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 adrenal cortex. The disease is caused by mutations in the ABCD1 gene, which encodes the peroxisomal ATP- binding cassette (ABC) transporter adrenoleukodystrophy protein (ALDP). ALDP deficiency leads to a characteristic accumulation of saturated very long-chain fatty acids (VLCFA) in tissues and causes various clinical phenotypes, ranging from adrenocortical insufficiency and neurodegeneration to severe cerebral inflammation and demyelination. The pathogenesis of the disease is unclear und underlying molecular mechanisms targeting the disease course have yet to be identified. Aldp-deficient mice, generated as a model for X-ALD, replicate metabolic dysfunctions of the human disease and develop late-onset axonopathy but lack histological signs of inflammation and demyelination. To test the hypothesis that subtle destabilization of myelin may initiate inflammatory demyelination in Abcd1 deficiency, we generated mice with a combined metabolic defect of X-ALD and the mild myelin abnormalities of myelin-associated glycoprotein (MAG) deficiency. Here we present behavioural and histopathological findings from different sex- and age-matched groups of mutant mice. A behavioural phenotype, impaired motor performance and tremor, developed in middle-aged Mag null mice independent of Abcd1 genotype. Routine histology revealed no signs of inflammation or demyelination in the CNS, but immunohistochemical analyses of spinal cord neuropathology revealed microglia activation and axonal degeneration in spinal cord of Mag and Abcd1/Mag double-knockout and, less severe and of later onset, in Abcd1 mutants. While combined Abcd1/Mag deficiency showed an additive effect on microglia activation, axonal degeneration, quantified by accumulation of amyloid precursor protein (APP) in axonal spheroids, was not accelerated. In cerebellar sections axonal injury was exclusively seen in Mag and double-knockout animals. Interestingly, abnormal APP reactivity was enhanced within compact myelin of Abcd1/Mag double-knockout mice compared with single mutants already at 13 months. These results suggest that ALDP deficiency enhances metabolic distress in oligodendrocytes that are compromised a priori by destabilized myelin. Furthermore, the age at which this occurs precedes by far the onset of axonal degeneration in Abcd1 deficient mice, implying that indeed oligodendrocyte/myelin disturbances precede axonopathy in X-ALD.

8 Zusammenfassung

ZUSAMMENFASSUNG

X-chromosomale Adrenoleukodystrophie (X-ALD) ist eine erbliche, progressive, peroxisomale Störung, die sich vornehmlich im Nervensystem und der Nebennierenrinde manifestiert. Die Erkrankung wird durch Mutationen im so genannten ABCD1-Gen ausgelöst, welches für den peroxisomalen ATP-binding-cassette (ABC) Transporter, Adrenoleukodystrophie Protein (ALDP), kodiert. Der funktionelle Verlust von ALDP führt zu einer charakteristischen Akkumulation von gesättigten, überlangkettigen Fettsäuren (VLCFA) in Geweben und zu einer Reihe verschiedener klinischer Phenotypen, die von Nebennierenrindeninsuffizienz, über langsam voranschreitende neurodegenerative Symptome, bis hin zu schwerer zerebraler Entzündung und Demyelinisierung reichen. Die Pathogenese der Erkrankung ist unklar und molekulare Mechanismen, die den Krankheitsverlauf bestimmen, gilt es noch zu identifizieren. Aldp-defiziente Mäuse, generiert als Modell für X-ALD zeigen, verglichen mit der humanen Erkrankung, ähnliche metabolische Veränderungen und entwickeln im späten Erwachsenenalter eine Axonopathie ohne Entzündung oder Demyelinisierung. Um die Hypothese zu testen, dass eine subtile Destabilisierung von Myelin zur entzündlichen Demyelinisierung in Abcd1- defizienten Mäusen führen könnte, haben wir Mäuse gezüchtet, die den metabolischen Defekt von X-ALD und milde Myelinschäden, die bei Verlust von Myelin-assoziiertem Glykoprotein (MAG) auftreten, in sich vereinen. In dieser Studie werden Ergebnisse von Verhaltens- und histopathologischen Untersuchungen präsentiert, die an mutanten Mäusen verschiedener Geschlechts- und Altersgruppen durchgeführt wurden. Motorische Beeinträchtigung und Tremor traten in Mag null Mäusen mittleren Alters auf unabhängig vom Abcd1-Genotyp. Die Routine Histologie zeigte keine Entzündungs- oder Demyelinisierungszeichen im ZNS, wohl aber Mikrogliaaktivierung und Axondegeneration im Rückenmark von Mag, Abcd1/Mag doppel-knockout und milder sowie später auch in Abcd1 knockout Mäusen. Während die kombinierte Abcd1/Mag Defizienz offenbar einen additiven Effekt auf die Mikrogliaaktivierung hatte, zeigte sich keine Erhöhung der Axondegeneration, gemessen an der Akkumulation von amyloid precursor protein (APP) in Axon-Spheroiden. Im Kleinhirn beschränkte sich die Axon- Schädigung auf Mag und Doppel-knockout Mäuse. Interessanterweise fand sich eine abnormal erhöhte APP Reaktivität auch in kompaktem Myelin von Abcd1/Mag Doppel-

9 Zusammenfassung knockout Mäusen ab einem Alter von 13 Monaten. Diese Ergebnisse deuten darauf hin, dass ALDP Verlust zu einem erhöhten metabolischen Stress in Myelin-vorgeschädigten Oligodendrozyten führt, der wesentlich früher in Erscheinung tritt als die Axondegeneration, was impliziert, dass in X-ALD tatsächlich Oligodendrozyten/Myelin Veränderungen der Axonopathie vorausgehen.

10 Introduction

CHAPTER 1

INTRODUCTION

11 Introduction

1. INTRODUCTION

1.1. Peroxisomes

Peroxisomes are small cellular organelles with an overall diameter of 0.1 to 0.5 µm, present in many eukaryotic cells. Their number varies from <100 to approximately 500 vesicles in human hepatocytes and renal tubule cells. They are surrounded by a single membrane and equipped with a unique set of enzymes to fulfil various physiological functions, but do not contain DNA (1, 2). Peroxisomal matrix proteins are encoded by nuclear genes, synthesized on free cytoplasmic ribosomes and targeted to the peroxisome by so called peroxisome-targeting- signals (PTS), which are cis-acting sequences present in the primary peptide sequence (3). Currently two different PTS are known: PTS1 is a C-terminal serine-lysine-leucine tripeptide or a conservative variant thereof (4), whereas PTS2 consists of an N-terminal nonapeptide sequence (5). Both signals are recognized by soluble receptors (Pex5 and Pex7), which target ligands to a docking machinery, located on the peroxisomal membrane (6). After binding to the docking machinery the receptor-ligand complex dissociates and the ligand is translocated across the peroxisomal membrane, meanwhile the receptor recycles back to the cytosol. A modified so-called “extended shuttle model” suggests the crossing of the receptor-cargo complex en block, which would then be followed by the back-transport of the empty receptor across the peroxisomal membrane to the cytosol (7, 8). Though peroxisomal membrane proteins are also synthesized on free polyribosomes in the cytosol, their import into peroxisomes is more complex and involves additional targeting information, other than PTS1 and PTS2, distributed in the polypeptide chain. Also the targeting process itself is different from that of peroxisomal matrix proteins and seems to be coupled to Pex9, 3 and 16 receptors (9). How, in detail, the biogenesis of peroxisomes occurs is unknown. Two, probably parallel, pathways are possible: The formation from pre-existing peroxisomes and a de novo synthesis from some endomembrane compartment such as the endoplasmic reticulum. Peroxisomes, once assembled, expand due to protein uptake until a critical size is reached and divide afterwards into two daughter peroxisomes, which may then undergo the same cycle of subsequent growth (5).

12 Introduction

Peroxisomes participate in a wide range of physiological processes, especially in those related to lipid metabolism. They contain a fatty acid β-oxidation machinery that is, at least in some aspects, comparable to the one in mitochondria (10). Via the four consecutive reactions: oxidation – hydration – oxidation – thiolytic cleavage, fatty acids are partly degraded to acetyl-CoA units. In peroxisomes, the FADH2 molecules produced during the first step of β-oxidation are re-oxidized by molecular oxygen producing H2O2, which is potentially a very dangerous oxidant and needs to be destroyed rapidly. This reaction is catalyzed by the peroxisomal enzyme catalase (1). As peroxisomes can only chain-shorten fatty acids, lack a citric acid (Krebs-) cycle and furthermore cannot degrade acetyl-CoA units to CO2 and H2O, intermediate products (like NADH, acetyl-CoA units and chain- shortened fatty acids) are believed to be exported, in the case of chain-shortened fatty acids by using a carnitine shuttle system (11). The main difference between mitochondrial and peroxisomal β-oxidation systems is the catalysis of different substrates. Peroxisomes specifically oxidize very long-chain fatty acids (VLCFA), pristanic acid and di- and trihydroxycholestanoic acid (DHCA and THCA). The latter two compounds are further used in the formation of bile acids (12). Another major function of the peroxisomal β- oxidation is the synthesis of polyunsaturated fatty acids (13) and the inactivation of many eicosanoids (14). Considering other metabolic functions, peroxisomes are also involved in α-oxidation of 3- methyl-branched fatty acids (15), the biosynthesis of cholesterol, other isoprenoids (16) and ether-phospholipids (17), notably plasmalogens and platelet-activating factor, the degradation of L-pipecolat (18), the glutaryl-CoA (19) and H2O2 metabolism (1) and the detoxification of glyoxylate, which would otherwise be converted to oxalate, a metabolite that rapidly precipitates as calcium oxalate with severe consequences for tissues (20). Currently 17 human diseases are known that result from the loss of peroxisomal functions. According to their molecular basis they are divided into peroxisome biogenesis disorders (PBD) and single peroxisomal enzyme deficiencies (8).

1.1.1 Peroxisome biogenesis disorders (PBD)

PEX genes are genes related to peroxisome assembly. Mutations within these genes give rise to the so-called “peroxisome biogenesis disorders” (PBD), which are characterized by a multiple loss of peroxisomal functions. Complementation analysis has

13 Introduction led to the identification of so far 12 different complementation groups, which result in four distinct PBD, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum’s disease (IRD) and rhizomelic chondrodysplasia punctata (RCDP) type 1 (8). ZS, NALD and IRD are assorted together to the so-called “Zellweger spectrum”. They are caused either by a defect in peroxisome membrane assembly leading to a complete loss of peroxisomes, or by a hindered import process of PTS1/PTS2-targeted matrix proteins into peroxisomes (8). ZS is the most severe disorder of the Zellweger spectrum. It goes along with craniofacial dysmorphism including a large anterior fontanel, a high forehead, micrognathia, external ear deformity, a broad nasal bridge and epicanthal folds, as well as profound neurological abnormalities. ZS children suffer from severe psychomotor retardation, hypotonia, neonatal seizures, impaired hearing, glaucoma and retinal degeneration and mostly die within the first year of life. Additionally calcific stippling of epiphyses, small renal cysts and micro-nodular cirrhosis of the liver are frequently found (6, 21). Patients with NALD also show hypotonia and seizures as well as polymicrogyria and progressive white matter disease. They usually die in late infancy (22). The phenotypic appearance of IRD, the least severe disease of the Zellweger spectrum seems to be quite heterogeneous. It may range from profound global handicaps to only moderate learning disabilities with deafness and visual impairment. Some patients even reach adulthood (23). All diseases of the Zellweger spectrum have in common the absence of functionally active peroxisomes. Peroxisomes are either completely lost, reduced in number and/or size, or appear as so called “ghosts”, which are aberrant peroxisomal structures that contain peroxisomal integral membrane proteins but lack most of the matrix enzymes (24). This loss of peroxisome function is reflected in elevated levels of plasma VLCFA, DHCA and THCA, pristanic acid and phytanic acid and L-pipecolic acid, whereas plasma docosahexaenoic acid levels and erythrocyte plasmalogens are reduced (8). RCDP type 1 is caused by mutations in the PEX7 gene, which leads to a segregated block in the PTS2 import pathway (25). It is clinically characterized by a disproportional short stature, a symmetrical shortening of femur and humerus, facial features such as a broad nasal bridge, dysplastic ears and micrognathia, a barrel-formed thorax, irregular, small spine discs, congenital contractures and severe mental retardation with spasticity.

14 Introduction

Regarding the biochemistry ether-phospholipid biosynthesis and fatty acid α-oxidation are defect, whereas peroxisomal β-oxidation remains normal. This explains the unaltered VLCFA profile in contrast to markedly reduced erythrocyte plasmalogen levels and, depending on dietary supply, partly high phytanic acid levels in plasma (8). Therapy options for PBD patients are still limited in so far as most abnormalities already develop in utero and postnatal treatment can be only supportive. Anticonvulsants, physical and orthopaedic therapy, correction of visual and auditory impairment as well as dietary measures are applied in order to improve quality of life (8).

1.1.2 Single peroxisomal enzyme deficiencies

This group of peroxisomal disorders is characterized by the loss of single peroxisomal proteins due to mutations in the corresponding genes. The peroxisomal structure is virtually intact. So far, eleven different diseases have been described and to simplify matters they are divided into the following subgroups: 1. disorders of peroxisomal β- oxidation, 2. disorders of fatty acid α-oxidation, 3. disorders of etherphospholipid biosynthesis, 4. disorders of glyoxylate detoxification, 5. disorders of glutaryl-CoA metabolism, 6. disorders of isoprenoid biosynthesis, 7. disorders of hydrogen peroxide metabolism and 8. other single protein disorders where the functions of the affected proteins are still unknown. Table 1 shows the single enzyme deficiencies belonging to each subgroup, the corresponding, defective proteins and some clinical aspects (see (26) for review). X-linked Adrenoleukodystrophy, the disease discribed in this thesis, is assorted to group number 8 due to the unknown function of the affected protein: Adrenoleukodystrophy protein (ALDP). Postnatal diagnosis of single peroxisomal enzyme deficiencies upon clinical suspicion involves the initial screening of plasma VLCFA levels and erythrocyte plasmalogens followed by the measurement of phytanic, pristanic and di- and trihydroxycholestanoic acid levels in plasma. The definitive diagnosis requires detailed analysis in fibroblasts including enzyme activity measurements, complementation studies and molecular investigations. Prenatal diagnosis can be done in chorionic villous material without the need to culture fibroblasts (26).

15 Introduction

Table 1: Classification of single peroxisomal enzyme deficiencies

DISORDER PROTEIN DEFECT CLINIC

Disorders of the peroxisomal β-oxidation* ƒ Acyl-CoA oxidase Straight-chain acyl-CoA PBD-like with neonatal hypotonia, deficiency oxidase psychomotor retardation, convulsions, deafness, retinopathy and accumulation of VLCFA ƒ D-Bifunctional protein D-BP/MF2/MFEII/DPBE PBD-like with hypotonia, seizures, deficiency craniofacial dysmorphism, polymicrogyria, adrenal atrophy, cysts in kidneys, fibrosis of liver and elevated levels of VLCFA, pristanic acid and bile acid intermediates ƒ Peroxisomal 2- AMACR Adult-onset sensory motor neuropathy methylacyl-CoA with elevated levels of 2-methyl racemase deficiency branched chain fatty acids, pristanic acid, DHCA and THCA Disorders of fatty acid α- oxidation* ƒ Refsum disease Phytanoyl-CoA hydroxylase Chronic polyneuropathy, retinitis pigmentosa, cerebellar ataxia, skeletal malformations, cardiac abnormalities, elevated cerebrospinal fluid protein levels and accumulation of phytanic acid Disorders of etherphospholipid biosynthesis* ƒ Rhizomelic Dihydroxyacetonephosphate RCDP1-like with craniofacial chondroplasia punctata acyltransferase (DHAPAT) dysmorphism, hypotonia, cataracts, type 2 shortening of upper limbs and decreased plasmalogen levels ƒ Rhizomelic Alkyldihydroxyacetone- RCDP1-like with contractures, chondroplasia punctata phosphate synthase (Alkyl cataracts, seizures, developmental type 3 DHAP synthase) delay and decreased plasmalogen levels Disorders of glyoxylate detoxification* ƒ Hyperoxaluria type 1 Alanine glyoxylate Nephrocalcinosis, urolithiasis, aminotransferase myocarditis, peripheral neuropathy, osteosclerosis, retinopathy, uremia and systemic oxalosis

16 Introduction

Disorders of glutaryl-CoA metabolism* ƒ Glutaric aciduria type 3 Glutaryl-CoA oxidase? Failure to thrive, postbrandial vomiting and glutaric aciduria Disorders of isoprenoid biosynthesis* ƒ Mevalonate kinase Mevalonate kinase Developmental delay, facial deficiency dysmorphism, cataract, hepatosplenomegaly and lymphadenopathy Disorders of hydrogen peroxide metabolism* ƒ Acatalasemia Catalase Ulcerating, gangrenous, oral lesions

Other single protein disorders* ƒ Mulibrey nanism Trim37p (unknown Growth failure, dysmorphic features, function) pericardial constrictions, hepatomegaly, muscular weakness, naevi flammei, enlarged cerebral ventricles ƒ X-linked ALDP (unknown function) At least 6 phenotypic variants with or adrenoleukodystrophy without inflammatory reactions in cerebral white matter leading to behavioural, cognitive and neurologic deterioration; adrenal insufficiency and accumulation of VLCFA in tissues (see below) * see reference (26) for details.

1.2. ABC Transporters

ATP-binding cassette (ABC) transporters form a large family of active transport systems and are found in archaea and eubacteria as well as in eukaryotes. They translocate a wide variety of substrates including ions, sugars, amino acids, vitamins, lipids, antibiotics and other drugs, oligosaccharides, oligopeptides and even high molecular weight proteins across cellular and subcellular membranes. Hence they play important roles in nutrient uptake, lipid trafficking, secretion of macromolecules, antigen presentation, cell volume regulation and protection from xenobiotics (27). Currently 48 human ABC genes are known which are divided into seven subfamilies based on amino acid sequence similarities

17 Introduction and phylogeny. Most of these ABC transporters are conserved in all vertebrates and thus seem to be of ancient origin (28). Mutations in ABC transporters are related to a number of different, partly complex, human diseases like, for instance, cystic fibrosis, retinal degeneration, hypercholesterolemia, cholestasis, lamellar ichthyosis and also X-linked adrenoleukodystrophy (X-ALD) (29). In contrast to prokaryotic species, where ABC transporters mainly function as influx systems, in eukaryotic organisms only ABC efflux pumps are found. They mediate the transport of compounds from the cytosol to the extracellular space or to the inside of intracellular compartments like the endoplasmic reticulum, mitochondria, peroxisomes or other vacuoles. Their architecture is somewhat less complex than ABC influx systems as they do not require so called substrate-binding proteins (SBP) for function. ABC efflux systems are composed of four core domains, two hydrophobic transmembrane domains (TMD) plus two water soluble nucleotide-binding domains (NBD) protruding into the cytosol. TMD are composed of α-helix bundles that transverse the membrane several times creating a channel through which the substrate passes. The NBD, in contrast, contain the ATP-binding cassettes and form the “engine” of the ABC transporter, enabling translocation by ATP-binding and hydrolysis (30). Frequently the four core domains of an ABC efflux pump are fused, forming one or two polypeptides. The most common organization is the so called “half-size” transporter in which a single TMD is merged to the N- or C-terminus of a NBD. Two half-transporters joined together would then form the functional complex either as a homo- or heterodimer. On the other hand all four core-domains can also be fused to one single polypeptide, which is then termed “full-length” transporter. In addition to the ubiquitous four core domain, accessory domains can be found in several ABC transporters, where they may carry out some important regulatory and catalytic functions (30, 31).

