homeostasis, and OPC authors declare that no competing interests exist.
    [Show full text]
  • Nucleotide Bound to Rab11a Controls Localization in Rod Cells but Not Interaction with Rhodopsin
    14854 • The Journal of Neuroscience, November 5, 2014 • 34(45):14854–14863 Cellular/Molecular Nucleotide Bound to rab11a Controls Localization in Rod Cells But Not Interaction with Rhodopsin Nicholas J. Reish,1,2 Evan R. Boitet,1,3 Katie L. Bales,1,3 and XAlecia K. Gross1,2,3 1Evelyn F. McKnight Brain Institute, 2Department of Neurobiology, and 3Department of Vision Sciences, School of Optometry, University of Alabama at Birmingham, Birmingham, Alabama 35294 Precise vectorial transport of rhodopsin is essential for rod photoreceptor health and function. Mutations that truncate or extend the C terminus of rhodopsin disrupt this transport, and lead to retinal degeneration and blindness in human patients and in mouse models. Here we show that such mutations disrupt the binding of rhodopsin to the small GTPase rab11a. The rhodopsin–rab11a interaction is a direct binding interaction that does not depend on the nucleotide binding state of rab11a. Expression of EGFP-rab11a fusion proteins in Xenopus laevis photoreceptors revealed that the nucleotide binding status of rab11a affects its subcellular localization, with GTP-locked mutants concentrated in the inner segment and GDP-locked mutants concentrated in the outer segment. shRNA-mediated knockdown of rab11a in rods led to shortened outer segments and retinal degeneration. Together, our results show the critical importance of direct rhodopsin–rab11a interactions for the formation and maintenance of vertebrate photoreceptors. Key words: protein interactions; protein trafficking; rab11a; rhodopsin Introduction Golgi and the distal inner segment (IS) in lipid vesicles (Deretic Rod photoreceptors sense light using a specialized organelle and Papermaster, 1991) and may traffic into the outer segment known as the outer segment, a unique structure consisting of a (OS) in vesicular form (Sung and Chuang, 2010) or through primary cilium and a stack of closely spaced membranous disks intraflagellar transport (Bhowmick et al., 2009), or by mecha- that are contained within a plasma membrane.
    [Show full text]
  • ID AKI Vs Control Fold Change P Value Symbol Entrez Gene Name *In
    ID AKI vs control P value Symbol Entrez Gene Name *In case of multiple probesets per gene, one with the highest fold change was selected. Fold Change 208083_s_at 7.88 0.000932 ITGB6 integrin, beta 6 202376_at 6.12 0.000518 SERPINA3 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 1553575_at 5.62 0.0033 MT-ND6 NADH dehydrogenase, subunit 6 (complex I) 212768_s_at 5.50 0.000896 OLFM4 olfactomedin 4 206157_at 5.26 0.00177 PTX3 pentraxin 3, long 212531_at 4.26 0.00405 LCN2 lipocalin 2 215646_s_at 4.13 0.00408 VCAN versican 202018_s_at 4.12 0.0318 LTF lactotransferrin 203021_at 4.05 0.0129 SLPI secretory leukocyte peptidase inhibitor 222486_s_at 4.03 0.000329 ADAMTS1 ADAM metallopeptidase with thrombospondin type 1 motif, 1 1552439_s_at 3.82 0.000714 MEGF11 multiple EGF-like-domains 11 210602_s_at 3.74 0.000408 CDH6 cadherin 6, type 2, K-cadherin (fetal kidney) 229947_at 3.62 0.00843 PI15 peptidase inhibitor 15 204006_s_at 3.39 0.00241 FCGR3A Fc fragment of IgG, low affinity IIIa, receptor (CD16a) 202238_s_at 3.29 0.00492 NNMT nicotinamide N-methyltransferase 202917_s_at 3.20 0.00369 S100A8 S100 calcium binding protein A8 215223_s_at 3.17 0.000516 SOD2 superoxide dismutase 2, mitochondrial 204627_s_at 3.04 0.00619 ITGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) 223217_s_at 2.99 0.00397 NFKBIZ nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta 231067_s_at 2.97 0.00681 AKAP12 A kinase (PRKA) anchor protein 12 224917_at 2.94 0.00256 VMP1/ mir-21likely ortholog
    [Show full text]