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Lack of Adrenoleukodystrophy Protein Enhances Oligodendrocyte Disturbance and Microglia Activation in Mice with Combined Abcd1/Mag Deficiency

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Lack of Adrenoleukodystrophy Protein Enhances Oligodendrocyte Disturbance and Microglia Activation in Mice with Combined Abcd1/Mag Deficiency Lack of Adrenoleukodystrophy protein enhances oligodendrocyte disturbance and microglia activation in mice with combined Abcd1/Mag deficiency Dissertation submitted for the academic qualification Doctor of Medical Science at the Medical University of Vienna Dr. Martina Dumser August 2007 1 This work was conducted under the supervision of Ao. Univ. Prof. Dr. Johannes Berger at the Center for Brain Research Medical University of Vienna Department of Neuroimmunology Spitalgasse 4, 1090 Vienna between January 2005 and June 2007 Supervisors: Ao. Univ. Prof. Dr. Johannes Berger Univ. Prof. Dr. Hans Lassmann Univ. Prof. Dr. Klaus-Armin Nave The work was financed by the European Adrenoleukodystrophy Association (ELA)/ The Myelin Project 2 We shall not cease from exploration And the end of all our exploring will be To arrive where we started And know the place for the first time. (T.S. Eliot, nobel price for literature 1948; Four quartets, quartet No. 4: “Little Gidding”) 3 Danksagung DANKSAGUNG Schon während der Schulzeit hat mich die komplexe und geniale Bauweise biologischer Systeme, speziell die des Wesens „Mensch“, fasziniert. Nach Abschluss des Studiums der Humanmedizin bin ich durch Zufall zur Medizinischen Wissenschaft gekommen. Die Zeit, die ich seither hier am Zentrum für Hirnforschung verbracht habe, war eine interessante und lehrreiche, die mich persönlich sehr bereichert und geprägt hat. Dass die vorliegende Arbeit überhaupt möglich wurde und letztendlich auch zu einem Abschluss gebracht werden konnte, verdanke ich einer Reihe von Personen, die hier nun Erwähnung finden sollen: An erster Stelle möchte ich mich bei meinen Eltern und Großeltern für ihre umfassende seelische, emotionale und finanzielle Unterstützung bedanken. Ohne Euren Zuspruch und Euren Ansporn wäre ich nie bis hierher gekommen! Danke! Ein ganz besonderer Dank geht außerdem an die gesamte Mannschaft der Abteilung Neuroimmunologie des Zentrums für Hirnforschung. Speziell bedanken möchte ich mich: Bei Thomas, Florian, Josa, Conny, Iris, Alexandra, Tadeja, Assunta und Christina für ihre Freundschaft, Kollegialität und soziale Kompetenz, sowie für die reibungslose Zusammenarbeit im Laboralltag. Bei Martina, dafür, dass sie mir den Einstieg ins „Laborleben“ so leicht gemacht und mir nicht nur einmal mit Rat sondern vor allem auch mit Tat zur Seite gestanden hat. Bei Marianne, Ulli und Angela dafür, dass sie mir ihr umfassendes Wissen und ihre Erfahrung im Histolabor zur Verfügung gestellt haben. Ohne Euch wäre vieles wesentlich schwerer gewesen! Ich danke Euch für die unzähligen „Lehrgänge“ in Probenaufbereitung, Schneiden und Färben, sowie für Eure Engelsgeduld angesichts der unzähligen Präparate. Bei Ao. Univ. Prof. Dr. Jan Bauer für all die vielen Stunden am Licht-, Konfokal- und Elektronenmikroskop, sowie für seine Hilfestellung beim Erstellen der Publikation. In kompetenten Diskussionen hab ich bei Dir sehr viel über Mikroskopie