1.2.1 Peroxisomal ABC transporters

Peroxisomal ABC transporters belong to the subfamily D, which comprises the four members: adrenoleukodystrophy protein (ALDP; encoded by the ABCD1 gene), adrenoleukodystrophy-related protein (ALDRP; encoded by ABCD2), the 70 kDa peroxisomal membrane protein (PMP70; encoded by ABCD3) and the 69 kDa peroxisomal membrane protein (PMP69; encoded by ABCD4) in humans and mice (32-35). All of them

18 Introduction are “half-size” transporters with the topology TMD-NBF (see above) and dimerize to form functional complexes (31). Though it has been demonstrated by co-immunoprecipitation of in vitro synthesized proteins and in yeast-two-hybrid systems that ALDP can form heterodimers with ALDRP and PMP70, it is still unclear whether these interactions occur in vivo (36, 37). The distinct expression patterns of ALDP, ALDRP and PMP70 in mice and humans, as well as the fact that the disease X-linked Adrenoleukodystrophy (X-ALD) is associated exclusively with mutations in the ABCD1 gene and not with defects in other peroxisomal ABC half-transporters, suggests that they all function independently as homodimers (38, 39). Moreover the stoichiometric expression of ABC transporters in vivo seems to be an important factor for homo- or heterodimerization and also for peroxisomal function. Unterrainer et al. demonstrated that altering the expression levels can have an impact on peroxisomal function, either by competing for membrane insertion sites or by forming malfunctioning heterodimers (40). Though peroxisomal ABC transporters have been studied extensively, many features concerning their roles in metabolism of substrates are still unknown. Latency experiments suggested that PMP70 mediates the transport of long-chain fatty acyl-CoA (LCFA-CoA) across the peroxisomal membrane by an ATP-dependent mechanism and may also be involved in the import of 2-methylacyl-CoA esters, like pristanoyl-CoA, DHCA- and THCA-CoA, into peroxisomes (41, 42). Based on sequence similarity to PMP70, PMP69 is thought to have analogue functions (35).

The disease, X-ALD, is linked to mutations in the ABCD1 gene (33). Since X-ALD patients show an accumulation of saturated, straight-chain VLCFA in plasma and tissues, a direct involvement of ALDP in VLCFA metabolism seemed evident. It has been variously suggested that ALDP might be involved in β-oxidation of VLCFA through transport of VLCFA, VLCFA-CoA or the cofactors CoA and ATP (43). Alternatively roles in the transport or stabilization of the VLCFA-CoA synthetase (VLACS) have been proposed. However, further studies revealed that immunoreactive VLACS was present in the peroxisomal matrix of X-ALD fibroblasts and that the peroxisomal β-oxidation of VLCFA was normal in tissues of X-ALD mice (44, 45). Based on these findings, it could be rejected that ALDP is necessary to maintain VLACS activity. Another hypothesis suggested a connection between ALDP deficiency and mitochondrial function. It was proposed that the loss of ALDP might impair mitochondrial function

19 Introduction causing an increase in long-chain fatty acids (LCFA). Since peroxisomal VLACS has a higher affinity for LCFA than for VLCFA, the net consequence would be saturation of peroxisomal VLACS by LCFA, resulting in VLCFA accumulation. It was further suggested that accumulation of VLCFA could cause mitochondrial damage (45). However, a detailed study of mitochondrial ultrastructure and function in muscle cells of X-ALD mice did not reveal abnormalities, indicating that mitochondrial dysfunction is not responsible for VLCFA accumulation and, vice versa, that VLCFA accumulation per se does not lead to mitochondrial damage (46). Recently it was found that X-ALD cells exhibit increased microsomal fatty acid elongation which could contribute to elevated VLCFA levels in vivo, but how these findings are linked to mutations in the ABCD1 gene is not yet known (47).

Human ALDRP shows approximately 63% sequence homology with ALDP at the amino acid level and shares the same half-transporter structure (48). This high degree of identity between ALDRP and ALDP suggested functional similarity and would, at least to some extent, explain the finding that VLCFA β-oxidation is not completely lost in ALDP deficient fibroblasts but shows some 30% residual activity (49). Furthermore several groups were able to demonstrate that overexpression of ABCD2 in cultured X-ALD fibroblasts can restore VLCFA β-oxidation and correct the pathognomonic accumulation of VLCFA (50, 51). Pujol and co-workers generated transgenic mice overexpressing Abcd2 under the control of the ubiquitously active β-actin promoter (52). When crossing these mice to Aldp deficient mice, a complete normalization of VLCFA levels in adrenal gland, peripheral (PNS) and central nervous system (CNS) of the latter was found. Taken together, all these results provide evidence, that ALDP and ALDRP share at least some functionality. Nevertheless Abcd2 mutant mice exhibit a distinct neurological phenotype compared with Aldp deficient mice. This perhaps reflects additional roles of ALDRP in vivo or may be caused by highly cell type-specific and somewhat contrary expression patterns of ABCD1 and ABCD2 in tissues (see Table 2 and Figure 1) (39, 53).

20 Introduction

Table 2: Gene expression profile of Abcd1 and Abcd2 in tissues of adult mice.

Abcd1 expression* Abcd2 expression*

brain mainly in white matter of the cerebrum mainly in neuronal cells of cerebrum, and cerebellum; cerebellum and retina; cell types: astocytes, microglial cells, cell types: pyramidal and granular cells of brain macrophages, choroids plexus, the cortex (especially of the endothelial cells and oligodendrocytes of hippocampus), Purkinje cells, bipolar and the corpus callosum photoreceptor cells testes spermatocytes, spermatides spermatogonias, Sertoli cells and interstitial cells adrenal gland adrenal cortex adrenal medulla * Tissue distribution based on results from In situ hybridization and immunohistochemical studies (39).

A ABCD1

140

120

100

80

60

40

20 relative expression level in % 0

rt d is er in x a a e m lan est liv u cord he t br rt s l g e co llo al na n ol ca i re ral s sp d wh u a ereb c corp

B ABCD2

140

120

100

80

60

40

20 relative expression level %

0

d is er n x n st ai rd a e liv rte sum heart gl t br l co l e co llo ol l a ina h s c p w u s drena p a erebra c cor

Figure 1: Gene expression profile of ABCD1 (A) and ABCD2 (B) in whole tissue samples of adult humans. Graphs taken from Berger et al. (53), represent data from RNA dot blot hybridizations.

21 Introduction

1.3. X-linked adrenoleukodystrophy (X-ALD)

1.3.1 Chronic

X-linked Adrenoleukodystrophy was first reported in 1910 by Haberfeld and Spieler and 13 years later again by Siemerling and Creutzfeld, who referred to the condition as „Bronzekrankheit und Sklerosierende Encephalomyelitis“ (54). They described a boy, 4 years of age, who developed progressive disturbances of behaviour, speech and gait, presented with hyperpigmentation and died only 3 years later. In 1963, pedigree analysis done by Fanconi et al. revealed, that the disorder has an X-linked mode of inheritance (55). The striking combination of white matter pathology found in the central nervous system and adrenal insufficiency gave rise to the designation “adrenoleukodystrophy” (56). Between 1973 and 1976, two different laboratories demonstrated characteristic inclusions in adrenal cortical cells and brain macrophages, which were found to consist of large amounts of cholesterol esterified with saturated VLCFA (mainly C24:0 and C26:0) (57, 58). This led to the recognition that X-ALD is a lipid storage disease. In 1976, Budka et al. described an adult onset form of X-ALD presenting with progressive paraparesis and primary adrenal insufficiency, which was referred to as “adrenomyeloneuropathy” (AMN) (59). Later on it was found that AMN has the same biochemical and genetic basis as the childhood form of X-ALD. Both phenotypes can co-occur in the same nuclear family. The gene responsible for X-ALD was mapped to Xq28 in 1981 (60) and Singh et al. demonstrated an impairment of VLCFA degradation in cultured X-ALD skin fibroblasts (61). Furthermore a decreased capacity to form the co-enzyme A (CoA) derivate of VLCFA was found, which led to the hypothesis that the peroxisomal enzyme VLCFA synthetase (VLACS) that catalyzes this reaction might be defect (62). All the more, it came to a surprise when Mosser et al. identified the disease-associated gene in 1993. It did not show any homology to known fatty acyl-CoA synthetases. ABCD1, as the gene is now called, codes for the peroxisomal membrane protein adrenoleukodystrophy protein (ALDP), which belongs to the ABC transporter protein family (33). Mouse models of X- ALD have been developed in order to be able to study the pathophysiology of X-ALD in more detail (63-65). However, the mechanism through which ALDP deficiency leads to accumulation of VLCFA and thus to brain and adrenal gland pathology remains unexplained.

22 Introduction

1.3.2 Clinical phenotypes X-ALD, caused by mutations in the ABCD1 gene, is inherited in an X-linked manner and leads to accumulation of saturated VLCFA in various tissues (60, 66). It mainly affects the nervous system white matter and the adrenal cortex. The phenotypic expression of the disease is heterogeneous and neither can be predicted by the nature of the mutation nor by the severity of VLCFA accumulation (67). Based on sex, age of onset, progression rate and main neuropathological symptoms patients are classified into 12 different clinical subtype groups, comprising cerebral forms starting in childhood, adolescence or adulthood, as well as milder spinal or neuropathic variants preferably occurring in young adulthood (reviewed in (68, 69)). All phenotypic variants of X-ALD can co-occur within one family, where all affected members carry the same mutation of the ABCD1 gene (70). The incidence is estimated to be 1:17,000, which would be approximately in the range of phenylketonuria (71).

Phenotypes in male carriers

ƒ Childhood cerebral ALD (cALD; 31-35% of X-ALD cases) The childhood cerebral phenotype is the most severe form of X-ALD. It goes along with intense inflammatory demyelination involving both cerebral hemispheres. In approximately 85% of the patients the initial lesion is located in parieto-occipital regions and secondary spreads rostrally. In 15% of the patients the initial lesion is found in the frontal lobes (72). Lesions are associated with breakdown of the blood brain barrier and accumulate contrast materials. MRI scans can be used for visualizing early abnormalities, which in many cases precede clinical symptoms (73). The mean age of onset of cALD has been calculated to 7.2 ± 1.7 years and usually lies within the range of 3 to 10 years. Psychomotor development is normal until the disease starts. Initial manifestations include emotional lability, withdrawn or hyperactive behaviour, defects in auditory discrimination and visual processing, impaired spatial coordination, dressing apraxia and occasionally seizures. Most patients already have an impaired adrenocortical function when the first neurological disturbances are recognized. Once the neurological symptoms have become manifest, progression is usually rapid. Patients develop bulbar dysfunction, spastic tetraparesis and dementia within two years. They become bedridden, are unable to eat, see or speak and remain in this vegetative state until they die months to years thereafter (72).

23 Introduction

ƒ Adolescent cerebral ALD (4-7% of X-ALD cases) Cerebral X-ALD may also occur in adolescence or young adulthood, meaning the age of onset lies between 11 and 21 years. Initial symptoms in these patients resemble those in childhood cALD but progression is somewhat slower. For unknown reasons the susceptibility for the inflammatory cerebral phenotype diminishes with age (74).

ƒ Adult cerebral ALD (2-5% of X-ALD cases) The adult cerebral form of X-ALD is relatively rare. It involves patients who develop cerebral symptoms after the age of 21 years and lack initial signs of spinal cord pathology (69). Most common manifestations resemble those of schizophrenia, dementia, mania or other psychiatric disturbances (79). Specific cerebral deficits like dysphasia, hemianopsia and distinct cranial nerve dysfunctions may occur. About 50% of the patients suffer from adrenocortical insufficiency. Disease progression in adult cerebral ALD parallels that of the childhood cerebral form, vegetative state leading to death is usually reached within 2-3 years (69).

ƒ Adrenomyeloneuropathy (AMN; 40-46% of X-ALD cases) Budka et al. first described this more slowly progressing adult form of X-ALD in 1976 (59). Patients initially present with stiffness or clumsiness in the legs, generalized weakness and occasional hyperpigmentation at a mean age of 28 ± 9 years. Urinary and sexual dysfunction may be present at that time. Also an unusual thinning of the hair is frequently found (75). About 50% of the patients suffer from Addison’s disease. The adrenocortical insufficiency usually precedes the onset of neurological symptoms (76). In some patients the partial loss of adrenocortical hormones can be compensated by abnormally high levels of adrenocorticotropic hormone (ACTH), which not only enhances the secretion of hormones from remaining adrenocortical cells but also stimulates melanocytes, resulting in hyperpigmentation of skin and mucosae. The neurological disability is slowly progressive and leads to spastic paraparesis, sensory loss and ataxia usually within years to decades (69). Extensive studies done by Powers et al. demonstrate that AMN is caused by a non-inflammatory axonopathy, which mainly affects the distal segments of the long spinal tracts (77). Abnormalities are found in the lower thoracic and lumbar segments of the descending corticospinal tracts and in the cervical ascending dorsal columns, as well as in the peripheral nervous system. Patients, in whom the neurological involvement is confined to spinal cord and peripheral nerves and, who do not have clinical

24 Introduction or MRI evidence of brain involvement are referred to as having “pure AMN” (77). About 50% of all AMN patients develop additional lobar cerebral lesions early in the disease process or at later stages. This form of AMN, presenting with a much more rapid progression and early cognitive function deficits, is referred to as “cerebral AMN” or “Adreno-leuko-myeloneuropathy” (ALMN) (78).

ƒ Addison disease only (up to 50% in childhood, declines with age) X-ALD can cause Addison’s disease. Laureti et al. identified X-ALD as the reason for adrenal insufficiency in 35% of otherwise normal, male patients who had previously been diagnosed as having primary idiopathic adrenocortical insufficiency (80). Most of the Addison-only patients are younger than 7.5 years of age. They may develop neurological symptoms typical for cALD or AMN at a later time point (69). Many asymptomatic boys who carry a mutation in the ABCD1 gene were found to have elevated levels of plasma ACTH, which is an indicator for an impaired adrenal reserve. Careful monitoring and occasionally appropriate adrenal hormone replacement therapy is essential in these cases (81).

ƒ Pre- or asymptomatic ALD (diminishes with age) The asymptomatic phenotype group comprises X-ALD patients who have the biochemical and gene abnormality of X-ALD, but lack any demonstrable adrenal or neurological deficits. Also the conventional brain MRI is normal. Only Magnetic Resonance Spectroscopy investigations may show subtle changes. Most of the patients in this category are younger than 7 years of age and still carry the risk of developing either cALD or AMN at a later time point (68, 69).

ƒ Olivo-ponto-cerebellar ALD (1-2% of X-ALD patients) Patients present with brain stem and cerebellar symptoms, like cerebellar ataxia. Brain MRI images show severe brain stem, cerebellar and cortical atrophy, often combined with coticospinal tract involvement and, later on, white matter changes similar to those found in the cerebral form of X-ALD. Onset of olivo-ponto-cerebellar ALD is more common in adulthood (68, 69, 82).

ƒ “Arrested” cerebral variant (rare) Some patients with childhood or juvenile onset of cerebral ALD presenting with typical symptoms like behavioural changes, school failure, withdrawal or hyperactivity, show a stop of disease progression or even partial recovery of cortical dysfunction. However, in

25 Introduction almost all cases reported remission was only temporary and more likely caused by steroid administration and consecutive reduction of brain oedema (69).

Phenotypes in female carriers

Female carriers are heterozygous for the mutated ABCD1 gene located on the X- chromosome. Approximately 85% of these heterozygous women show elevated levels of VLCFA in plasma; the remaining 15% can only be detected by mutation analysis. More than 50% of female carriers develop at least slight neurological abnormalities, independently from the status of VLCFA accumulation (68, 83).

ƒ Asymptomatic phenotype (diminishes with age) Some of the female X-ALD carriers do not show evidence of adrenal or neurological involvement. Most of these women are younger than 30 years of age. The frequency of asymptomatic heterozygous carriers diminishes when the mean age of disease onset, calculated to 37.8 ± 14.6 years, is exceeded (69).

ƒ Mild myelopathy (approximately 50% in female carriers older than 40 years of age) Mild myelopathic symptoms, comprising increased deep tendon reflexes and distal sensory changes in the lower extremities, get more frequent with age, but usually patients demonstrate no or only mild disability. Their brain MRI images are normal (68).

ƒ Adrenomyeloneuropathy (AMN; approximately 15% in female carriers older than 40 years of age) Nature of symptoms and pathology in this group of female X-ALD carriers resemble those in male AMN patients, but disease onset is later, the grade of disability is somewhat less severe and progression seems to be slower when compared with affected males. Only about 15-20% of heterozygous women classified in this category, develop significant spastic paraparesis and require walking aids (68).

ƒ Adult cerebral ALD or Adreno-leuko-myeloneuropathy (extremely rare, 1-2%) Cerebral involvement in female X-ALD carriers is almost not at all seen in childhood and only slightly more common in middle age or later (68).

26 Introduction

ƒ Addison only (extremely rare, 1%) Most, even neurologically impaired, heterozygous women have normal adrenocortical function. Only 8 out of more than 1600 female carriers reported in literature showed overt adrenocortical insufficiency (84).

Unlike most other X-linked, recessive disorders a high percentage of female X-ALD carriers have characteristic biochemical and clinical manifestations. Clonal analysis and segregation studies revealed that the ABCD1 locus at Xq.28 is subjected to X-inactivation (60). This suggested that non-random X-inactivation in heterozygous females might be related to the phenotypic variability of clinical and biochemical features in carriers. Maier et al. analyzed the patterns of X-inactivation in a series of female X-ALD carriers and provided evidence for a significantly higher frequency of skewing of X-inactivation in peripheral blood cells of female patients that is correlated with the clinical but not with the biochemical phenotype (85). However, it was also found that skewing not inevitably favours the mutated ABCD1 allele as had been proposed.

