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Characterization of Tspo 18 Kda

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Characterization of Tspo 18 Kda CHARACTERIZATION OF TSPO 18 KDA IN NEUROSTEROID SYNTHESIS AND MITOCHONDRIAL METABOLISM CHARAKTERISIERUNG VON TSPO 18 KDA IN DER NEUROSTEROIDSYNTHESE UND DEM MITOCHONDRIALEN STOFFWECHSEL DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN FAKULTÄT IV CHEMIE UND PHARMAZIE DER UNIVERSITÄT REGENSBURG vorgelegt von Luisa Scheck, geb. Wolf aus Schmalkalden im Februar 2017 Promotionsgesuch eingereicht am: 24. Februar 2017 Die Arbeit wurde angeleitet von: Prof. Dr. med. Dr. rer. nat. Ekkehard Haen Table of contents ACKNOWLEDGEMENTS .................................................................................................. 1 ABSTRACT ................................................................................................................... 2 TRANSLATION OF THE ABSTRACT (GERMAN) .................................................................. 4 1 TSPO 18KDA - A CONTROVERSIAL PROTEIN OF THE OMM ....................................... 6 1.1 Expression of TSPO ...................................................................................... 6 1.2 TSPO structure and resulting functions ......................................................... 7 1.3 Recent TSPO structure ................................................................................. 8 1.4 TSPO ligands ................................................................................................ 9 2 NEW PERSPECTIVES FOR TSPO ........................................................................... 11 2.1 TSPO in neurosteroid synthesis of brain mitochondria ................................ 12 2+ 2.2 TSPO in mitochondrial Ca homoeostasis .................................................. 15 2.3 TSPO in mitochondrial respiration ............................................................... 17 2.4 TSPO in mitochondrial energy metabolism, inflammation and protection .... 19 2.5 TSPO in the brain ........................................................................................ 21 3 AIM OF THE THESIS .............................................................................................. 23 4 MATERIALS AND METHODS ................................................................................... 24 4.1 Cell culture procedure of BV-2 mouse microglia cells ................................. 24 4.2 TSPO knock-down in BV-2 mouse microglia cells ....................................... 24 4.3 Immunohistochemistry ................................................................................. 28 4.4 TSPO distribution in different mouse tissues ............................................... 29 4.5 Western Blotting .......................................................................................... 29 4.6 Pregnenolone Quantification ....................................................................... 30 4.6.1 Enzyme-Linked Immunosorbent Assay (ELISA) ................................... 32 4.6.2 GC/MS analysis .................................................................................... 32 4.7 The relationship of TSPO ligand pharmacology and neurosteroidogenesis 34 4.7.1 Western Blot ......................................................................................... 34 4.7.2 Pregnenolone ELISA ............................................................................ 34 4.7.3 [³H]PK11195 radioligand binding assay ................................................ 34 2+ 4.8 Analysis of intracellular Ca levels with FURA-2 dye ................................. 35 4.9 Mitochondrial membrane potential analysis with JC-1 dye .......................... 39 4.10 Mitochondrial respiration experiments ......................................................... 41 4.10.1 Analysis of mitochondrial respiration in intact cells ............................ 42 4.10.2 High resolution respirometry (HRR) with the oxygraph-2k (O2k) ....... 45 5 RESULTS ............................................................................................................. 47 5.1 Confirmation of the BV-2-TSPOknock-down ...................................................... 47 5.2 Low TSPO expression in mouse wild-type brain ......................................... 49 5.3 Regulation of TSPO protein expression ...................................................... 50 5.4 Pregnenolone quantification with ELISA technique ..................................... 52 5.4.1 Neurosteroid quantification with GC/MS analysis ................................. 54 5.5 TSPO ligand pharmacology in relationship to neurosteroidogenesis........... 56 5.5.1 Analysis of TSPO protein expression after TSPO ligand treatment ...... 56 5.5.2 Analysis of the TSPO binding affinity after TSPO ligand treatment ....... 