allgemein, sowie über Neurohistopathologie im speziellen gelernt. Mit Deiner Hilfe sind auch schwierigere Färbungen sehr gut gelungen. Danke, dass Du mir und meinem Projekt so viel Zeit geopfert hast! Bei Gerti für all die unzähligen kleinen und großen Hilfestellungen während der vergangenen Jahre, sowie für ein jederzeit offenes Ohr. Bei Univ. Doz. Dr. Monika Bradl, für ihre „Entwicklungshilfe“ in Sachen APP, Mac-3 und Co.. Last but not least danke ich ganz herzlich meinen Betreuern: Ao. Univ. Prof. Dr. Johannes Berger und Dr. Sonja Forss-Petter für die interessante Themenstellung, die finanzielle Ermöglichung der Arbeit, die Weitergabe ihres Fachwissens, ihre unermüdliche Diskussionsbereitschaft sowie ihre tatkräftige Unterstützung beim Erstellen der zu dieser Arbeit gehörenden Publikation. Univ. Prof. Dr. Hans Lassmann für wiederholte Lehrgänge am Diskussionsmikroskop, unzählige wertvolle Vorschläge und Ideen, die diese Arbeit vorangebracht haben, seine Hilfestellung bei der Ergebnisinterpretation sowie für das Überarbeiten der Publikation. Und Univ. Prof. Dr. Klaus-Armin Nave und seinem Team, für nützliche Tipps bezüglich „mouse behaviour testing“, sowie seine Kooperation im Rahmen eines parallel laufenden Projekts. 4 Index INDEX Page List of abbreviations……………………………………………………………...…… 7 Summary………………………………………………………………………………. 8 Zusammenfassung……………………………………………………………….…….. 9 CHAPTER 1: INTRODUCTION…………………………………………….……… 11 1.1 Peroxisomes…………………………………………………………………… 12 1.1.1 Peroxisome biogenesis disorders (PBD)…………………………………… 13 1.1.2 Single peroxisomal enzyme deficiencies…………………………………… 15 1.2 ABC transporters……………………………………………………………... 17 1.2.1 Peroxisomal ABC transporters……………………………………………... 18 1.3 X-linked adrenoleukodystrophy (X-ALD)…………………………………… 22 1.3.1 Chronic……………………………………………………………………... 22 1.3.2 Clinical phenotypes………………………………………………………… 23 1.3.3 Diagnosis…………………………………………………………………… 27 1.3.4 Pathology and inflammation………………………………………………... 29 1.3.5 Therapeutic strategies………………………………………………………. 34 1.4 Abcd1 knockout mice – the X-ALD mouse model………………………….. 38 1.4.1 Accumulation of VLCFA and β-oxidation…………………………………. 39 1.4.2 Behavioural and neurological phenotype…………………………………... 39 1.4.3 Electrophysiology…………………………………………………………... 40 1.4.4 Peripheral nerve pathology…………………………………………………. 41 1.4.5 Pathology of the central nervous system…………………………………… 41 1.4.6 Adrenal gland pathology………………………………………………….... 42 1.4.7 Testis and ovary pathology…………………………………………………. 42 1.5 Mag knockout mice…………………………………………………………… 43 1.5.1 Myelin………………………………………………………………………. 43 1.5.2 Myelin-associated glycoprotein…………………………………………….. 44 1.5.3 Mag knockout mice………………………………………………………… 46 CHAPTER 2: AIM OF THE THESIS………………………………………………. 52 CHAPTER 3: MATERIALS AND METHODS…………………………………….. 56 3.1 Animals………………………………………………………………………… 57 3.2 Genotyping…………………………………………………………………….. 57 3.2.1 Isolation of DNA from mouse tail biopsies………………………………… 57 3.2.2 Genotyping by PCR……………………………………………………….... 59 3.2.3 Agarose gel electrophoresis……………………………………………….... 60 5 Index Page 3.3 Behavioural analysis…………………………………………………………... 61 3.3.1 Climbing test………………………………………………………………... 61 3.3.2 Hind limb reflex extension………………………………………………….. 62 3.3.3 Tremor………………………………………………………………………. 