1.3.3 Diagnosis

Up to now awareness among neurologists about X-ALD and other leukodystrophies has been low and many patients are still initially misdiagnosed. Due to early behavioural changes the most frequent misdiagnosis in young X-ALD carriers is an attention deficit or hyperactive disorder, often leading to therapies with stimulants. AMN patients and, even more, heterozygous women lacking any signs of adrenocortical insufficiency are frequently misdiagnosed for having multiple sclerosis. Other apparently wrong initial diagnoses include major depression, schizophrenia, spastic spinal paralysis, Friedreich ataxia, olivo-ponto-cerebellar atrophy, tumors and autoimmune adrenalitis (68, 69).

ƒ VLCFA levels and genetic analysis The recommended postnatal diagnostic procedure in X-ALD involves the assessment of neurological and psychiatric symptoms combined with the measurement of plasma VLCFA levels using gas chromatography techniques (86). The level of hexacosanoic acid (C26:0), the ratio of C26:0 to docosanoic acid (C22:0) and the ratio of C26:0 to tetracosanioc acid (C24:0) are determined and compared to normal control values. Elevation of plasma VLCFA levels equivalent to those seen in X-ALD also occurs in other peroxisomal disorders. However, usually they can easily be distinguished from X-ALD

27 Introduction due to the strikingly different clinical presentation. Advanced diagnostic procedures include the assay of VLCFA levels (87) and β-oxidation in cultured skin fibroblasts (88), immunocytochemical analysis of ALDP expression in cultured skin fibroblasts or white blood cells of patients (89) and mutation analysis (90). More than 400 mutations within the ABCD1 gene have been identified so far. As plasma VLCFA levels are not elevated in 15% of the female X-ALD carriers, DNA based diagnosis is recommended to identify heterozygous women. Boehm et al. developed a reliable procedure that uses a non-nested genomic amplification of the ABCD1 gene, followed by fluorescent dye-primer sequencing and analysis (90). Mutation analysis in fetal cells is done for prenatal diagnosis. Further more increased VLCFA levels as well as a decreased expression of ALDP can be demonstrated in cultured amniocytes or chorion villus cells (91).

ƒ Magnetic resonance imaging of CNS lesions In cerebral forms of X-ALD, neuroimaging studies often show characteristic changes. A number of advanced MRI techniques have been developed that allow a comprehensive anatomical and functional evaluation of the central nervous system and detection of early abnormalities that often precede neurological and neuropsychological changes in X-ALD. Moreover MRI can serve as a predictive parameter for disease progression and helps to select candidates for bone marrow transplantation (92). Typical cALD lesions seen on MRI scans are symmetrical, located parieto-occipitally in the periventricular white matter and appear hyperintense in T2-weighted images (93). Loes et al. elaborated a severity scoring system based on these T2 signal hyperintensities (94). Enhancement of the contrast material gadolinium-diethylenetriamine pentaacetic acid, shown on T1-weighed MRI images indicates breakdown of the blood-brain-barrier and corresponds to areas of inflammation (95). Proton MR spectroscopic imaging (MRSI) permits the evaluation of several nervous system metabolites. An increase of choline-containing compounds (Cho) and myo-inositol (Ins) indicates the onset of demyelination, the increase of Cho, Ins and glutamine peaks reflects active demyelination and glia proliferation and a reduction of N- acetyl aspartate and glutamate is associated with neuronal loss (68, 96). MRSI, Diffusion tensor Imaging (DTI) and Magnetization Transfer Imaging are advanced MR applications that might be helpful in the assessment of the long descending and ascending tracts affected in AMN (68).

28 Introduction

ƒ Neurophysiological examinations Neurophysiological abnormalities are present in all symptomatic and most presymptomatic X-ALD patients. Tibial nerve somatosensory evoked potentials (SSEP), transcranial magnetic stimulation (TMS) and brain stem auditory-evoked responses (BAER) are used to detect clinical and subclinical spinal cord and brain involvement in X-ALD patients. A characteristic pattern of mostly symmetric abnormalities is found in AMN patients as well as in heterozygous women (68). Male AMN patients show pathological SSEP responses of peripheral and central pathways, whereas the majority of abnormal SSPEs in female carriers are confined to the central pathways (97). TMS often reveals prolonged corticomotoneuronal latencies in both male and female patients (98). Prolonged I-V interpeak latencies, as well as a decrease of amplitudes are found in BAER (99). Abnormalities of visual evoked potentials (VEP) are less frequent and mainly observed in patients with advanced cerebral involvement (100). Nerve conduction velocity patterns change depending on the disease stage. In presymptomatic and early AMN, latency is increased indicating a predominance of demyelinating patterns. In later stages of AMN an additional reduction of amplitudes is found as a sign of progressive axonal damage (68, 101). The electroencephalogram frequently shows irregular large amplitude slow activity, prominent over the posterior brain regions of cALD patients (102).

1.3.4 Pathology and inflammation

Pathologic changes in X-ALD are mainly confined to the CNS, the PNS, the adrenal cortex and testes. Abnormalities in cerebral disease forms differ from those found in AMN and heterozygous women. While cerebral forms of X-ALD are associated with severe inflammatory reactions in brain white matter, pure AMN is mainly an axonopathy that involves the long spinal cord tracts and the PNS. An inflammatory response is mild or absent in AMN (77, 103).

ƒ Pathology of the nervous system in cerebral X-ALD forms The hallmark of childhood cerebral X-ALD is the symmetric loss of myelin, usually found most prominently in parieto-occipital regions, spreading caudorostrally. In post mortem examinations the grey matter appears macroscopically intact whereas in the centrum semiovale sclerotic changes as well as occasional cavitation and calcification are seen. In

29 Introduction cerebellar white matter similar, but smaller, lesions can be found. The spinal cord is usually spared except for secondary descending corticospinal tract degeneration (69, 72). Histopathological analysis normally shows marked losses of myelin and oligodendrocytes combined with hypertrophic reactive astrocytosis. Axons are initially spared. The most numerous participants in the inflammatory reaction are reactive astrocytes, macrophages and lymphocytes. Plasma cells and natural killer cells appear much less frequently. Lymphocytes are mainly found just within the edge of demyelination, where myelin and oligodendrocytes have already been lost. The majority of these lymphocytes belong to the T-cell population (104). Many of them are CD8+ and express the α/β-type of the T-cell receptor as well as granzyme B (105). Reactive astrocytes and macrophages also infiltrate morphologically normal or mildly affected white matter areas, adjacent to the demyelinative edge and are immunoreactive for tumor necrosis factor alpha (TNF α), interleukin 1 (IL-1) and 6 (IL-6), interferon γ (IFN γ) and transforming growth factor β (TGF β). Intracellular adhesion molekule 1 (I-CAM) was found to be upregulated in endothelial cells (104, 105). Beyond the active edge a splitting and fragmentation of myelin sheath has been observed, as well as an enlarged extracellular space due to oedema (72). Though there is a severe loss of oligodendrocytes, classical apoptotic changes in these cells are rare (105). Only few oligodendrocytes were found to be immunoreactive for typical apoptosis markers, like activated caspase-3, FAS/FAS-L, TNF receptors or MHC class I molecules. MHC class I has been identified on endothelial cells, many macrophages, microglia, lymphocytes and astrocytes. Microglia cells were also MHC class II positive. CD44, which can mediate MHC-unrestricted target cell death, is present on many lymphocytes and macrophages. CD1 molecules, which play major roles in MHC- unrestricted lipid antigen presentation, are noted on hypertrophic astrocytes, perivascular lymphocytes, microglia and macrophages (105). On ultrastructural examination macrophages containing myelin debris, lipid droplets (consisting of cholesterol esters) and lamellar lipid profiles were found (69). The peripheral nervous system (PNS) in cALD is typically unremarkable, despite from Schwann cells containing lamellar or lamellar-lipid inclusions (68).

ƒ The immunologic scenario in cerebral X-ALD X-ALD is associated with accumulation of VLCFA (predominantly C24:0 and C26:0) in various tissues (66). These VLCFA are also known to be incorporated into many lipids of

30 Introduction cell membranes including myelin. In particular phosphatidylcholine, gangliosides (in lesioned areas) and myelin proteolipid protein (PLP) were shown to assimilate VLCFA (106). This incorporation of VLCFA into myelin was proposed to have an adverse impact on myelin stability and function (72, 105, 107). As a result dysmyelination, myelinolysis and liberation of myelin constituents might occur triggering a natural (innate) immune response. TNFα and IL-1 secreted from astrocytes, macrophages and microglia cells could possibly set in motion a cascade of events resulting in enhanced vascular permeability and augmented damage of myelin mediated by cytokines (104). Furthermore TNFα is known to inhibit peroxisomal β-oxidation in oligodendrocytes which would aggravate the biochemical abnormality of X-ALD by increasing the accumulation of VLCFA in membranes (108). Prior to the second stage of fulminant inflammatory demyelination at least three additional events are thought to occur: the MHC class II restricted presentation of peptide antigens by microglia, the CD1-restricted presentation of lipid antigens and the cytolytic killing of oligodendrocytes by CD8+ cytotoxic T-lymphocytes. VLCFA-PLP has been viewed as a plausible candidate to serve as a CNS specific antigen. It has been speculated that it might be presented to CD8+ cells by CD1b on astrocytic or by CD1c on microglial processes in close proximity to oligodendroglia cells (105). Oligodendrocytes are then killed due to the release of granzyme B from CD8+ cells via a bystander effect or via a CD44 mediated MHC class I-independent mechanism. Superantigen presentation and antigenetic determinant spreading have been implicated in the extensive nature and rapid progression of CNS lesions in cALD (105, 109). Alternatively, the destruction of oligodendrocytes may also be indirect, due to the recruitment of additional effector cells and induction of deleterious cytokines or chemokines. Despite from proinflammatory cytokines (TNFα, IL-1, IL-6, IL-2, IL-12A), also colony-stimulating cytokines (IL-3, GM-CSF) and β family chemokines (CCL2, -4, - 7, -11, -16, -21, -22) were shown to be upregulated within the plaque area and to a lower extent in normal appearing white matter (110). Moreover, the incorporation of VLCFA in gangliosides could confine their normally immunosuppressive activity and thus enhance inflammation (111). Evidence of nitric oxide participation in X-ALD has been reported by Gilg et al. and by Paintlia et al. (110, 112). Mediated by microglia and astrocytes nitric oxide may at least contribute to the lytic death of oligodendrocytes. Powers et al. found profound oxidative

31 Introduction damage resulting from lipid peroxidation in inflammatory lesions of X-ALD patients (113). Signs of oxidative stress were also seen in CNS white matter beyond the active lesion edge where early myelin breakdown was occurring. When peroxinitrite, one of the most diffusible and damaging reactive oxygen species (ROS) is produced, it can kill cells in a few seconds via a non-apoptotic mode of cell death (114). There is also evidence that nictric oxide (NO) can cause necrotic death of oligodendrocytes (115, 116). The role of B-lymphocytes and immunoglobulins in the inflammatory demyelination of X- ALD still remains unclear. Although there is evidence of B-cells, plasma cells and (intrathecally synthesized) immunoglobulins presence in the CNS of cALD patients, it is unknown whether these immunoglobulins target specific CNS antigens or whether they are produced by an unspecific stimulation of B-cells (114). Investigations of blood serum samples from several X-ALD patients revealed the occurrence of autoimmune antibodies against CNS antigens such as myelin oligodendrocyte glycoprotein (MOG) and ganglioside GM1, but these antibodies are not specific for X-ALD (116, 117). Moreover, it is unknown how they originate and under which conditions peripheral antibody responses would affect the CNS.

ƒ Pathology of the nervous system in AMN patients and female heterozygotes Other than in cALD, pathological changes involving the CNS of AMN patients and female heterozygotes are mainly found in spinal cord regions. Loss of myelinated axons combined with a milder loss of oligodendrocytes has been observed in the long ascending and descending spinal cord tracts (118). Cervical fasciculi graciles and lumbal lateral corticospinal tracts are usually most severly affected, indicating a distal (dying-back) axonopathy. The myelin loss is equivalent to the axonal loss. Lesions are found to be bilateral and symmetrical. In contrast to cALD, signs of inflammation are minimal or completely absent in AMN (77). Only activated microglia cells were observed to a greater extent. Astrogliosis is moderate and lipophages are mainly seen in perivascular regions, particularly in relatively preserved tracts. Ultrastructural examinations revealed axonal atrophy, infrequent segmental thinning of myelin sheaths, myelin corrugation and myelin figures with little to no evidence for demyelination (69).

32 Introduction

Peripheral nerve lesions may occur, but are usually milder, more variable and more non- specific than the myelopathy. The largest myelinated fibers seem to be most severely affected (118). Also endoneurinal fibrosis and thinning of myelin sheaths has been observed in sural and peroneal nerves (69). Lamellar inclusions in Schwann cells that are diagnostic for AMN or ALD have been documented (119). In lumbar dorsal root ganglia no obvious neuronal loss, necrosis or apoptosis was found. However, morphometric studies revealed neuronal atrophy with a decrease in the number of large neurons and a corresponding increase of smaller neurons (< 2,000 µm²) (118). Powers et al. reported that many mitochondria in AMN neurons contain lipidic inclusion (118). They hypothesized that an impaired mitochondrial function may contribute to the myelopathy in AMN through a failure of ATP-dependent axoplasmic transport with consequent dying-back axonal degeneration. Summing up, the fundamental defect in AMN myeloneuropathy was proposed to be an axonal or neuronal membrane abnormality that leads to neuronal dysfunction and atrophy.

ƒ Pathology of adrenal cortex, testes and pituitary Most male ALD and AMN patients develop atrophy of the adrenal cortex and the seminiferous tubules (57, 120, 121). Adrenocortical cells, especially those of the inner fasciculata reticularis become ballooned and show accumulations of lamellar profiles and fine lipid clefts. These structures were found to contain cholesterol esterified with VLCFA. The lamellae are thought to consist of bilayers of these abnormal fatty acids. Intersitial cells of Leydig and their precursors also display accumulation of lipid profiles (122). As neither inflammatory cells nor antibodies seem to be involved in the loss of endocrine cells, it has been proposed, that the overload with saturated VLCFA might be toxic to adrenocortical and Leydig cells, resulting in apoptotic cell death (123). Moreover it has been demonstrated that VLCFA interfere with the ACTH receptor on the surface of adrenocortical cells which possibly aggravates hormone deficits. Similar pathology of the adrenal cortex can also be seen in heterozygous females. The striking difference is that striated adrenocortical cells in female X-ALD patients are limited to small, multifocal clusters whereas in males these lesions are more diffuse (122). The pituitary gland, where ACTH production takes place, often shows an increase in basophilic adrenocorticotrophins with varying numbers of stainable granules, but these

33 Introduction findings seem to depend on the degree and duration of glucocorticoid insufficiency and replacement therapy (69).

1.3.5 Therapeutic strategies

Depending on the phenotypic expression of X-ALD, the progression rate of the disease and the individual handicaps, X-ALD patients may need special treatment and supportive care. Symptomatic therapies include special teaching programs, physical therapy, psychological counselling and individual medications tempting to disburden muscle spasms, pain, urinary incontinence, erectile dysfunctions, seizures and psychiatric disorders. Currently no cure for manifest X-ALD is available and although many attempts have been made, no satisfying overall treatment for all different disease forms has been found so far (68, 69).

ƒ Adrenal and gonadal hormone replacement As the majority of the male X-ALD patients develop primary adrenocortical insufficiency, steroid replacement must be provided. It improves well-being and may be life saving, though it does not appear to alter the progression of the neurological disease (124). Measurement of plasma cortisol levels alone is not sufficient for surveillance, for it may fail to detect a borderline insufficiency corrected by elevated levels of ACTH. ACTH stimulation tests should be performed at least once a year in order to start therapy with hydrocortisone early. Few patients additionally require administration of mineralocorticoids when they present clinically with postural hypotension, hyponatremia and hyperkalemia (69). Subnormal levels of testosterone that need substitution are found in less than 10 % of all male ALD patients. Impotence, in most instances, is due to spinal cord involvement or neuropathy and can be treated with Sildenafil (124).

ƒ Bone marrow transplantation (BMT) To date, bone marrow transplantation is the only proven effective therapy for cerebral forms of X-ALD in children and adolescents, at least when it is performed at early stages of the disease. In these cases, median 5-year survival rate after BMT is stated with 62%. Most patients that are not rescued by the procedure die within the first year after BMT due to progression of X-ALD, when no engraftment of the donor cells had occurred, graft versus host disease or infections (125).

34 Introduction

Because of the high risk of the procedure, indications for BMT must be considered carefully. It is generally agreed that the prime indication for transplantation is in boys or adolescents who have mild progressive brain involvement detected by MRI examination (126). Patients in advanced stages often benefit little from BMT as the performance procedure is known to aggravate neurologic symptoms initially (127). These patients tend to continue to deteriorate following the transplant. BMT is also not indicated in young patients whose brain is uninvolved as about half of them may escape the inflammatory form of X-ALD even without therapy. It is unclear whether BMT will be beneficial for AMN as the basic pathology in this form of the disease seems to be different from cALD and patients usually lack an inflammatory response (69). In a 5-10 year follow-up study of transplanted patients basically suffering from cALD, none developed clinical signs of spinal cord involvement, suggesting that BMT may also prevent the onset of axonal degeneration and therefore AMN (128). However, since the onset of AMN may be delayed until late in life longer follow-up studies are required. The mechanism by which BMT improves the condition of cALD patients is still unknown. After BMT plasma VLCFA levels were shown to be decreased by 55% (128). In many patients the disease course is arrested and clinical improvement has been documented 6-12 months later. However, BMT has no effect on the abnormal adrenocortical function in X-ALD. Krivit et al. proposed that the beneficial effect of BMT is mediated by brain microglia, which are, at least in part, bone marrow derived (129). Microglia cells are thought to be involved in the metabolism and recycling of myelin components released during myelin breakdown in X-ALD (130). They take up myelin lipids but cannot oxidize VLCFA due to the genetic deficiency of ALDP. VLCFA accumulate, may be secreted in lipoproteins and furthermore could be taken up by remyelinating oligodendroglia cells, where they are incorporated again into myelin sheaths, making them increasingly unstable. This circulus vitiosus could be interrupted by BMT when host microglia cells are replaced by healthy donor cells.

ƒ Dietary therapy The concept of dietary therapy was induced by Kishimoto et al. in 1980 (131). They found that a substantial proportion of brain C26:0 fatty acids in X-ALD patients were of dietary origin and developed a diet that restricted C26:0 to less than 15% of the normal intake.