57 5.5.3 Neurosteroid synthesis ......................................................................... 58 2+ 5.6 Ca levels via FURA-2 fluorescence technique .......................................... 59 2+ 5.6.1 Ca imaging of BV-2scramble cells .......................................................... 59 5.7 Analysis of the mitochondrial membrane potential ...................................... 63 5.8 The bioenergetic profile of intact BV-2scramble cells ....................................... 65 5.9 The bioenergetic profile of intact BV-2scramble and BV-2-TSPOknock-down cells 67 5.10 High resolution respirometry in permeabilized cells..................................... 69 6 DISCUSSION ........................................................................................................ 73 7 CONCLUSION ....................................................................................................... 82 REFERENCES .............................................................................................................. 84 Acknowledgements 1 Acknowledgements Schreiben musste ich diese Arbeit zwar allein – doch an ihrem guten Gelingen waren einige mir nahestehender Menschen beteiligt, denen ich dafür Dank schulde. An erster Stelle muss mein Doktorvater Prof. Dr. Ekkehard Haen genannt werden. Herzlichen Dank für die Unterstützung. Ein besonderer Dank geht an Prof. Dr. Christian Wetzel und Dr. Vladimir Milenkovic, die mir die technischen Möglichkeiten und das technische Know-how gegeben und vermittelt haben, die Arbeit in dieser methodischen Vielfalt durchzuführen. Darüber hinaus möchte ich Prof. Rupprecht und Dr. Nothdurfter für die Zusammenarbeit danken. Auch Dr. Markus Reichold, Dr. Michael Gruber und Prof. Dr. Marina Kreutz als meine Kooperationspartner sind hier besonders zu benennen. Für mich war es äußerst wertvoll, dass es ehrliche und offene Kritik gab und eine sehr gute Kooperationsarbeit. Für diese Ehrlichkeit, die Kollegialität und die tolle Zusammenarbeit der Institute bin ich sehr dankbar. Ein persönlicher Dank gilt meinen Mann Johannes Scheck. Wenn ich mit dem Kopf mal wieder nicht zuhause, sondern bei der Arbeit war, ist er es gewesen, der mich dabei immer unterstützt und motiviert hat. Dieser Beistand ist für mich nicht selbstverständlich, sondern ein sehr besonderes Gut. Darüber hinaus möchte ich meinen Laborassistentinnen Heike Haloff-Buestrich, Tatjana Jahner, Doris Melchner, Petra Störtebecker, Martina Domani und Regina Lindner sowie meiner Kollegin Angelika Bauer danken, die mich jahrelang motiviert haben und mir Rückhalt boten. Abstract 2 Abstract The translocator protein (18 kDa) (TSPO) is a highly conserved, ubiquitous protein with a high affinity in binding cholesterol. Located in the outer mitochondrial membrane (OMM), TSPO has been discussed as the key protein for the cholesterol transport and therefore as a critical factor in steroidogenesis for a long time. Since recent in vivo and in vitro studies using TSPO knock-out models have been refuting its role in steroidogenesis, it is still an open question if TSPO is involved in neurosteroidogenesis. Furthermore, studies of the last two years started to characterize TSPO as a key protein in mitochondrial metabolism, assuming that TSPO has a regulatory function in calcium (Ca2+) homoeostasis, glucose homoeostasis, mitochondrial respiration and ROS (reactive oxygen species) production. In this thesis the link between TSPO and the regulation of mitochondrial metabolism and neurosteroid synthesis was analyzed to characterize a possible role of TSPO. In order to ascertain a possible involvement of TSPO in neurosteroidogenesis, a TSPO knock-down in mouse brain cells was performed to measure pregnenolone concentration as a precursor of neurosteroid synthesis by means of ELISA technique (enzyme-linked immunosorbent assay) and GC/MS (gaschromatography/mass spectroscopy). I was able to show that TSPO regulates neurosteroid synthesis of brain microglia cells. The regulatory function of TSPO in mitochondrial Ca2+ homoeostasis was analyzed by means of Ca2+ imaging via FURA-2 dye. The results showed no significant differences in Ca2+ homoeostasis of TSPO knock-down and scramble cells. Using drug treatment, both TSPO agonists XBD173 and PK11195 showed TSPO unrelated effects. Only LPS and Ro5-4864 treatment lead to the assumption that the presence of TSPO might be necessary to protect the cell from cell death under special conditions. Considering TSPO as a regulatory protein of the mitochondrial energy metabolism, the mitochondrial membrane potential was analyzed under different conditions using the ratiometric JC-1 dye. My results show a direct link between the TSPO knock- down and the significantly reduced energy state of the cell. To further
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