63 3.3.4 Rotarod test for motor coordination and balance…………………………… 63 3.4 Histopathology………………………………………………………………… 64 3.4.1 Mouse tissue dissection…………………………………………………...... 64 3.4.2 Tissue processing for light microscopy…………………………………….. 65 3.4.3 Standard staining methods………………………………………………...... 66 3.4.4 Immunohistochemistry……………………………………………………... 69 3.4.5 Confocal microscopy………………………………………………….……. 73 3.4.6 Electron microscopy………………………………………………………... 79 3.5 Quantitative evaluation of (immuno-) labelled structures and statistical analysis………………………………………………………………….……… 80 3.5.1 Bielschowsky silver impregnation: torpedoes in cerebellar granular layer.... 80 3.5.2 Mac-3 staining: activation of macrophages/microglia cells………………… 81 3.5.3 APP-staining: axonal bulbs and extra-axonal, diffuse APP accumulation.... 82 CHAPTER 4: RESULTS…………………………………………………………….. 84 4.1 Behavioural analysis………………………………………………………….. 85 4.1.1 Climbing test……………………………………………………………….. 85 4.1.2 Tremor…………………………………………………………………….... 86 4.1.3 Hind limb reflex……………………………………………………………. 88 4.1.4 Motor coordination and balance……………………………………………. 90 4.2 Neuropathological analysis…………………………………………………… 91 4.2.1 Gross histopathological examination of brain, spinal cord and sciatic nerves……………………………………………………………….. 92 4.2.2 Microglia activation in spinal cord and cerebellum………………………… 97 4.2.3 Axon pathology in spinal cord………………………………………………100 4.2.4 Myelin pathology in spinal cord……………………………………………. 103 4.2.5 APP accumulation in compact myelin……………………………………… 109 4.2.6 Light microscopy of semithin sections of spinal cord white matter……....…113 4.2.7 Electron microscopy of spinal cord white matter…………………………... 115 4.2.8 Light microscopy of semithin sections of sciatic nerves…………………. 117 CHAPTER 5: DISCUSSION…………………………………………………………. 118 CHAPTER 6: REFERENCES……………………………………………………….. 125 Curriculum vitae……………………………………………………………………… 139 Outcome of the thesis…………………………………………………………………..140 6 List of Abbreviations LIST OF ABBREVIATIONS ABCD1/Abcd1 ATP-binding cassette transporter subfamily D member 1 gene, human/mouse Abcd1 ko (in figures) Abcd1-knockout mice ABCD2/Abcd2 ATP-binding cassette transporter subfamily D member 2 gene, human/mouse ACTH adrenocorticotropic hormone ALDP/Aldp adrenoleukodystrophy protein, human/mouse ALDR/Aldr adrenoleukodystrophy related protein, human/mouse ALMN adreno-leuko-myeloneuropathy AMN adrenomyeloneuropathy APP amyloid precursor protein BMT bone marrow transplantation cALD cerebral adrenoleukodystrophy CMAP compound muscle action potential CNP 2’,3’-cyclic nucleotide 3’-phosphodiesterase CNS central nervous system Double ko (in figures) Abcd1/Mag-double-knockout mice DHCA, THCA di- and trihydroxycholestanoic acid GFAP glial fibrillary acidic protein LCFA long-chain fatty acids MAG/Mag myelin-associated glycoprotein, human/mouse Mag ko (in figures) Mag-knockout mice MBP myelin basic protein MHC major histocompatibility complex MOG myelin oligodendrocyte glycoprotein PBD Peroxisome biogenesis disorders PLP proteolipid protein PNS peripheral nervous system VLACS very long-chain fatty-acyl CoA synthetase VLCFA very long-chain fatty acids X-ALD X-linked adrenoleukodystrophy 7 Summary SUMMARY X-linked adrenoleukodystrophy (X-ALD) is an inherited, progressive, peroxisomal disorder, which primarily affects the nervous system as well as the
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