35 Introduction

However, clinical trials using this diet were not successful, suggesting that the decreased exogenous supply of C26:0 was overruled by the endogenous synthesis of VLCFA (132). Six years later Rizzo et al. reported that addition of oleic acid (C18:1) to the medium of cultured X-ALD fibroblasts was able to reduce the levels of saturated VLCFA in these cells (133). The same effect could be seen in patients who received glyceryl trioleate orally (134). Their plasma VLCFA levels were reduced by 50%. The administration of a 4:1 mixture of glyceryl trioleate and glyceryl trierucate (known as “Lorenzo’s oil”) turned out to be even more effective (135). Plasma VLCFA levels were normalized within 4 weeks of treatment. This striking effect was explained by the theory that glyceryl trioleate and glyceryl trierucate as mono-unsaturated fatty acids compete with saturated long chain fatty acids for the microsomal fatty acid elongation system, thus decreasing the endogenous production of saturated VLCFA (136). Large “open” clinical trials using this diet were carried out. The absence of a placebo treated control group hampered evaluation but today’s experience indicates that benefit of this treatment is limited. Though plasma VLCFA levels are reduced the clinical status of patients who are already symptomatic continues to deteriorate (137). Post mortem examinations revealed that C26:0 levels in brain tissues of patients treated with Lorenzo’s oil did not differ from the levels in untreated patients, indicating that Lorenzo’s oil is not able to penetrate the brain in substantial amounts (138). Nevertheless it has been proposed that dietary treatment with Lorenzo’s oil may have preventive effects in previously unaffected boys. A prevention study done by Moser et al. demonstrated a two-fold reduction of the risk of developing cALD in patients treated with Lorenzo’s oil (139). An open study on the effects of Lorenzo’s oil treatment in AMN patients was conducted by Koehler et al. and indicates that therapy with Lorenzo’s oil combined with a C26:0 low diet may influence the progression of neurodegeneration in non-cerebral X-ALD phenotypes (124). Occurrence of inflammatory demyelination seems to be reduced or at least delayed in AMN patients, who are treated with Lorenzo’s oil.

ƒ Immunosuppression and Plasma exchange Many attempts have been made to develop an immunosuppressive or immunomodulatory therapy that is able to reduce the inflammatory response in cerebral forms of X-ALD or to convert the rapidly progressive forms into the milder non-cerebral phenotype. Cyclophosphamide, interferon-β, corticosteroids, glatiramer-acetate, thalidomide,

36 Introduction intravenous immunoglobulins, cyclosporine and plasma exchange therapy have been tried without clear benefits for patients (reviewed in (124)). TNFα-blockers have been proposed to possibly influence the inflammatory response in X- ALD because many macrophages and astrocytes in cALD lesions are immunoreactive for TNFα and elevation of TNFα seems to be correlating with the clinical phenotype (140). Infliximab, a TNFα-receptor blocker shows significant anti-inflammatory effects in various animal models and a halt of progression in 2 out of 3 adult X-ALD patients suffering from rapidly progressing cerebral ALD (124). However, further studies are needed to determine long term as well as clinical side effects.

ƒ Clofibrate, Carnitine and dextrane sulfate adsorption It is known that Clofibrate enhances the proliferation of peroxisomes in rat liver and influences their oxidizing system. Therapy with Clofibrate temporarily lowered plasma C26:0 levels in patients but had no effect on their clinical status (132). Reduction of plasma VLCFA levels in X-ALD patients by plasma exchange does not alter the course of disease (124). However, Bambauer et al. reported a significant improvement of neurological symptoms in one adult cALD patient after therapy with dextrane sulfate adsorption (141). Administration of L-carnitine seems to have no effects, neither on plasma VLCFA levels, nor on clinical status (132).

ƒ Lovastatin and 4-Phenylbutyrate The HMG-CoA reductase inhibitor lovastatin, a cholesterol-lowering drug, was proposed to be a possible therapeutic agent for X-ALD because it is able to normalize the levels of VLCFA in cultured X-ALD cells (142). Two small studies demonstrated a beneficial effect of Lovastatin in combination with a low fat diet in X-ALD patients (143). The treatment resulted in a successful reduction of total plasma VLCFA. However, results from placebo controlled clinical trials and information about long term effects are not yet available. Apart from reducing plasma LDL cholesterol by inhibiting the Cholesterol biosynthesis and by up-regulating the LDL receptor on hepatocytes, statins have been shown to block the expression of inducible nitric oxide synthase (iNOS) and of proinflammatory cytokines in astrocytes and microglia (144). This may contribute to their clinical benefit. 4-Phenylbutyrate increases the capacity of cultured X-ALD fibroblasts to oxidize VLCFA (53). This effect may be a result of the induction of peroxisomal proliferation and an

37 Introduction enhanced expression of ALDRP initiated by the substance. Further investigations revealed that 4-Phenylbutyrate is also able to lower VLCFA levels in brain and adrenal gland tissues of X-ALD mice (51). However, in a study with AMN patients the drug failed to decrease plasma VLCFA levels (69).

1.4. Abcd1 knockout mice – the X-ALD mouse model

Mice deficient for ALDP have been generated in 1997 by three different laboratories (63-65). The rational was to create a model system, which enables us to study the pathophysiology of X-ALD in more detail. The respective constructs used for targeted inactivation of the Abcd1 gene are depicted in figure 2. Forss-Petter et al. and Kobayashi et al. deleted most of exon 1 (including the translation start site) of the Abcd1 gene and replaced it by a neomycin resistance gene (neo). Lu et al. inserted the neo cassette within exon 2. The first results derived from these models were quite humbling. Although elevated levels of VLCFA in total lipid fractions were found in all tissues examined, the mice did not show any overt neurological symptoms or adrenal insufficiency up to the age of 6 months. Their life span was normal. Examinations done 5 years later by Pujol et al. revealed that Abcd1 knockout mice, which had been backcrossed to C57BL/6J mice, develop a late onset phenotype with similarities to AMN, the human adult onset form of X-ALD. (145)

38 Introduction

Figure 2: Constructs used to inactivate the Abcd1 gene in mice. (A) Wild type allele; (B) Inactivation constructs used by Forss-Petter et al., Lu et al. and Kobayashi et al..

1.4.1 Accumulation of VLCFA and β-oxidation

X-ALD mice have biochemical defects that resemble those found in human X-ALD patients but to a milder degree. Total levels of VLCFA have been measured in several tissues. A 5-fold increase of C26:0 levels is usually found in brain. Adrenal gland, spinal cord, heart, liver, lung and kidney show a 2- to 4-fold increase (63-65). Strikingly, in contrast to human X-ALD patients, Abcd1 knockout mice do not display an elevation of C24:0 and C26:0 in plasma. VLCFA β-oxidation activity in skin fibroblasts derived from X-ALD mice was reported to be reduced by 60%, whereas in human X-ALD fibroblasts the reduction ranges up to 80% (63, 65).

1.4.2 Behavioural and neurological phenotype

Abcd1 knockout mice on a mixed 129Sv/ C57BL/6 background did not show signs of abnormal motor development, loss of motor functions, or other neurological deficits up to the age of 6 months (63). To exclude the possibility that one of the two genetic backgrounds could have a masking effect on the results gained by a rotarod test procedure,

39 Introduction a cohort of both 129Sv and C57BL/6 wild type animals had also been tested. No differences between the two strains could be detected. Pujol and coworkers analysed 15 and 20 months old Abcd1 knockout mice generated by Lu and collaborators and which had been backcrossed to C57BL/6 (145). Up to these time points home cage behaviour remained indistinguishable from that of wild type littermates and no obvious signs of motor uncoordination, ataxia or other apparent neurological abnormalities were found. A rotarod test was performed in order to detect subtle differences in motor coordination ability. Mice were challenged at a constant speed of 10 rpm and the time until they fell off the apparatus was recorded. At the age of 15 months still no difference in performance between wild type and knockout mice could be detected. However, at the age of 20 months severe impairment was seen. Abcd1 knockout mice fell off the rotarod in less than 20 seconds. These results were quite surprising as they indicate a late onset form of neurological disease in ALDP deficient mice with some features of AMN. A video-controlled open field mobility test, also done by Pujol and coworkers showed that the initial spontaneous motor activity is reduced in 15 months old Abcd1 knockout mice. At 20 months of age the mice presented with hypoactivity during the whole testing period. Episodes of rearing behaviour were reduced in both age groups compared with wild type animals. No significant differences could be found in gait pattern analysis (145). Our laboratory also generated Abcd1 knockout mice but although the Abcd1 gene disruption in our mice and in the ALDP deficient strain used by Pujol and coworkers are functional null mutations with similar biochemical features of X-ALD, the impairment described by Pujol at al. was not reproduced in our mice. The reason for this conflicting result is still unclear. Differences in genetic backgrounds (although in both cases a major contribution of C57BL/6 is expected), in animal housing conditions or in test procedures have been speculated to account for the variable findings.

1.4.3 Electrophysiology

Compared with age matched control animals, ALDP deficient mice exhibit a significant increase of compound muscle action potential (CMAP) latency recorded in sciatic nerve and gastrocnemius muscle at the age of 15 months (145). CMAP latencies provide information about the function of fast conducting (motor) fibres. As CMAP

40 Introduction amplitudes remain normal in Abcd1 knockout mice compared with their wild type littermates, myelin abnormalities seem to be more likely than substantial axonal loss. A slowing of sensitive nerve conduction velocities was found in 20 months old Abcd1 knockout mice (145). In contrast spinal reflex, measured by H-wave latency and amplitude, remains normal (147).

1.4.4 Peripheral nerve pathology

Light microscopy of sciatic and trigeminal nerves of 6-month-old Abcd1 knockout mice did not show any pathologic alterations (63). Myelin of the peripheral nervous system appeared intact. At the age of 16 months, minor myelin and axon abnormalities were seen in light and electron microscopy investigations of sciatic nerves. Axons with thick and disorganized myelin sheaths were found (145). These focal myelin swellings are termed “tomaculae” (148). Electron microscopy studies revealed that these tomaculae consist of infoldings of myelin loops. Schwann cell processes are enlarged and contain myelin-like figures as well as fibrillary and paracristalline osmiophilic inclusions. However, the characteristic lamellar and lamellar-lipid inclusions often seen in Schwann cells and macrophages in the PNS of X-ALD patients are absent in Abcd1 knockout mice (145).

1.4.5 Pathology of the central nervous sytem (CNS)

Luxol-fast blue staining did not reveal generalized or focal demyelination in brain and spinal cord sections of 6 months old Abcd1 knockout mice (63-65). Bielschowsky silver impregnation showed a normal distribution of axons in the CNS and no signs of inflammation could be detected by hemotoxilin eosin staining. Also in 16 and 21 months old knockout animals no infiltrates of immune cells, vacuolar degeneration or myelin loss could be found (145). In semithin and ultrathin sections of the fiber tracts of corpus callosum, internal capsule and anterior commisure (regions that are primarily affected in human cALD) no obvious pathological features were determined. Semithin and ultrathin sections of spinal cords of 16 and 21 months old mice revealed focal white matter abnormalities including redundant myelin sheaths and degenerating axons. Macrophages containing fibrillary and paracrystalline, spicular needle-like structure and engulfing myelin debris were found within the CNS (145).

41 Introduction

Using immunohistochemical markers, signs of microglia and astrocyte activation were detected (52). Staining for amyloid precursor protein (APP) and synaptophysin, two marker proteins that accumulate along axonal swellings revealed axonal degeneration in 22 and in 12 and 22 months old animals respectively. The lesions are Ubiquitin positive but do not colocalize with myelin lesions. Moreover, axonal degeneration seems to precede myelin disturbances. Classical apoptotic changes, estimated by biotinylated UTP nick end labelling and caspase-3 immunohistochemistry, are missing (52). Examination of the cerebellum revealed normal appearance of molecular, Purkinje cell and granular layers, normal foliation and cytoarchitecture up to 20 months of age. Thereafter atrophy of Purkinje cells, detected by immunostaining against calbindin was seen, as well as local reactive gliosis (147).

1.4.6 Adrenal gland pathology

Under the electron microscope lipid clefts were found in adrenocortical cells of 3- month-old Abcd1 knockout mice (65). These lipid clefts contain some spicular lamellar material and are located either freely in the cytoplasm or in lysosome-like organelles of cortical cells and macrophages. Medullary cells, in contrast, appear normal. At 6 months of age, alterations in adrenocortical cells are also visible at light microscopy level (63). Cortical cells appear swollen and contain fewer lipid droplets, compared with wild type animals. Adrenals of 18-month-old Abcd1 knockout mice show pronounced fibrosis of reticular and fascicular cell laye (52).

1.4.7 Testis and ovary pathology

In testis and ovary the number of interstitial cells is increased. Also some atrophy of seminifereous tubules has been reported (63). Interstitial cells contain the same needle-like inclusions like adrenocortical cells. Hemizygous males also show severe testicular atrophy. Fertility seems to be somewhat reduced in inbreed homozygous and hemizygous ALDP deficient mice. These findings are similar to the infertility and testicular atrophy seen in human AMN patients (120).

42 Introduction

1.5. Mag knockout mice

1.5.1 Myelin

The development of myelin sheeths insulating axolemmal membranes is a major evolutionary achievement. It facilitates rapid signal conduction required in organisms with complex nervous systems. Specialized glial cells, oligodendrocytes in the CNS and Schwann cells in the PNS, elaborate cytoplasmic wrappings around axons, that appear rather unstructured in invertebrate species but are highly organised in vertebrates (149, 150). Oligodendrocytes differ from Schwann cells in that they have the ability to form multiple myelin internodes by extending several processes to diverse axons rather than enveloping only one single axon. Schwann cells in turn form only one myelin internode, thus making contact to only one axon. Ultrastructurally myelin internodes were shown to consist of two distinct domains, compact and noncompacted myelin. Compact myelin surrounds the axon, whereas noncompacted myelin forms the exterior of the internode. Schmidt-Lantermann incisures found in PNS myelin are translucent cytoplasmic channels, which radially transverse compact myelin and connect outer and inner regions (151). Most myelin components are synthesized in oligodendrocyte and Schwann cell perinuclear cytoplasm and are secondarily transported via radial and logitudinal cytoplasmic channels to their target location. For this purpose, the cytoplasmic channels contain mitochondria for energy supply as well as microtubules and other cytoskeletal components that mediate transport functions. Inner cytoplasmic channels and paranodal loops, moreover, are sites of myelin-axon interaction (151). In contrast to other membranes that contain a more or less 1:1 distribution of lipids and proteins, myelin is enriched in lipid fraction, which ranges up to 75% and comprises phospholipids, glycolipids and cholesterol (152). Major proteins found within compact myelin include P0 protein, myelin basic protein (MBP), P2 protein and peripheral myelin protein-22 (PMP-22) in the PNS and proteolipid protein (PLP) and MBP in the CNS (153, 154). P0, PMP-22 and PLP have been shown to be integral membrane proteins, whereas MBP and P2 are extrinsic membrane proteins. PLP and MBP seem to be required for normal spacing of compact CNS myelin, while P0 is essential for normal spacing of PNS compact myelin (151, 155, 156). Stoichiometric distribution of these myelin proteins seems to be important. Alterations in myelin protein gene dosage have been shown to

43 Introduction cause chronic diseases like hereditary neuropathy (PMP-22) (157), Charcot-Marie-Tooth disease type 1 (PMP-22) (158) and Pelizaeus Merzbacher disease (PLP) (159). Other important myelin proteins located in the noncompact myelin include Myelin associated glycoprotein (MAG) (160), 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) (161) and myelin oligodendrocyte protein (MOG) (162). MOG was shown to be a mammalian specialization. It is a minor myelin component preferentially found at the outermost surface of myelin sheeths in PNS and CNS. MAG, CNP and microfilaments in contrast are located in periaxonal regions of myelin. These periaxonal membranes are separated from the axon by a 12-14 nm cleft, the periaxonal space (see figure 3) (151).

abaxonal membrane: MOG, CNP noncompact myelin periaxonal membrane: MAG, CNP compact myelin: P0, P2, MBP, PMP-22 (PNS) axon PLP, MBP (CNS) axolemma periaxonal space Abasal lamina

Figure 3: Schematic cartoon of a myelinated axon and the distribution of myelin proteins in compact and non-compact myelin.

1.5.2 Myelin-associated glycoprotein (MAG, siglec-4a)

MAG, a minor constituent of CNS and PNS myelin, is a glycoprotein that belongs to the so called “siglec family” of sialic acid–binding immunoglobulin-like lectins (163). It consists of a short cytoplasmic domain, a single transmembrane domain and an extracellular N-terminal domain, which comprises five immunoglobulin-like domains (164). MAG expression is almost exclusively restricted to myelin-forming cells. On Schwann cells and oligodendrocytes it can be detected prior to the onset of myelination but in

44 Introduction contrast to oligodendrocytes Schwann cells seem to require at least some neuronal contact to express MAG and other myelin proteins (165, 166). During the myelination process MAG immunoreactivity is found all around the cell surface, including cell body and processes. It stays present also in wrapped myelin layers, until the compaction process has been completed (167). After axons have been fully myelinated and compact myelin has been formed MAG expression is restricted to noncompacted myelin, in the CNS to periaxonal regions and to the inner mesaxon. In the PNS it can also be detected in paranodal loops, Schmidt-Lantermann incisures and, to some low extent, at the external (abaxonal) surface of myelinating Schwann cells (167). Two isoforms of MAG exist. The small 67 kDa form (S-MAG) and the large 72 kDa form (L-MAG) result from alternative splicing (168). Their expression is regulated temporally and spatially. In the CNS, L-MAG predominates during development and myelination process, whereas S-MAG accumulates at later stages. In the PNS, L-MAG is always rare (169). Functional consequences resulting from the different expression patterns seem likely. Moreover, it has been shown that S-MAG and L-MAG have distinct signalling capacities. There is evidence that MAG fulfils various functions. It is a cell recognition molecule involved in neuron-glial interaction and has been implicated in the formation and maintenance of myelin. It also affects axonal viability and cytoskeletal organisation. In adult individuals it seems to inhibit axonal sprouting and regeneration in PNS and CNS (reviewed in (167)). However, during early development MAG is not inhibitory. On the contrary it has been shown to increase outgrowth of cerebellar, spinal cord and also neonatal dorsal root ganglion neurons (170). Several groups found that MAG binds to the Nogo Receptor (NgR) expressed on neuronal membranes and that this interaction mediates MAG inhibition on axonal growth in adults (171). Since NgR is virtually absent during early development and levels increase only later on, this may explain why MAG does not inhibit growth of young neurons. Upon injury to the adult nervous system, myelin-forming glia cells downregulate MAG. Moreover, several mediators have been identified, which are able to override the inhibitory activity of MAG, for instance: laminin, present in basal lamina of Schwann cells; the extracellular matrix glycoprotein, tenascin-R; and neutrophins, such as brain-derived neurotrophic factor (BDNF) and glia cell line-derived neurotrophic factor (GDNF) (167). In addition, MAG binds to gangliosides (sialic acid-containing glycosphingolipids) GD1a

45 Introduction and GT1b present on the nerve cell surface (172). This interaction contributes to the inhibitory effects of MAG on neurite outgrowth and has been shown to enhance axon- myelin stability. MAG and its ganglioside receptors are frequent targets of auto-antibodies in immune- mediated paraproteinemia and peripheral neuropathies, which lead to neuronal or axonal degeneration and demyelination (173). However, no human disease is known that affects the MAG gene, possibly because the phenotype is not severe enough to be detected easily.

