The role of endocannabinoid system in brain aging PDF
Preview The role of endocannabinoid system in brain aging
The role of endocannabinoid system in brain aging Thesis Submitted for a Doctoral Degree in Natural Sciences (Dr. rer. nat.) Faculty of Mathematics and Natural Sciences Rheinische Friedrich Wilhelms University, Bonn submitted by Anastasia Piyanova from Moscow Bonn, 29.03.12 Prepared with the consent of the Faculty of Mathematics and Natural Sciences, Rheinische Friedrich Wilhelms University, Bonn 1. Reviewer: Priv.-‐Doz. Dr. Andras Bilkei-‐Gorzo 2. Reviewer: Prof. Dr. Gerhard von der Emde Examination date: 28.06.12 Year of publication: 2012 1-2 Disclosure statement I hereby declare that I prepared this thesis entitled: “The role of endocannabinoid system in brain aging” by myself except where otherwise stated. All text passages that are literally or correspondingly taken from published or unpublished papers are indicated as such. All materials or services provided by other people are equally indicated. Part of this thesis was published as listed: Albayram O, Alferink J, Pitsch J, Piyanova A, Neitzert K, Poppensieker K, Mauer D, Michel K, Legler A, Becker A, Monory K, Lutz B, Zimmer A, Bilkei-‐Gorzo A. Role of CB1 cannabinoid receptors on GABAergic neurons in brain aging. Proc Natl Acad Sci U S A 2011 Jul 5; 108(27):11256-‐61. Epub 2011 Jun 20 Bonn, den 29.03.12 Anastasia Piyanova 1-3 All diseases run into one, old age. (Ralph Waldo Emerson) 1-4 Abbreviations 2-‐AG = 2-‐arachidonoylglycerol 2D-‐PAGE = two-‐dimensional polyacrylamide gel electrophoresis AA = arachidonic acid ACN = acetonitrile AEA = anandamide Akt (protein kinase B, PKB) = alpha serine/threonine-‐protein kinase APP/PS1 = amyloid precursor protein/presenilin 1 BCA = bicinchoninic acid BDNF = brain-‐derived neurotrophic factor BSA = bovine serum albumine BrdU = 5-‐bromo-‐2'-‐deoxyuridine CA1/2/3 = cornu ammonis areas 1,2,3 CCD = charge-‐coupled device CB1, CB1R, Cnr1 = cannabinoid receptor type 1 CB2, Cnr2 = cannabinoid receptor type 2 cDNA = complementary DNA CO = carbon dioxide 2 Cnr1+/+ = wildtype (for cannabinoid receptor 1) Cnr1-‐/-‐ = knockout (for cannabinoid receptor 1) 1-5 Ct = cycle threshold CuSO = copper (II) sulfate 4 DAPI = 4’,6-‐diamidino-‐2-‐phenylindole DAGLα = diacylglycerol lipase alpha DAGLβ = diacylglycerol lipase beta DEPC = diethylpyrocarbonate DG = dentate gyrus DNA = deoxyribonucleic acid DNPH = 2,4-‐dinitrophenylhydrazine DTT = dithiothreitol EC = endocannabinoid ECL = enhanced chemiluminescence EDTA = ethylenediaminetetraacetic acid e.g. = for example FA = formic acid FAAH = fatty acid amide hydrolase FADD = Fas-‐associated protein with death domain GABA = gamma-‐aminobutyric acid GAPDH = glyceraldehyde 3–phosphate dehydrogenase h = hours (s) 1-6 H O = water 2 H O = hydrogen peroxide 2 2 H PO = phosphoric acid 3 4 HCl = hydrochloric acid HRP = horseradish peroxidase HTPLC = high-‐performance thin-‐layer chromatography Iba1 = ionized calcium binding adaptor molecule 1 IEF = isoelectric focusing IL6 = interleukin 6 IPG = immobilized pH gradient Kir = inwardly rectifying potassium channels LAMP2 = lysosomal-‐associated membrane protein 2 LC-‐ESI/QTOF-‐MS = high performance liquid chromatography coupled with electrospray ionization-‐quadripole/time of flight hybrid mass spectrometry LC3 = microtubule-‐associated protein light chain 3 LC-‐MS/MS = liquid chromatography/tandem mass spectrometry LDS (buffer) = lithium dodecyl sulfate MAGL = monoacylglycerol lipase MES (buffer) = 2-‐(N-‐morpholino)ethanesulfonic acid milliQ (TM Millipore) = ultrapure water 1-7 MOPS (buffer) = 3-‐(N-‐morpholino)propanesulfonic acid MRM = multiple-‐reaction monitoring mRNA = messenger RNA mtDNA = mitochondrial DNA mTOR = mammalian target of rapamycin NeuN = neuronal nuclear antigen n.s. = not significant OEA = oleoylethanolamide p62 (SQSTM1) = sequestome 1 PARP = Poly (ADP-‐ribose) polymerase PBS = phosphate buffered saline PC = personal computer PFA = paraformaldehyde PEA = palmithoylethanolamide qPCR (RT-‐PCR) = quantitative (reverse-‐transcriptase) polymerase chain reaction RIPA (buffer) = radioimmunoprecipitation assay RNA = ribonucleic acid ROS = reactive oxygen species RT = reverse transcriptase SDS = sodium dodecyl sulphate 1-8 TBARS = thiobarbituric acid reactive substances TBS = Tris-‐buffered saline TBST = Tris-‐buffered saline with Tween 20 TCA = trichloroacetic acid TLC = thin-‐layer chromatography SDS-‐PAGE = sodium dodecyl sulphate polyacrylamide gel electrophoresis SEM = standard error of mean WT = wildtype 1-9 1. ABSTRACT (SUMMARY)......................................................................................................1-‐13 2. INTRODUCTION..................................................................................................................2-‐14 2.1 Aging of the brain: theories and mechanisms.......................................................................................2-15 2.2 The endocannabinoid system.....................................................................................................................2-19 2.3 The emerging role of the endocannabinoid system in brain aging...............................................2-21 2.4 Aims of this work............................................................................................................................................2-25 3. MATERIALS AND METHODS................................................................................................3-‐27 3.1 Equipment.........................................................................................................................................................3-27 3.2 Software and databases................................................................................................................................3-29 3.3 Antibodies.........................................................................................................................................................3-30 3.3.1 Primary Antibodies..................................................................................................................................................3-‐30 3.3.2 Secondary Antibodies.............................................................................................................................................3-‐30 3.4 Kits.......................................................................................................................................................................3-31 3.5 Animals...............................................................................................................................................................3-31 3.6 Tissue preparation methods.......................................................................................................................3-32 3.6.1 Brain isolation and punch technique (isolation of brain areas)...........................................................3-‐32 3.6.2 Transcardial perfusion...........................................................................................................................................3-‐32 3.6.3 Preparation of frozen brain slices for histology..........................................................................................3-‐33 3.7 Genotyping........................................................................................................................................................3-33 3.7.1 Sample preparation.................................................................................................................................................3-‐33 3.7.2 Polimerase-‐chain reaction (PCR)......................................................................................................................3-‐33 3.7.3 Detection of PCR products: agarose gel electrophoresis and gel staining with ethidium bromide 3-‐34 3.8 Oxidative stress determination: colorimetric assays and 2D-Western blots.............................3-34 3.8.1 Lipid peroxidation assay.......................................................................................................................................3-‐34 3.8.2 Protein carbonylation assay................................................................................................................................3-‐35 3.8.3 Derivatization of protein carbonyls for 2D-‐Western blotting...............................................................3-‐36 3.9 Protein isolation..............................................................................................................................................3-37 3.9.1 Protein isolation from frozen brain tissue.....................................................................................................3-‐37 1-10
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