1.5.3 Mag knockout mice

Mice deficient for Mag have been generated by two separate groups in 1994. Li et al. created Mag knockout mice on a mixed background of C57BL/6, 129Sv inbred strains and the CD1 random bred strain (174). Montag et al. injected 129Sv ES cells into B6CBAF1 hybrid blastocytes and crossed male chimeras to C57BL/6 or 129Sv females (175). The first results obtained were derived from these mice comprising a mixture of different backgrounds. Other mouse mutants, which have alterations in major myelin proteins like P0, MBP, PLP or PMP-22, usually develop severe impairments. In contrast Mag knockout mice, when first analysed, displayed a phenotype that was far from being severe. They had only minor defects in myelin and signs of axonal degeneration were exclusively encountered in PNS. Pan et al. reinvestigated Mag knockout mice in 2005 (176). For their study they used animals, which had been extensively backcrossed to C57BL/6 and showed a 99.5 % strain purity. They found clear evidence of axonal degeneration in PNS and CNS as well as marked behavioural deficits. Thus, it seems that the genetic background of Mag knockout mice influences the CNS pathology and phenotype.

ƒ Behavioural and neurological phenotype Montag et al. investigated the coordinated movement and learning capacity of 2-month-old Mag knockout mice (comprising a mixture of genetic backgrounds) using a water maze test (175). No genotype related differences were found concerning learning and spatial memory. Swim speed was equal in Mag knockout mice and age-matched wild type controls. No episodes of circling, indicating hind limb malcoordination, could be detected. Li et al. performed open field and bar-cross tests in 2-month-old mice (174). Locomotion, grooming and rearing parameters in the open field test were the same in Mag knockout and

46 Introduction wild type mice. In the bar cross test, which requires finer motor coordination mutant mice showed a decreased motor activity. Exploratory sniffling and grooming were less frequent. Moreover the mice exhibited a mild, transient (intention-) tremor on the bar. Pan et al. investigated Mag knockout mice on a C57BL/6 background, at the age of 6 months (176). Rotarod-testing revealed impaired motor coordination and balance in mutant mice. Also marked deficits in hind limb reflex (scored from 0 = paralysis to 4 = normal extension of hindlimbs >90°) were seen. Whole body tremor during rest and during movement was encountered in some of the Mag knockout mice. Despite motor behavioural deficits mutant mice showed hyperactivity.

ƒ Pathology of the PNS

Electron microscopy studies of sciatic nerves of 2-month-old Mag knockout mice (on a mixed background) did not show any pathological changes in myelin sheaths (175). Periaxonal space, myelin thickness, axon-Schwann cell interface, inner and outer mesaxon, Schmidt-Lanterman incisures and paranodal loops appeared normal. Large calibre axons were consistently myelinated. The myelination process, investigated in pectineus nerve preparations at early developmental stages (postnatal day 4 and 10) was also normal. No significant delay, neither at onset nor during myelin formation, could be detected (175). The amount of nonmyelinated axons per microscopic field was the same in Mag knockout and wild type control mice. Also immunohistochemical distribution and presence of other Schwann cell related marker proteins like P0, MBP, L2/HNK-1, L3 and tenascin appeared unaltered (175, 177). Neuronal cell adhesion molecule (N-CAM) was shown to be down regulated in sciatic nerve cross sections in one study (175). However, this result could not be verified by western blot analysis (174). On cross sections of lumbar ventral spinal roots derived from 3-month-old Mag knockout mice some myelinated fibers displayed a dilated periaxonal space. In others, the periaxonal space as well as the oligodendrocyte cytoplasmic collar was missing (174). Yin et al. found few axon-Schwann cell profiles with features of Wallerian-like degeneration in sciatic nerve sections of 35 days old Mag knockout mice (178). Their number increased in 3-, 9- and 16 months old mice. A decrease in the calibres of myelinated axons that correlated with a reduction in interneurofilament spacing was seen at the age of 3 months and thereafter. Western blot analysis revealed that these alterations

47 Introduction were caused by a decreased neurofilament phosphorylation, which normally is responsible for enhanced lateral extension of neurofilament sidearms. Axonal atrophy was most prominent in paranodal regions, where it resulted in the collapse of myelin and tomacula formation (179). Tomacula are described as myelin thickenings measuring more than 50% of the fiber diameter (148). They are caused by overproduction of myelin and complex in- and outfolding. Electron microscopic investigations of brachial, sciatic and femoral nerves derived from 8- 10-month-old Mag knockout mice also showed abnormal appearance of some axon- Schwann cell units (180). Degenerating axons, characterized by unusual small diameter, high electron density and an axoplasm containing vesicles and/or cellular debris were found. So called “onion bulbs”, Schwann cells forming centrically arranged, crescent- shaped processes were discovered. Such structures have been described in various human peripheral neuropathies where they are indicative for demyelination-induced Schwann cell proliferation (181). Furthermore an upregulation of the extracellular matrix protein tenascin-C, which is a marker for degeneration events occurring in the PNS, was detected by immunhistochemistry (180). Electron micrographs of sciatic nerves of 12-14-months-old Mag knockout mice revealed substantial myelin redundancy, though myelin compaction remained normal (177). Disruption of the periaxonal Schwann cell membrane, degeneration of periaxonal myelin as well as considerable degeneration of axons was seen.

ƒ CNS myelin formation The onset of myelin formation in optic nerve of Mag knockout mice is retarded during development. Investigations in 10 and 11 days old animals revealed a decrease in the number of myelinated axons per microscopic field of 48% and 39% respectively (182).

ƒ Pathology of the CNS Investigations of optic nerves derived from 2- and 4-months-old Mag knockout mice revealed some morphological changes in a couple of myelin sheaths (175). Cytoplasm was found within compact myelin. Closer ultrastructural inspections showed that some axons were encircled by more than one myelin sheath. Moreover a reduction in the number of myelin sheaths carrying an oligodendrocyte cytoplasmic collar at the inner region was found. The 12-14 nm cleft between axon and myelin sheath was not preserved in many

48 Introduction myelinated axons, indicating that Mag indeed participates in the formation and maintenance of the periaxonal space in the CNS. The total number of nonmyelinated axons in optic nerves, detected by L1 (a neural adhesion molecule that stains unmyelinated axons) immunoreactivity, was increased (175). Similar results were obtained by Bartsch et al. who investigated ultrathin sections of optic nerves derived from 2 months and 9 months old Mag knockout mice (165). They found that the number of unmyelinated axons diminishes with age indicating a slowing of myelination in mutant mice. Light microscopic examination of brain and spinal cord sections did not show any gross abnormalities, especially no areas of demyelination or tissue distruction (174). Expression of myelin antigens, like PLP, MBP, MOG and CNP, detected by immunochytochemistry, was found to be normal (174). High magnification analysis of semithin sections of spinal cords revealed scattered, thickened myelinated fibers with some osmiophilic material located between the myelin sheath and the axon (183). In light microscopy this periaxonal material was immunoreactive for CNP, but not for neuronal or astrocytic markers. Electron microscopy studies showed that axons were normal. Pathological alterations could be found only in periaxonal and intramyelinic oligodendrocyte processes, which were enlarged and contained myelin-like vesicles and multivesicular bodies. Amorphous and granular osmiophilic material was seen side by side with these vesicular inclusions or with mitochondria. More severe involvement was represented by deposition of paracrystalline material, lipofuscin granules and large depots of densely packed mitochondria in oligodendrocyte processes. Perikaryal cytoplasm and proximal oligodendrocyte processes remained intact (183). Similar changes were described as dying back oligodendropathy in a model of cuprizone toxicity, multiple sclerosis and Theiler’s virus enduced encephalomyelitis (184-186). However, in these disease models dying back oligodendropathology was associated with widespread demyelination. Though axons with abnormally thin myelin sheaths were encountered occasionally, Mag knockout mice lacked signs of acute demyelination. Thus it has been proposed that the dystrophic process in oligodendrocytes develops at a very slow pace. No evidence was found for apoptosis of oligodendrocytes (187). Loers et al. described increased numbers of CD3+ T-cells and Mac-3+ activated microglia cells in spinal cords of Mag knockout mice, that did not strictly correlate with the severity of the dysmyelinating phenotype, but seemed to be influenced to a larger extent by the age

49 Introduction of the animals (levels increased from 5- to 15-18-month-old mice) (188). Also APP- positive axonal spheroids which are indicative for acute, ongoing axonal injury were encountered (in 5- and in 15-18-month-old animals), but axonal injury was not locally restricted to axons with damaged myelin sheaths (188). In cerebellum alterations of Purkinje cells were described. Bielschowsky’s silver impregnation revealed increased numbers of torpedoes (enlarged axons) already in 5- month-old mice. The Purkinje cell axonal injury increased with age (188).

ƒ Biochemical changes Weiss et al. investigated the expression levels of myelin related proteins in whole brain homogenates and isolated myelin fractions of 2- and 14-months-old Mag knockout mice (187). Levels of MBP, PLP and carbonic anhydrase II (CAII), examined by western blot analysis, were normal compared to age matched control animals. CNP was reduced in all preparations. Neural cell adhesion molecule (NCAM), β-tubulin and Na+/K+-ATPase were decreased in the myelin fraction. However, semiquantitative RT-PCR-measurements did not reveal any differences in mRNA levels of CNP, NCAM, MBP and PLP between Mag knockout and wild type mice. Western blot analysis of sciatic nerve homogenates revealed a statistically significant reduction of CNP, high molecular weight neurofilament protein and phosphorylated high molecular weight neurofilament protein in PNS, whereas the levels of compact myelin proteins remained more or less unaltered. Loers et al. described alterations in the lipid profile of membrane, rafts (lipid-rich microdomains) and myelin fractions of total brains derived from 5- and 15-18-month-old Mag knockout mice (188). They found increased levels of arachidonic acid in raft and myelin fractions and increased levels of lysophospholipids in membrane fractions. The levels of gangliosides and sphingomyelin were not altered. A 2-4 fold increase in cholesterol levels was detected in membrane, raft and myelin fraction. However normal cholesterol levels in serum as well as in cytosol and membrane fractions of liver indicate that this is not a generalized abnormality. Western blot analysis showed that apolipoprotein E, a molecule that is critical for shuttling of cholesterol, was also increased in the raft fraction, thus indicating an imbalance of lipid metabolism. Other cholesterol interaction partners like low-density lipoprotein receptor,

50 Introduction low-density receptor-related protein, very low-density lipoprotein receptor and scavenger receptor BI were not increased in any of the fractions.

ƒ Electrophysiology Nerve conduction studies done in facial and sciatic nerves of 4-10 weeks old Mag knockout mice did not reveal any pathology (175). Facial nerve compound muscle action potential (CMAP) response latencies and amplitudes were normal. Also facial nerve refractory time and M-response latencies and amplitude remained within the normal range. Waveforms of sciatic nerve CMAPs did not indicate temporal dispersion. Mild reductions in CMAP amplitude and conduction velocity as well as mildly prolonged F-wave latencies that did not reach significance were found in Mag knockout mice at the age of 12-14 months (177). These mild abnormalities could be explained by a reduction in axonal calibre of large myelinated nerve fibers.

51 Aim of the Thesis

CHAPTER 2

AIM OF THE THESIS

52 Aim of the Thesis

2. AIM OF THE THESIS

X-linked Adrenoleukodystrophy is an inherited disease caused by mutations in the ABCD1 gene (60). The functional loss of the ABCD1 gene product, the peroxisomal transporter protein Adrenoleukodystrophy protein (ALDP), results in an accumulation of saturated very long-chain fatty acids (VLCFA) in plasma and tissues, the diagnostic hallmark of X-ALD (66). However, there is no general correlation between the type of mutation, or the level of VLCFA and the clinical phenotypes (67). Though the pathology of cerebral X-ALD differs fundamentally from that of AMN, both disease forms can be caused by the same mutation. The link between the loss of ALDP, the peroxisomal defect and the accumulation of VLCFA remains to be elucidated. Today it is unknown how ALDP deficiency affects the metabolism of VLCFA, how it leads to morphological and functional alterations in specific organs and, how the different clinical manifestations of X- ALD are accomplished. The trigger, which sets off the fulminant, inflammatory, demyelinating, cerebral form of X-ALD has not been found yet. Obviously the accumulation of VLCFA alone is not sufficient to cause demyelination and inflammation, since patients with similar biochemical perturbations can develop AMN and/or cALD and also the age of onset varies. A number of environmental factors and modifier genes have been proposed to impact the development of different clinical phenotypes. A viral infection, for instance, could possibly initiate inflammation. However, no clear association between specific virus antigens and clinical phenotypes has been encountered so far (105). Other peroxisomal ABC transporters, in particular ALDRP, may, at least in part, compensate for the loss of ALDP (50, 52). Furthermore, genes influencing immune responses like CD1 or HLA-haplotypes could possibly trigger or prevent an inflammatory phenotype (189, 190). Mutations in neurotrophic factors such as CNTF or BDNF are also candidates for modulating clinical manifestation or at least might play a role in determining the age of disease onset (190). Abcd1 knockout mice have been generated as a model for X-ALD (63-65). They replicate metabolic dysfunctions of the human disease and develop axonal degeneration, as well as some neurological and behavioural abnormalities, but they lack signs of cerebral inflammation and demyelination. The reason for this phenotypic divergence in mice and humans is unknown. Perhaps differences in immune response mechanisms, like the CD1

53 Aim of the Thesis repertoire, may account for the absence of cerebral inflammation in mice. Moreover, Powers and co-workers reported signs of oxidative stress in brain sections of human X- ALD patients, which were clearly absent in Abcd1 knockout mice (113). A 2-stage pathogenesis for the CNS white matter lesions of human cALD has variously been suggested. According to this hypothesis, the primary defect in cALD is a dysmyelination that results from the incorporation of VLCFA into myelin lipids and proteolipid protein (PLP), rendering myelin unstable and susceptible to spontaneous breakdown. In a second step, a vehement inflammatory demyelinating process is set off, associated with CD8+ cytotoxic T-lymphocytes, microglia/macrophages, reactive astrocytes, a wide range of cytokines and possibly also CD1-restricted lipid antigen presentation (104, 105, 107). However, since Abcd1-knockout mice lack any signs of severe myelin alterations, they may thus also lack the initial trigger for developing cerebral inflammation. The goal of the present study was to answer the question whether a superimposed subtle destabilization of myelin would be able to trigger demyelination in Abcd1 knockout mice, and, whether this leads to the onset of an inflammatory response in the CNS. We generated mice in which the metabolic defect of X-ALD was combined with an inherited myelin protein deficiency. Mice carrying a null mutation for myelin-associated glycoprotein (MAG) (175) were chosen for cross-breeding to our Abcd1 knockout mice (63) because, in addition to perturbations in axon integrity, they also exhibit ultrastructural patterns of dysmyelination in the CNS and PNS (174, 175). Thus we considered Mag knockout mice a good “indicator strain” for assessing the impact of myelin disturbances on the CNS pathology in Abcd1 deficiency. Behavioural analysis should clarify if the combination of both pathologies in one mouse model leads to an enhanced (multiplicative) phenotype, thus indicating a mutual interaction between myelin disturbance and the development of cerebral X-ALD. Approved test procedures were used to detect gross as well as subtle impairments. Histopathological analysis, involving standard staining methods, immunohistochemistry, confocal laser microscopy and electron microscopy, was carried out in order to determine the status of tissue damage in the central and in the peripheral nervous system of single- and double-knockout mice.

54 Aim of the Thesis

Still a matter of controversy in X-ALD, is the question how axonal degeneration fits into the overall concept of pathogenesis. Is axonal degeneration the primary defect and the impaired myelin integrity only an epiphenomenon in X-ALD, as it has been proposed for AMN? Or, does multifocal dys- and demyelination precede and axons become secondarily involved, like it seems to be the case for cALD? Another possibility is that there exists a mixture of myelin and axonal involvement ab initio and myelin abnormalities and axon degeneration develop independent from each other due to different triggers. We intended to investigate the basic relationships by comparing the two pathological features in single- and double-mutants. Since Abcd1 mutant mice do not develop neurological deficits before the age of 15 months and signs of motor incoordination have first been noticed in 20-month-old animals, in this study we primarily concentrated on aged individuals (18-19 months) and secondarily extended our investigations to younger animals (13-14 months and 6-8 months).

55 Materials and Methods

CHAPTER 3

MATERIALS AND METHODS

56 Materials and Methods

3. MATERIALS AND METHODS

3.1. Animals

Abcd1-knockout mice, generated by Forss-Petter et al. (63), as well as all C57BL/6 wild type controls were bred and housed at the local animal facility of the Center of Brain Research. Mice with a null mutation in the gene for Mag were kindly provided by Klaus- Armin Nave (Max-Planck Institute for Experimental Medicine, Göttingen, Germany) and Melitta Schachner (Zentrum für Molekulare Neurobiologie, University of Hamburg, Hamburg, Germany) (175). Both Abcd1 and Mag knockout mice originally comprised a mixed genetic background of C57BL/6 and 129Sv, but had been backcrossed for at least ten and seven (estimated) generations respectively to the C57BL/6 strain before producing the cohorts for this study. Double-mutant mice (Abcd1/Mag knockout mice) were obtained by crossing Abcd1 and Mag knockout strains. Mice were housed in a temperature- (22°C) and humidity-controlled room. They were maintained in a constant 12:12 hour light-dark- cycle, had free access to food and water and were exposed to constant low level acoustic background noise. All subsequent care and handling procedures were in accordance with Principles of Laboratory animal care (NIH publication No. 86-23 revised 1985) and European Communities Council Directives (86/609/EEC) as well as local institutional and governmental guidelines. Behaviour tests were conducted in the housing facility, so that mice did not need to acclimatise to a new surrounding prior to the testing. All experiments were performed between 2 p.m. and 6 p.m. (in the second half of the light phase).

3.2. Genotyping

3.2.1 Isolation of DNA from mouse tail biopsies

Mice used for subsequent studies were genotyped twice, once 2-3 weeks after birth and again, when they were sacrificed. For this purpose tail biopsies (snips<5mm) were collected in microcentrifuge tubes and rapidly frozen on dry ice. Biopsies were digested by incubating in 600 µl TEN-buffer containing proteinase K and SDS over night at 55°C on a mixing device. After cooling to room temperature, proteins were removed by phenol extraction using 1 volume TEN-saturated phenol, mixing gently and the phases separated in a refrigerated microcentrifuge at 14,000 rpm for 15 minutes. The aqueous (top) phase

57 Materials and Methods was collected into a new tube and extracted with 500 µl of a phenol-chloroform- isoamylalcohol-solution as in the previous step. After transferring the aqueous phase again into a new tube, samples were mixed with 800 µl 96% ethanol, mixed briefly and the precipitated DNA collected by centrifugation for 10 minutes at 14.000 rpm. The supernatant was removed and discarded. DNA pellets were washed with 500 µl 70% ethanol and centrifuged for 5 minutes at 14.000 rpm. The supernatant was again removed carefully. DNA pellets were air-dried briefly and re-dissolved in 200 µl TE buffer over night and stored at 4°C.

TEN buffer: 50 mM Tris pH 8.0 (Merck, No. 1.08387) 10 mM EDTA (AppliChem, No. A2937) 100 mM NaCl (Merck, No. 1.06404) 1 % SDS (Sodium dodecyl sulfate, Promega, No. H5114) 500 µg/ml proteinase K (Merck, No. 1.2456)

TEN-saturated phenol: 40 ml phenol (equilibrated phenol, pH 8, VWR, No. 75829) 10 ml TEN buffer mixed and centrifuged for 15 min at 5,000 rpm, water (top) phase discarded

Phenol/chloroform/isoamylalcohol solution: 25 volumes TEN-saturated phenol 24 volumes chloroform (Sigma, No. 36.691-9) mixed with 1 volume isoamylalcohol (Merck, No. 818969)

96% ethanol: Merck, No. 100983

TE buffer: 10 mM Tris (Merck, No. 1.08387) 1 mM EDTA (AppliChem, No. A2937) adjusted to pH 8.0

Eppendorf microcentrifuge 5415D

58 Materials and Methods

3.2.2 Genotyping by PCR

A standard PCR procedure using RedTaq DNA polymerase from Sigma was performed to identify mutant and wild type mice.

PCR reaction mix to identify the wild type and knockout alleles of the Abcd1 gene: 2 µl RedTaq buffer (10x) 0,4 µl dNTPs (10 mM) 1,2 µl forward primer 226 (10 pmol/µl) 0,8 µl reverse primer 229 (10 pmol/µl) 0,8 µl reverse primer 108 (neomycin resistance gene, neo) (10 pmol/µl) 0,8 µl RedTaq polymerase (1 U/µl) 0,8 µl DNA (~10 ng/µl)

13,2 µl bidestilled, autoclaved H2O 20 µl total volume

PCR reaction mix to identify wild type and knockout alleles of the Mag gene: 2 µl RedTaq buffer (10x) 0,4 µl dNTPs (10 mM) 1,2 µl forward primer 606 (10 pmol/µl) 1 µl reverse primer 108 (neomycin resistance gene, neo) (10 pml/µl) 0,8 µl reverse primer 680 (10 pmol/µl) 0,8 µl RedTaq polymerase (1 U/µl) 0,8 µl DNA (~10 ng/µl)

13 µl bidestilled, autoclaved H2O 20µl total volume

For both reactions the same PCR cycling parameters were used: 94°C 1 minute 94°C 20 seconds 60°C 20 seconds 35 cycles 72°C 50 seconds 72°C 7 minutes 4°C ∞

59 Materials and Methods

RedTaq PCR reaction buffer, Sigma, No. B5926

RedTaqTM DNA Polymerase, Sigma, No. D4309 dNTPs: 100 mM dNTP Set, PCR Grade, Invitrogen, No. 10297-018

Murine Abcd1 specific primers (purchased from MWG-Biotech AG): Oligonucleotide No. Sequence wt or ko locus 226 forward 5’-TGT-CGG-GCG-TAG-ACG-CTG-TCG-T-3’ wt/ko 229 reverse 5’-CAG-GAC-CAC-AGC-TGT-GCG-CTT-C-3’ wt 108 reverse (neo) 5’-GCC-TTC-TAT-CGC-CTT-CTT-GAC-GAG-3’ ko

Product sizes: OLI 226 + OLI 229 (wt): 597 bp OLI 226 + OLI 108 (ko): 210 bp

Murine Mag specific primers (purchased from MWG-Biotech AG): Oligonucleotide No. Sequence wt or ko locus 606 forward 5’-CAC-CCT-GCC-GCT-GTT-TTG-GAT-AAT-3’ wt/ko 108 reverse (neo) 5’-GCC-TTC-TAT-CGC-CTT-CTT-GAC-GAG-3’ ko 680 reverse 5’-AAC-GGC-AGG-GAA-TGG-AGA-CAC-A-3’ wt

Product sizes: OLI 606 + OLI 680 (wt): 227 bp OLI 606 + OLI 108 (ko): ~600 bp

PCR cycler: Perkin Elmer, Gene Amp PCR system 2400 Bio Rad, MyCyclerTM Thermal cycler

3.2.3 Agarose gel electrophoresis

Agarose gel electrophoresis was used to visualize PCR-amplified DNA fragments. Agarose (1.5 %) was dissolved in 1x TAE buffer by heating in a microwave oven. After cooling to ~60°C, ethidium bromide (50 µg/100 ml gel, final conc.) was added and the gel casted. 15 µl PCR product (already containing loading dye from the RedTaq polymerase solution) were loaded onto the gel next to a size standard (100-bp DNA ladder, Invitrogen). Electrophoresis was routinely done in 1x TAE buffer at 80-90 V until bands of interest were clearly separated (1-2 hours). Bands were visualized by using transmitted UV light.

60 Materials and Methods

0.5 M EDTA solution, pH 8.0: 186.1 g EDTA (AppliChem, No. A2937) 800 ml d H2O adjust to pH 8.0 with NaOH (Merck, No. 1.06498) dH2O to 1 l final volume

50x TAE buffer stock: 242 g Tris (Merck, No. 1.08387) 51.1 ml acetic acid (Merck, No. 1.0063) 100 ml 0.5 M EDTA pH 8.0 dH2O to 1 l final volume

→ diluted 1:50 in dH2O for 1x TAE buffer solution

1.5 % agarose gel: 1.5 g agarose (Biozym, No. 50004E) 100 ml 1x TAE buffer

Ethidium bromide stock : 10 mg ethidium bromide (USB, No. US75808) 1 ml dH2O stored in a dark bottle → add 5 µl per 100 ml agarose gel solution

Imaging apparatus and software: Bio Rad Gel Doc 2000 Bio Rad Quantity One 4.2.3

3.3. Behavioural analysis

Behavioural analysis was conducted in a blinded fashion with respect to the individuals’ genotype. Male and female animals were tested independently. Climbing, hindlimb reflex extension and tremor was investigated in two different age groups (13-14 months and 18-19 months), whereas the rotarod test for motor coordination was performed only in 18-19 months old mice. Each age- and sex-matched genotype-group comprised 5- 17 animals (as indicated).

3.3.1 Climbing test

To test gross motor performance and muscular strength, mice were placed on the grid- lid of a cage, which was slowly inverted, leaving the mice hanging upside down 20 cm above the bottom of a box filled with sawdust. The latency to fall (up to 60 seconds) was registered in three consecutive trials. Best performance values were taken for statistical analysis and scored for whether or not they exceeded 60 seconds. As mice climb upside down, as a natural behaviour, only one training trial was performed prior to the test series. Between each trial, animals had 5 minutes time to rest.

61 Materials and Methods

For statistical analysis individual results of animals belonging to one genotype group were pooled and subjected to Chi Square test. Calculations were performed using SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL). Due to small sample size (7-9 age- and sex-matched animals per genotype group), Fisher’s exact test (two-sided) was used for evaluation. As more than two groups were compared, Schaffer’s procedure was performed in a second step to correct significant values for multiple testing. Final p-values ≤ 0.05 (5%) were considered as statistically significant.

3.3.2 Hind limb reflex extension

Each mouse was gently lifted by the tail for 10 seconds in three consecutive trials separated by 5 minutes rest. The hind limb reflex was analysed. When lifted by their tail wild type mice typically hold their hind limbs extended steadily at an angle of ≥ 90°. When the motor system becomes affected the hind limb reflex gets impaired. Scoring was done from 0, paralysis, to 4, normal reflex, according to the classification suggested by Pan et al. (176). For detail see table 3. The best performance score of each animal was taken for statistical analysis.

Table 3: Scoring of hind limb reflex extension score description 0 Paralysis of both hind limbs 0,5 Paralysis of one hind limb 1 Clasping of hind limbs 1,5 Alternate clasping and flexion of hind limbs 2 Flexion of hind limbs 2,5 Alternate flexions and extension of hind limbs < 90° 3 Extension of hind limbs < 90° 3,5 Alternate extension of hind limbs < and ≥ 90° 4 Extension of hind limbs ≥ 90°

Values of age- and sex-matches animals belonging to one genotype group were pooled and assessed using SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL). As no normal distribution of values was encountered, results were subjected to nonparametric statistical test procedures. Kruskal Wallis test was applied (in order to reveal differences between any of the genotype groups), followed by pairwise comparison of genotype

62 Materials and Methods groups using Wilcoxon rank sum test (Mann-Whitney U test). Due to the small sample size (7-9 sex- and age-matched individuals per genotype group) the exact two-sided test form was used for evaluation. Significant values (p < 0.05) were corrected according to Shaffer’s procedure for multiple testing. Final p-values ≤ 0.05 (5%) were classified as statistically significant. Results are represented in box-plot graphics showing median, 1st (lower) and 3rd (upper) quartiles (box) comprising 50 % of the values, ranges (smallest and largest non-outlier observations) and outliers (mild, 1.5-3x the interquartile range; extreme, > 3x the interquartile range away from the box).

3.3.3 Tremor

Each mouse was scored for the presence or absence of whole body tremor during rest and during movement. Animals were placed in an observational plastic cage and afterwards on the grid-lid of a cage and watched each time for 1 minute. Statistical analysis was performed using Fisher’s exact Chi Square test followed by Schaffer’s procedure as described above for climbing test.

3.3.4 Rotarod test for motor coordination and balance

Rotarod test was performed with cohorts of age- and sex-matched animals using the Accelerating Rota-rod 7650 treadmill for mice. Each mouse was placed in a separate lane of the apparatus while the cylinder was rotating at a constant speed of 2 rpm. Animals had 1-2 minutes time to get accustomed to the motion, before the accelerating mode was switched on and time measurement started. In a period of 5 minutes the rotor accelerated in 30 seconds-intervals from 2 to 20 rpm (see table 4). The time elapsed from the start to the fall (latency to fall in seconds) was recorded. The experiment was designed according to literature (191, 192). We used a 3 day training/test scheme: On the first day mice were trained twice, on the second day three times and on the third day the test was performed in three consecutive trials. Between each trial mice were allowed to rest for at least 10 minutes in order to avoid exhaustion. Mean values of the three trials performed by each mouse were taken for statistical analysis. Kruskal Wallis test and exact two-sided Wilcoxon rank sum test were performed using SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL). Significant values were corrected according to Schaffer’s procedure for

63 Materials and Methods multiple testing and final p-values ≤ 0.05 (5%) were considered as statistically significant. Results are represented in box-plot graphics as described above.

Table 4: Acceleration configuration of the rotarod. Speed increases within 5 minutes (300 seconds) from 2 to 20 rpm in 30 seconds-intervals. Time 120- 150- 180- 210- 240- 270- 0-30 30-60 60-90 90-120 (sec) 150 180 210 240 270 300 Speed 12.8- 14.6- 16.4- 2-3.8 3.8-5.6 5.6-7.4 7.4-9.2 9.2-11 11-12.8 18.2-20 (rpm) 14.6 16.4 18.2

Rotarod: Rota-Rod treadmill for mice No.7650, accelerating model, Ugo Basile, Italy

3.4. Histopathology Histopathological analysis was conducted in three different age groups (6-8, 13-14 and 18-19 months). Each sex- and age-matched genotype group intended for light microscopic investigations comprised tissues of 4-7 individuals (as indicated). Electron microscopy was done only in 18-19 months old mice. Two animals of each genotype group (separated for sex) were sacrificed and processed.

3.4.1 Mouse tissue dissection

Mice were deeply anesthetized by intraperitoneal injection of Vetanarcol® (Phenobarbital Sodium) and afterwards transcardially perfused, first with 15-20 ml PBS buffer and secondly with either 15-20 ml 4 % paraformaldehyde solution (for light microscopy) or 4 % paraformaldehyde/2,5 % glutaraldehyde solution (for electron microscopy). Skullcaps were opened and sciatic nerves exposed. Whole mice bodies were immersed in respective perfusion solutions for another 7-14 days before tissues (brain, spinal cord, sciatic nerves and trigeminal nerves) were harvested and cut for processing and embedding.

64 Materials and Methods

Vetanarcol® ad us. vet. Injektionslösung: Phenobarbital Sodium, Veterinaria AG, Zürich, Switzerland Dilution 1:10 in PBS Application: 0,15-0,2 ml of the dilution i.p.

PBS: Phosphate-buffered saline, Cambrex Bio Science, No. BE17-516F

8 % paraformaldehyde solution (stock): 40 g paraformaldehyde (Merck, No. 1.04005) 500 ml dH2O dissolve by heating to 60°C ad 1M NaOH (Merck, No. 1.06498) in drops until the solution clears adjust pH to 7.2 using HCl (Merck, No. 1.00317) stored at -20°C

Sörensen buffer: (pH 7.4, 0.2 M) 13.8 g NaH2PO4 (Merck, No. 1.06346) 71.2 g Na2HPO4 (Merck, No. 1.06580) 2.5 l dH2O

4 % paraformaldehyde solution for light microscopy: 2 volumes 8 % paraformaldehyde solution 1 volume Sörensen buffer 1 volume dH2O sterile filtered

0.08 M Sodium-Cacodylate buffer: 8.56 g Cacodylic acid- Sodium (Serva, No. 15540) 500 ml bi-distilled H2O adjust pH to 7.2 using 1M HCl (Merck, No. 1.00317) sterile filtered

2,5 % glutaraldehyde/4 % paraformaldehyde solution for electron microscopy: 125 ml 8 % paraformaldehyde solution 25 ml 25 % glutaraldehyde solution (Merck, No. 104239) 25 ml bi-distilled H2O . 0.165 g CaCl2 2H2O (Merck, No. 1.02382) 75 ml 0.08 M Sodium-Cacodylate buffer sterile filtered

3.4.2 Tissue processing for light microscopy

Tissues were processed by using the Tissue-Tek® V.I.P.™ Vacuum Infiltration Processor. Thereby samples are fixed, dehydrated by ethanol, cleared by xylol and impregnated by paraffin automatically according to standard protocols (see table 5).

65 Materials and Methods

Table 5: Tissue processing by Tissue-Tek® V.I.P.™ Vacuum Infiltration Processor Substance Duration Temperature in °C 50% Ethanol 20min 40 70% Ethanol 1h 40 70% Ethanol 1:30h 40 80% Ethanol 1h 40 80% Ethanol 1:30h 40 96% Ethanol 1h 40 96% Ethanol 1:30h 40 96% Ethanol 2h 40 Xylol 30min 40 Xylol 1h 40 Paraffin 1h 60 Paraffin 1h 60 Paraffin 1h 60 Paraffin 2h 60

Afterwards tissues were embedded in paraffin. 2-5 µm sections were cut, mounted on microscope slides and dried at 56°C for 6 hours.

Ethanol: 96% denaturated ethanol, Neuber/Brenntag

Xylol: Xylenes, Fluka, No. 95692

Paraffin: Histosec pastilles, Merck, No. 1.11609

Tissue-Tek® V.I.P.TM vacuum Infiltration Processor

Microtomes: Reichert Austria No. 31998 and No. 9530

Microscope slides: Star frost, Laborchemie, No. E5501-G

3.4.3 Standard staining methods

ƒ Hematoxylin-Eosin Mounted, 2-3µm thick, sections were deparaffinized in xylol substitute XEM-200 two times for 15 minutes and afterwards hydrated in a graded, descending, series of ethanol ending in distilled water. Following the staining procedure sections were then incubated in Mayer’s Hämalaun staining solution for 5 minutes, rinsed in tap-water 4 times, differentiated with HCl-ethanol solution (5 times), again rinsed in tap-water (2 times) and

66 Materials and Methods then exposed to Scott’s solution for 5 minutes. After another wash step in tap-water sections were counterstained with eosin solution for 3 minutes. At last samples were dehydrated in graded, ascending, series of ethanol, incubated in butylacetate and mounted in Eukit solution under glass coverslips.

ƒ Luxol fast blue-periodic acid Schiff (Klüver Barrera-PAS) Mounted, 5 µm thick, sections were deparaffinized in xylol-substitute XEM-200 two times for 15 minutes, rinsed in 96% ethanol and incubated in 0.1 % Luxol Fast Blue solution overnight (12-14 hours) at 56°C. After cooling the slides to room temperature, they were washed in 96 % ethanol, rinsed in distilled water, exposed to 0.1 % lithium carbonate solution for 5 minutes and finally differentiated in 70 % ethanol until the background was discoloured to light blue and myelin protruded dark blue. As murine tissues sometimes differentiate poorly, incubation in lithium carbonate and 70 % ethanol was occasionally repeated. Sections were again rinsed in distilled water and thereafter counterstained with PAS (Periodic Acid Schiff). For this, sections were placed in 0.8 % periodic acid for 10 minutes, rinsed in distilled water, incubated in Schiff’s reagent for 20 minutes and at last placed in sulfit solution 2 times for 3 minutes. After washing the sections for 10 minutes in running tap-water, samples were dehydrated in graded series of ethanol, incubated in butylacatate and mounted in Eukit solution under glass coverslips.

ƒ Bielschowsky silver impregnation Mounted, 2-3 µm thick, sections were deparaffinized in xylol substitute XEM-200 two times for 15 minutes and hydrated in graded series of ethanol ending in distilled water. After incubating the sections in 10% silver nitrate for 20 minutes, they were rinsed in distilled water. Used silver nitrate solution was collected in a tube and mixed with ammonia solution until precipitates disintegrated and the solution cleared. Then another 2 drops ammonia were added and sections exposed to this solution for 15 minutes, under exclusion of light. Samples were transferred into distilled water mixed with 3 drops ammonia for 3 minutes. 3 drops developer were added to the silver nitrate-ammonia solution and sections incubated in this solution until nerve fibres stained black and background yellowish-brown. After another wash step in distilled water sections were fixed in 5 % sodium thiosulfate for 3 minutes, again washed in distilled water, dehydrated

67 Materials and Methods in graded series of ethanol, incubated in butyl acetate and mounted in Eukit solution under glass coverslips.

Xylol substitute: XEM-200, Vogel, No. ND-HS-200

Ethanol: 96% denaturated ethanol, Neuber/Brenntag

Mayer’s Hämalaun: Merck, No.109249

HCl-ethanol solution: 100 ml 70 % ethanol 0.5 ml conc. HCl (Fluka, No.30721)

Scott’s solution: 2 g KHCO3 (Merck, No.104854) . 20 g MgSO4 7H2O (Merck, No. 105886) 1000 ml dH2O

Eosin solution: Stock: 10 g Eosin Y (Merck, No. 115935) 100 ml dH2O

Used Eosin solution: 2.5 ml Eosin stock solution 250 ml dH2O 12 drops glacial acetic acid (Fluka, No. 33209)

0,1% Luxol Fast Blue solution: 1 g Luxol Fast Blue (Chroma, No. 1B389) 1000 ml 96% ethanol dissolved overnight at 57°C

0,1% lithium carbonate solution: 1 g lithium carbonate (Merck, No. 105680) 1000 ml dH2O

Periodic acid: 4 g periodic acid (Merck, No. 100524) 500 ml dH2O

Schiff’s reagent: Merck, No. 109033

Sulfite solution: 5 ml conc. HCl (Fluka, No. 30721) 20 ml 10% potassium disulfite (Fluka, No. 31268) 500 ml dH2O

Silver nitrate: 10 g silver nitrate (Merck, No. 101512) 100 ml dH2O

Ammonia solution: 25%, Merck, No. 105432 opened 20 minutes before use

Developer: 20 ml formaldehyde solution (Merck, No. 1.04003) 100 ml dH2O 1 drop conc. nitric acid (Merck, No. 100456) 0.5 g citric acid monohydrate (Fluka, No. 2749)

5% Sodium thiosulfate: 25 g sodium thiosulfate pentahydrate (Merck, No. 106516) 500 ml dH2O

Butyl acetate: Fluka, No. 45860

68 Materials and Methods

Eukit solution: Eukit® mounting medium for microscopic preparations; O. Kindler GmbH&Co (Siebert)

Light microscopes: Nikon Optiphot-2 Olympus BX50 Reichert Polyvar 2

3.4.4 Immunohistochemistry

Standard protocol Mounted, 2-3 µm thick, sections were deparaffinized in xylol substitute XEM-200 (two times for 20 minutes), rinsed in 96 % ethanol and incubated in hydrogen peroxide/methanol-solution for 30 minutes, in order to saturate any endogenous peroxidase activity. Afterwards sections were washed in 96 % ethanol and rehydrated in graded, descending, series of ethanol ending in distilled water. Pre-treatment for antigen retrieval: Using a household food steamer device sections were pre-treated by boiling them in either citrate buffer or EDTA buffer for 60 minutes (see table 8 for antibodies and according pre-treatment procedures). After cooling the slides to room temperature they were rinsed with PBS and either loaded onto cover plates using DAKO buffer or further treated in humidity chambers. Sections were incubated with FCS/DAKO-solution for 15 minutes, in order to minimize protein background reactions. Afterwards sections were incubated with specific primary antibodies overnight at 4°C. For detection a biotin/avidin-technique was used. Sections were washed with DAKO buffer (cover plates) or PBS (humid chamber) and thereafter incubated with a biotinylated secondary antibody for 1 hour. After another wash step in either DAKO buffer or PBS, sections were incubated with horseradish-peroxidase (HRP)- conjugated avidin (diluted 1:100 in FCS/DAKO solution) for 1 hour. Avidin binds to biotin present on secondary antibodies and enhances signals. Finally sections were transferred into PBS buffer and washed three times in PBS. Development was conducted by incubating sections in 3,3’ diaminobencidine (DAB)/H2O2-solution for 0.5 to approximately 3 minutes (until a satisfactory staining was achieved). Thereby the chromogen DAB is converted to a brown pigment, which precipitates. The development reaction was stopped by another wash step in PBS. To intensify signals sections were optionally treated with 2 % copper sulfate solution for 5 minutes. At last sections were counterstained with Mayer’s Hämalaun solution for 5-25 seconds, rinsed in distilled water,

69 Materials and Methods differentiated with HCl/ethanol solution, transferred into Scott’s solution, washed in tap- water, dehydrated and mounted in Eukit solution as described before.

Some antibodies needed additional signal amplification (see table 8). In these cases treatment with a specific amplification reagent, containing biotinylated tyramine (CSA) was added to the standard procedure. After sections had been incubated with HRP/avidin- conjugate (diluted 1:100 in FCS/DAKO solution) for 1 hour, they were rinsed in PBS and incubated with CSA/H2O2-solution for 20 minutes. Sections were then washed in PBS and incubated with HRP/avidin-conjugate (diluted 1:200 in FCS/DAKO solution) for another 30 minutes. Subsequent development reactions were conducted as described.

In order to be able to compare individuals, tissue sections of one sample group were always stained at the same time and subjected to the same staining procedure. As a control, one slide of each genotype group was treated with FCS/DAKO-solution instead of a primary antibody and afterwards processed as described before.

Double-staining

ƒ Amyloid precursor protein (APP) + Glial fibrillary acidic protein (GFAP) 2-3 µm sections were deparaffinized, incubated in hydrogen peroxide/methanol-solution and hydrated as described. Using a household food steamer device sections were pre- treated by boiling them in citrate buffer for 1 hour. After cooling the slides to room temperature they were rinsed in PBS and then transferred into TBS buffer. All subsequent treatments were performed in humidity chambers. After blocking sections with FCS/DAKO-solution for 15 minutes, they were incubated with the primary antibody composite (APP + GFAP) overnight at 4°C. On the next day sections were rinsed in TBS buffer and incubated with the secondary antibody composition (bi-anti mouse + anti rabbit alkaline phosphatase) for 1 hour at room temperature. After another wash step in TBS buffer, sections were incubated with HRP/avidin-conjugate (diluted 1:100 in FCS/DAKO solution) for 1 hour and again rinsed in TBS buffer. Sections were then primarily developed with Fast Red solution for about 20 minutes. The development reaction was stopped by rinsing sections in TBS. DAB-Ni solution was used for the second development reaction. Sections were incubated in DAB-Ni solution for 2 minutes. The developmental reaction was stopped by rinsing sections in TBS buffer.

70 Materials and Methods

Finally sections were washed in distilled water, mounted in Geltol solution under glass coverslips and stored at 4°C.

ƒ Amyloid precursor protein (APP) + Carbonic anhydrase (CA II) 2-3 µm sections were deparaffinized, incubated in hydrogen peroxide/methanol-solution and hydrated as described. Using a household food steamer device sections were pre- treated by boiling them in EDTA buffer for 1 hour. After cooling the slides to room temperature they were rinsed in PBS buffer. All subsequent treatment was performed in humidity chambers. Sections were incubated with FCS/DAKO-solution for 15 minutes and afterwards incubated with APP monoclonal antibody overnight at 4°C. For detection a biotin/avidin technique was used. Sections were washed in PBS buffer and incubated with biotinylated anti-mouse secondary antibody for 1 hour. After another wash step in PBS buffer, sections were incubated with HRP/avidin-conjugate (diluted 1:100 in FCS/DAKO solution) for 1 hour and again rinsed in PBS buffer. Sections were then developed with DAB-Ni solution for 2 minutes. The development reaction was stopped by another wash step in PBS. Afterwards sections were incubated with CA II polyclonal antibody over night at 4°C. On the next day sections were rinsed in TBS buffer and incubated with biotinylated anti-goat secondary antibody (diluted in FCS/DAKO solution) for 1 hour at room temperature. After another wash step in TBS buffer, sections were incubated with streptavidine/alkaline phosphatase-conjugate for 1 hour and again rinsed in TBS buffer. Fast Red was used for development. Sections were incubated in Fast Red solution for 20 minutes. The development reaction was stopped by washing sections in TBS. Finally sections were transferred into distilled water, mounted in Geltol solution under glass coverslips and stored at 4°C.

ƒ Amyloid precursor protein (APP) + Ionized calcium binding adaptor molecule I (IBA-1) 2-3 µm sections were deparaffinized, incubated in hydrogen peroxide/methanol-solution and hydrated as described. Using a household food steamer device sections were pre- treated by boiling them in EDTA buffer for 1 hour. After cooling the sections to room temperature they were rinsed in PBS and then transferred into TBS buffer. All subsequent steps were performed using humid chambers. After blocking sections with FCS/DAKO-

71 Materials and Methods solution for 15 minutes, they were incubated with the primary antibody composite (APP + IBA-1) overnight at 4°C. On the next day sections were rinsed in TBS buffer and incubated with the secondary antibody composition (bi-anti mouse + anti rabbit alkaline phosphatase) for 1 hour at room temperature. After another wash step in TBS buffer, sections were incubated with HRP/avidin-conjugate (diluted 1:100 in FCS/DAKO solution) for 1 hour and again rinsed in TBS buffer. Sections were then primarily developed with Fast Red solution for about 20 minutes. The development reaction was stopped by washing sections in TBS. DAB-Ni solution was used for the second developmental reaction. Sections were incubated in DAB-Ni solution for 2 minutes. The developmental reaction was stopped by rinsing the sections in TBS buffer. Finally sections were washed in distilled water, mounted in Geltol solution under glass coverslips and stored at 4°C.

ƒ Amyloid precursor protein (APP) + 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) 2-3 µm sections were deparaffinized, incubated in hydrogen peroxide/methanol-solution and hydrated as described. Using a household food steamer device sections were pre- treated by boiling them in EDTA buffer for 1 hour. After cooling the slides to room temperature they were rinsed in PBS buffer, transferred into humidity chambers and incubated with FCS/DAKO-solution for 15 minutes. Afterwards sections were incubated with APP monoclonal antibody overnight at 4°C. For detection a biotin/avidin technique was used. Sections were washed in PBS buffer and incubated with biotinylated anti mouse secondary antibody for 1 hour. After another wash step in PBS buffer, sections were incubated with HRP/avidin-conjugate (diluted 1:100 in FCS/DAKO solution) for 1 hour and again rinsed in PBS buffer. Sections were then developed with DAB-Ni solution for 2 minutes. The development reaction was stopped by another wash step in PBS. Afterward sections were incubated with CNP monoclonal antibody (diluted in FCS/DAKO solution) over night at 4°C. On the next day sections were rinsed in TBS buffer and incubated with biotinylated anti- mouse secondary antibody (diluted in FCS/DAKO solution) for 1 hour at room temperature. After another wash step in TBS buffer, sections were incubated with streptavidine/alkaline phosphatase-conjugate for 1 hour and again rinsed in TBS buffer. Fast Red was used for development. Sections were incubated in Fast Red solution for 20

72 Materials and Methods minutes. The development reaction was stopped by washing the sections in TBS. Finally sections were transferred into distilled water, mounted in Geltol solution under glass coverslips and stored at 4°C.

3.4.5 Confocal microscopy

Standard protocol 2-3 µm sections were deparaffinized, incubated in hydrogen peroxide/methanol- solution and hydrated as described. Using a household food steamer device sections were pre-treated by boiling them in citrate buffer or EDTA buffer (pH 8.5) for 1 hour. After cooling the slides to room temperature they were rinsed in PBS buffer. All subsequent steps were performed in humidity chambers. Sections were incubated with DAKO diluent solution for 15 minutes and afterwards incubated with the primary antibody composite (exception: antibodies against APP and SMI 31+32 were applied separately on two consecutive days), diluted in DAKO diluent solution, overnight at 4°C. On the next day sections were rinsed in PBS and afterwards incubated with a composition of secondary antibodies (exception: secondary antibodies to anti-APP and anti-SMI 31+32 were applied separately), labelled with fluorescence dyes, for 2h at room temperature under exclusion of light. All further steps were carried out exposing sections to light as little as possible, in order to conserve the fluorescent labelling. Sections were washed again in PBS and once more incubated with the primary antibody composite (diluted in DAKO diluent solution) for 2h at room temperature. Afterwards sections were rinsed in PBS and incubated with the secondary antibody composite (diluted in DAKO diluent solution) once again for 1h. Sections were rinsed in PBS and finally incubated with streptavidine carrying fluorescence markers for 1h. After a final wash step in PBS sections were transferred into distilled water, mounted in Gallate/Geltol solution under glass coverslips and stored at 4°C under exclusion of light.

Xylol (xylene) substitute: XEM-200, Vogel, No. ND-HS-200

Ethanol: 96% denaturated ethanol, Neuber/Brenntag

Hydrogen peroxide-methanol solution: 1.2 ml 30% hydrogen peroxide (Merck, No.107210) 180 ml methanol (Fluka, No. 65543)

73 Materials and Methods

Citrate buffer: stock (1 M, pH 6.0): 210.14 g citric acid monohydrate (Fluka No. 27490) 1000 ml dH2O

Used citrate buffer (10 mM, pH 6.0): citrate buffer stock diluted 1:100 in dH2O

EDTA buffer: stock (pH 9.0): 1.21 g Tris base (AppliChem No. A1379) 0.37 g EDTA (Merck, No. 1.08454) 50 ml dH2O

EDTA working solution (pH 8.5): 2.5 ml EDTA stock solution 47.5 ml dH2O

Sörensen buffer: (pH 7.4, 0.2 M) 13.8 g NaH2PO4 (Merck, No. 1.06346) 71.2 g Na2HPO4 (Merck, No. 1.06580) 2.5 l dH2O

PBS buffer: 9 g NaCl (Merck, No. 1.06404) 250 ml Sörensen buffer 750 ml dH2O

TBS stock: 60.57 g Tris base(AppliChem, No. A1379) 500 ml dH2O 400 ml 1M HCl (Fluka, No. 35328) adjust to pH 7.5 ad dH2O to a final volume of 1 l ad 180 g NaCl (Merck, No. 1.06404)

Used TBS buffer: 50 ml TBS stock 950 ml dH2O

DAKO buffer: 100 ml DAKO® cytomation wash buffer 10x (DAKO, No. S3006) 900 ml dH2O

DAKO/FCS-solution: 10% FCS (fetal bovine serum, FBS, Cambrex BioScience, No. DE-14-802F) in DAKO buffer

DAKO diluent solution: ChemMateTM Antibody Diluent, DAKO, No. S2022

Primary and secondary antibody composites: were diluted in FCS/DAKO-solution (light microscopy) or DAKO diluent solution (confocal microscopy) and combined as indicated in table 8-10; for vendors see table 6 and 7

Table 6: Primary antibodies Primary antibody Host Vendor Cat. No. Mac-3 rat BD biosciences 553322 NF 150 kD rabbit Chemicon AB1981 CM1 rabbit Idun APP mouse Chemicon MAB348 PLP rabbit Serotec AHP261 GFAP rabbit DAKO Z0334 IBA-1 rabbit Wako 019-19741 CNP (SMI-91) mouse Sternberg monoclonals Inc. SMI-91 CA II sheep The binding site PC076 MBP rabbit DAKO A0623 BiP/GRP78 mouse Transduction Laboratories G73320 PDI mouse StessGen Biotechnologies SPA-891

74 Materials and Methods

Table 7: Secondary antibodies Secondary Host Target Conjugate Vendor Cat. No. antibody antibody bi-α-m sheep mouse biotin Amersham RPN1001 bi-α-rat goat rat biotin Amersham RPN1005 bi-α-Rab donkey rabbit biotin Amersham RPN1004 bi-α-sg donkey goat/sheep biotin Amersham RNP1025 α-Rab-AP donkey rabbit alkaline phosphatase Jackson ImmunoResearch 711-055-152 α-m-cy3 donkey mouse fluorochrome cy3 Jackson ImmunoResearch 715-165-151 α-Rab-cy2 goat rabbit fluorochrome cy2 Jackson ImmunoResearch 111-225-144 α-m-AF 660 goat mouse Alexa fluor 660 Molecular Probes A 21055

HRP-conjugated avidin: (Sigma, No. A3151-2MG) diluted 1:100 or 1:200 in DAKO/FCS solution

Streptavidin/alkaline phosphatase-conjugate: DAKO LSAB® 2 System, DAKO, No. 002786

Streptavidin conjugated with fluorescence markers: Streptavidin-cy2 (Amersham, No. PA42001) Streptavidin cy3 (Jackson ImmunoResearch, No. 016- 160-084) diluted 1:75 in DAKO diluent solution

Biotinylated tyramine (CSA) stock: Borate buffer: 0.1545 g boric acid (Merck, No. 1.00165) 50 ml dH2O adjust pH to 8.0 using NaOH (Merck, No. 6498)

CSA: 6 ml borate buffer 15 mg sulpho-NHS-LC-Biotin (Pierce, No. 21335) 4.5 mg tyramin (Sigma, No. T-7255) stir at room temperature over night filtrate, aliquot and store at -20°C

CSA/H2O2-solution: CSA stock solution 1:500 in PBS buffer ad H2O2 1:500 (Merck, No. 1.07210)

Diaminobenzidine (DAB) stock: 25 mg DAB (DAKO, No. S3000) 1 ml PBS buffer stored at -20°C

DAB/H2O2 solution: 1 ml DAB stock solution 49 ml PBS for one cuvette 16.5 µl H2O2 (Merck, No. 1.07210) filtrate

Fast Red solution: 0.1 M Tris, pH 8.2: 12.1 g Tris base (AppliChem, No. A1379) 920 ml dH2O adjust to pH 8.2 using 1 M HCl (Fluka, No. 35328)

APAAP substrate: 200 mg naphtol ASMX-phosphate (Sigma, No. N4875-1G) 20 ml N,N-Dimethylformamide (Fluka, No. 40250) 980 ml 0,1 M Tris, pH 8.2 1 ml Levamisol (Sigma, No. L-2768)

75 Materials and Methods

Fast Red solution: 50 mg Fast Red salt (Sigma, No. F-2768) 50 ml APAAP substrate for one cuvette filtrate

DAB-Ni solution: 1 ml DAB stock solution 49 ml PBS 16.5 µl H2O2 (Merck, No. 1.07210) filtrate for one cuvette add 1 ml 1 % nickelammoniumsulfate (Fluka, No. 09885) add 0.6 ml 1 % cobalt II chloridhexalhydrate (Merck, No. 2539)

Copper sulfate solution: 2 % copper sulfate (Merck, No. 1.02791) in 0.9 % NaCl (Merck, No. 1.06404)

Mayer’s Hämalaun: Merck, No.109249

HCl/ethanol solution: 100 ml 70 % ethanol 0.5 ml conc. HCl (Fluka, No.30721)

Scott’s solution: 2 g KHCO3 (Merck, No. 1.04854) 20 g MgSO4x7H2O (Merck, No. 1.05886) 1000 ml dH2O

Butyl acetate: Fluka, No. 45860

Eukit solution: Eukit® mounting medium for microscopic preparations; O. Kindler GmbH&Co (Siebert)

Geltol solution: 6 g glycerine (Life Technologies, No. 15514-011) 2.4 g Mowiol (Calbiochem, No. 475904) 6 ml dH2O expand for 2h ad 12 ml 0.2 M Tris pH 8.5 stir for 10 minutes at 50°C centrifuge 15 minutes at 5,000 g store at -20°C

Gallate/Geltol solution: 10 ml Geltol solution 2.1 g gallate (Sigma, No. P-3130)

Household food steamer: MultiGourmet FS 20, Braun, Kronberg/Taunus, Germany

Light microscopes: Nikon Optiphot-2, Olympus BX50, Reichert Polyvar 2

Confocal microscope: Zeiss LSM-410, Carl Zeiss, Jena, Germany

76 Materials and Methods

Table 8: Antibodies for standard immunohistochemistry Primary Target Monoclonal (mc)/ Dilution Pre- Secondary Dilution Cover plates (cp)/ Amplification Counter- antibody Polyclonal (pc) and Host treatment antibody Humid chamber (hc) staining Mac-3 Monocyte mc (rat) 1:200 citrate buffer bi-α-rat* 1:200 cp - 5 sec chemoattractant protein NF 150 kD neurofilament pc (rabbit) 1:2000 EDTA bi-α-Rab* 1:200 cp - 10 sec (neurons/axons) pH 8.5 CM1 caspase 3 pc (rabbit) 1:1000 citrate buffer bi-α-Rab* 1:200 hc CSA 1:200 25 sec

APP amyloid precursor protein mc (mouse) 1:1000 citrate buffer bi-α-m* 1:200 cp - 10 sec

APP amyloid precursor protein mc (mouse) 1:1000 citrate buffer bi-α-m* 1:200 hc 2% copper - sulfate PLP proteolipid protein pc (rabbit) 1:1000 - bi-α-Rab* 1:200 cp - 10 sec (myelin) BiP BiP chaparone in the mc (mouse) 1:100 citrate buffer bi-α-m* 1:200 hc - 20 sec endoplasmic reticulum PDI protein disulfide mc (mouse) 1:100 citrate buffer bi-α-m* 1:200 hc - 20 sec isomerase in ER * bi-α-rat: biotinylated goat anti rat antibody; bi-α-Rab: biotinylated donkey anti rabbit antibody; bi-α-m: biotinylated sheep anti mouse antibody

Table 9: Antibody composits for immunohistochemical double-staining Primary antibody Target Monoclonal (mc)/ Dilution Pre- Secondary Dilution compositions Polyclonal (pc) and host treatment antibody APP amyloid precursor protein mc (mouse) 1:2000 citrate buffer bi-α-m* 1:200 + GFAP glial fibrillary acidic protein (astrocytes) pc (rabbit) 1:1500 α-Rab-AP* 1:200 APP amyloid precursor protein mc (mouse) 1:2000 EDTA bi-α-m* 1:200 + pH 8.5 IBA-1 microglia/macrophages pc (rabbit) 1:1500 α-Rab-AP* 1:200 APP amyloid precursor protein mc (mouse) 1:2000 EDTA bi-α-m* 1:200 + pH 8.5 CNPase (SMI-91) oligodendrocytes + non-compact myelin mc (mouse) 1:1000 bi-α-m* + Streptav.-AP* 1:200 APP amyloid precursor protein mc (mouse) 1:2000 EDTA bi-α-m* 1:200 + pH 8.5 CA II carbonic anhydrase II (oligodendrocytes) pc (sheep) 1:500 bi-α-gs* + Streptav.-AP* 1:200 * bi-α-m: biotinylated sheep anti mouse antibody; α-Rab-AP: alkaline phosphatase-conjugated donkey anti rabbit antibody; bi-α-gs: biotinylated donkey anti goat/sheep antibody; Streptav.- AP: alkaline phosphatase conjugated streptavidin

77 Materials and Methods

Table 10: Anitbody composits for confocal microscopy Primary Target Monoclonal (mc)/ Dilution Pre- Secondary antibody systems Dilution antibody Polyclonal (pc) and host treatment compositions APP amyloid precursor protein mc (mouse) 1:500 citrate buffer bi-α-m* + Streptav.-cy3* 1:200+1:75 + GFAP glial fibrillary acidic protein (astrocytes) pc (rabbit) 1:1500 α-Rab-cy2* 1:100 APP amyloid precursor protein mc (mouse) 1:500 citrate buffer bi-α-m* + Streptav.-cy3* 1:200+1:75 + PLP proteolipid protein (compact myelin) pc (rabbit) 1:500 α-Rab-cy2* 1:100 APP amyloid precursor protein mc (mouse) 1:500 citrate buffer bi-α-m* + Streptav.-cy3* 1:200+1:75 + MBP myelin basic protein (compact myelin) pc (rabbit) 1:1250 α-Rab-cy2* 1:100 APP amyloid precursor protein mc (mouse) 1:500 citrate buffer α-m-cy3* 1:200 + CA II carbonic anhydrase II (oligodendrocytes) pc (sheep) 1:500 bi-α-gs* + Streptav.-cy2* 1:200+1:75 APP amyloid precursor protein mc (mouse) 1:500 EDTA α-m-cy3* 1:200 + pH 8.5 SMI 31+32 phosphorylated and nonphosphorylated both mc (mouse) 1:10000+ α-m-AF 660* 1:100 neurofilaments respectively 1:500 * bi-α-m: biotinylated sheep anti mouse antibody; Streptav.-cy3: streptavidin conjugated with fluorochrome cy3; α-Rab-cy2: goat anti rabbit antibody conjugated with fluorochrome cy2; α-m- cy3: donkey anti mouse antibody conjugated with fluorochrome cy 3; bi-α-gs: biotinylated donkey anti goat/sheep antibody; Streptav.-cy2: streptavidin conjugated with fluorochrome cy2; α-m- AF 660: goat anti mouse antibody conjugated with fluorochrome Alexa fluor 660.

78 Materials and Methods

3.4.6 Electron microscopy

ƒ Pre-treatment with osmium tetraoxide Thin sections of glutaraldehyde fixed tissues (see above) were incubated with osmium tetraoxide/ferrocyanide-solution over night at 4°C. On the next day osmium tetraoxide/ferrocyanide solution was discarded and sections were rinsed in phosphate buffer 3 times. Afterwards they were dehydrated in graded series of ethanol, using 70 %, 80 % and 96 % solutions (10 minutes each). At last sections were incubated in “100%” ethanol (ethanol pre-treated with coppersulfate) two times for 15 minutes. After incubating them in propylene oxide (again 2 times 15 minutes) sections were placed in propylene oxide/resin-solution for 2h. Finally they were mounted in resin solution and cured over night at 60°C.

ƒ Semithin sections Tissue samples embedded in resin were cut using an Ultracut-microtome. Semithin 0.5 µm sections were mounted on microscope slides, dried using a hot plate and afterwards stained with toluidine blue. Sections were incubated with toluidine blue solution until the staining solution started to dry. Then sections were rinsed in hot tap-water, blotted dry using filter paper and further dried on a hot plate. Finally sections were mounted in immersion oil.

ƒ Ultrathin sections Osmicated, resin-embedded tissues were cut into 0.07 µm sections using an ultra- microtome and were mounted onto copper grids (designed for electron microscopy investigations). To enhance signals sections were contrasted using uranyl acetate and lead citrate. Sections were incubated with 2 % uranyl acetate solution for 5 minutes, rinsed in destilled water (3 times) and afterwards incubated in lead citrate for 1-2 minutes. They were rinsed again in distilled water and finally dried on filter paper at 60°C.

Osmium tetraoxide/Ferrocyanide solution: 1 volume 2 % osmium tetraoxide (Merck, No. 124505) 1 volume 3 % potassium ferrocyanide (Loba Chemie, No. 19157)

Phosphate buffer “Sörensen” (0,2M): 71.2 g Natrium Hydrogenphosphate Dihydrate (Merck, No. 106580) 13.8 g Natrium dihydrogenphostphate Monohydrate (Merck, No. 106346) 2.5 l dH2O

79 Materials and Methods

100 % ethanol: 96 % ethanol copper(II) sulfate (Merck, No. 102791)

Propylene oxide: Fluka, No. 82320

Resin: 20 ml Vinylclohexendioxide (Gröpl., No. 18306) 52 ml Nonenyl succinic anhydride (Gröpl., No. 18301) 10 ml Diglycide ether of polypropylene glycol (Gröpl., No. 18310) 2 ml Dimethylamineethanol S1 (Gröpl) stir for 1h stored at -20°C

Toluidine blue: 2 g borax (di-sodiumtetracarbonate, Merck, No. 6310) 1 g toluidine blue (Merck, No. 1.15930) 100 ml 10% acetone (Fluka, No. 32201) dissolve and filtrate

Immersion oil: Merck, No. 104699

Uranyl acetate: 2 % uranyl acetate (Merck, No. 8473) in dH2O

Lead citrate: 1.33 g lead nitrate (Fluka, No. 31137) 1.76 g sodium citrate (Merck, No. 6448) 30 ml dH2O dissolve for 30 minutes add 8 ml NaOH (Merck, No. 6498) add dH2O to a final volume of 50 ml leave solution for 3-5 days, until it clears

Microtome: Ultracut 365885

Copper grids: Square mesh copper, 3.05 mm, Gröpl, No. G2200C

Electron microscope: Jeol 1010

3.5. Quantitative evaluation of (immuno-) labelled structures and statistical analysis

Quantitative evaluation was conducted in a blinded fashion with respect to the individuals’ genotype.

3.5.1 Bielschowsky silver impregnation: torpedoes in cerebellar granular layer

Axonal torpedoes (“en passant spheroids”) of Purkinje cells were evaluated in Bielschowsky silver stained cerebellar sections of 18-19 months old male mice. We counted torpedoes in 10-11 standardized microscopic fields of 62.5 µm². For this we used

80 Materials and Methods an ocular morphometric grid and placed it within granular cell layers at random. Results are represented as torpedoes/mm² granular layer. For statistical analysis SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL) was used. Individual values of animals belonging to one genotype group were pooled and subjected to nonparametric statistical test procedures. Kruskal Wallis test was applied, followed by pairwise comparison of genotype groups using exact two-sided Wilcoxon rank sum test (Mann-Whitney U test). Significant values (p < 0.05) were corrected according to Shaffer’s procedure for multiple testing. Final p-values smaller or equal to 0.05 (5%) were classified as statistically significant. Results are represented in box-plot graphics showing median, 1st (lower) and 3rd (upper) quartiles (box) comprising 50 % of the values, ranges (smallest and largest non-outlier observations) and outliers (mild, 1.5-3x the interquartile range; extreme, > 3x the interquartile range away from the box).

3.5.2 Mac-3 staining: activation of macrophages/microglia cells

From each mouse, 3-4 randomly selected complete spinal cord cross sections, including cervical, thoracal and lumbal regions, were screened for activated microglia/macrophages. Mac-3 positive labels were counted only, when they were associated with a nucleus and thus distinct cells could be identified. No differentiation was made between Mac-3 positive macrophages and Mac-3 positive microglia cells. In Mac-3 positive cell clusters, the nuclei within the clusters were counted. Also perivascular Mac-3 positive cells were included. Areas of complete cross sections were calculated using a morphometric grid. Intersection points lying within the sections were counted and translated into mm² using an ocular scale. Results are represented as Mac-3 positive cells/mm² spinal cord. Basically the same evaluation was done in cerebellar sections of 18-19 months old male mice. Here we counted 5-6 standardized microscopic fields of 62.5 µm². For this we used an ocular morphometric grid and placed it in white matter areas at random. Results are represented as Mac-3 positive cells/ mm² white matter. For statistical analysis SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL) was used. Kruskal Wallis test, exact two-sided Wilcoxon rank sum test (Mann-Whitney U test) and Schaffer’s procedure for multiple testing were applied as described above. Final p-

81 Materials and Methods values smaller or equal to 0.05 (5 %) were considered as statistically significant. Results are represented in box-plot graphics.

3.5.3 APP staining: axonal bulbs and extra-axonal, diffuse APP accumulation

APP positive spheroids, indicating acute disturbance of axonal transport and thus ongoing axonal impairment, were counted in at least 6 complete spinal cord cross sections, comprising cervical, thoracal and lumbal regions, of each mouse. Total areas of the cross sections were calculated using a morphometric grid as described above. Results are represented as APP-positive spheroids/mm² spinal cord. Diffuse APP labelling detected in spinal cord sections was initially subjected to semi- quantitative evaluation. White matter areas of at least 6 spinal cord cross sections from each mouse were scored for severity of diffuse/small dotted, most likely extra-axonal, accumulation of APP according to the following criteria:

-/+ score ordinal ranking description - 0 Virtually no diffuse/ small dotted APP staining + 1 Some staining around nuclei ++ 2 Some staining around nuclei and several diffuse APP dots +++ 3 Heavy, diffuse, small dotted APP accumulation

Mean scores calculated for each animal were subjected to statistical analysis.

APP-positive, small dotted, circular, myelin-like structures were counted in white matter areas of at least 6 spinal cord cross sections of 18-19 months old animals and related to the corresponding enumerated expanse (calculated by using a morphometric grid as described above). Results are represented as APP-positive myelin-like structures/mm² white matter. Finally APP accumulation was evaluated by subjecting photographs of spinal cord sections to computer-aided analysis. For digital assessment of APP accumulation, spinal cord sections were immunolabelled for APP but not counterstained with Mayer’s Hämalaun. In a blinded fashion respective to genotype, but in accordance with semiquantitative results, one distal (lumbar) spinal cord cross section of each animal was chosen. Dorsal columns of these sections were photographed and pictures digitized. Using Adobe Photoshop CS (Version 8.0.1) pictures were adjusted to one another regarding to brightness and contrast. They were translated into greyscale and adapted to a size of 15 cm x 10 cm and 200 dpi.

82 Materials and Methods

Processed pictures were imported into Scion Image (Release Alpha 4.0.3.2), an image processing and analysis program based on NIH Image developed at the National Institutes of Health (www.scioncorp.com/pages/scion_image_windows.htm). White matter areas (dorsal columns) were selected by freehand mode and their size measured. By using the “density slice” option, dark APP dots were charted. Their overall expanse was measured and related to the calculated corresponding white matter area. Results are represented as the ratio of dark area (APP): white matter area. For statistical analysis SPSS 14.0 statistical software system (SPSS Inc., Chicago, IL) was used. Kruskal Wallis test, exact two-sided Wilcoxon rank sum test (Mann-Whitney U test) and Schaffer’s procedure for multiple testing were applied as described above. Final p- values smaller or equal to 0.05 (5 %) were considered as statistically significant. Results are represented in boxplot graphics.

83 Results

CHAPTER 4

RESULTS

84 Results

4. RESULTS

4.1. Behavioural analysis

Previous studies demonstrated that Abcd1 as well as Mag knockout mice develop some behavioural deficits in adult life. Mag knockout mice display a decreased motor activity already at the age of 2 months and are clearly impaired concerning motor coordination and balance at the age of 6 months (174, 176). Abcd1 knockout mice remain normal at least until the age of 15 months, when they in one study showed reduced initial spontaneous motor activity and signs of motor incoordination became apparent at the age of 20 months (145). To assess the neurological consequences of a combined loss of Aldp and Mag we compared motor skills of double-knockout, single-knockout and wild type littermates. Mice with combined Abcd1/Mag deficiency were generated by crossbreeding Abcd1 (63) and Mag (175) knockout mice that had been extensively backcrossed to C57BL/6 background. Abcd1/Mag double-mutants were viable, as indicated by a normal Mendelian distribution. The general physical appearance, homecage behaviour, including fertility, of young adults was normal. Although ageing Mag and double-knockout mice developed body tremor, they were not severely impaired and appeared to have a normal life span. Because there was no phenotypic indication of early-onset cerebral ALD resulting from the added myelin perturbation in double-mutant mice, the further study was designated to primarily evaluate the impact of combined deficiency on the late-onset neuropathology in the spinal cord.

4.1.1 Climbing test

To assess gross motor skills and muscular strength, we performed a climbing test, in which mice were forced to move, hanging upside down on the grid lid of a cage. The latency to fall was scored for whether or not it exceeded 60 seconds and data was analyzed using Chi square test (figure 4). No differences between wild type, single- and double- knockout mice could be detected at the age of 18-19 months, neither in male nor in female animals. Same results were encountered in 13-14 months old male mice. However, overall motor activity seemed to be reduced in Mag and double-knockout mice, as they stayed at

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the same place for longer time periods, whereas wild type and Abcd1 knockout mice were inquisitively climbing around, exploring the environment and trying to pass the edge of the grid to get to the top side (not scored observation).

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genotype latency to fall total genotype latency to fall total < 60 sec ≥ 60 sec < 60 sec ≥ 60 sec wt 1 6 7 wt 0 8 8 Abcd1 ko 1 6 7 Abcd1 ko 0 8 8 Mag ko 3 6 9 Mag ko 0 7 7 double ko 2 7 9 double ko 1 6 7

C genotype latency to fall total < 60 sec ≥ 60 sec wt 1 7 8 Abcd1 ko 2 4 6 Mag ko 4 2 6 double ko 4 3 7

Figure 4: Climbing test. Number of individuals showing a latency to fall <60 seconds and ≥60 seconds related to the total number of animals tested. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months.

4.1.2 Tremor

Li et al. described a mild, transient (intention) tremor in 2 months old Mag knockout mice (174). This result was verified by Pan et al. who encountered whole body tremor during rest and during movement in some of the 6 months old Mag knockout mice (176). We looked for whole body tremor during rest and during movement independently in single- and double-knockout mice. All double-knockout mice (male and female, 18-19 and 13-14 months) displayed severe episodes of whole body tremor during movement. Tremor during movement was also recorded in most Mag knockout mice of both sex and age groups. On the other hand, no consistant signs of tremor could be detected in either wild type animals or Abcd1 knockout mice of all sex and age groups, movement tremor was noted for a single Abcd1 knockout mouse at 18-19 months of age. To quantify this observation, whole body tremor during movement was estimated as present or absent and data was analyzed using Chi square test. Double-knockout mice were

86 Results significantly impaired compared to wild type animals (p < 0.01 in all sex and age groups) and compared to Abcd1 knockout mice (male, 18-19 months: p < 0.05; female, 18-19 months and male, 13-14 months: p < 0.01). Significant differences were also found between Mag knockout mice and wild type animals (male and female, 18-19 months: p < 0.01; male 13-14 months: p < 0.05) and between 18-19 months old Mag knockout mice and age- and sex-matched Abcd1 knockout mice (male: p < 0.05; female: p < 0.01).

A B genotype tremor during total genotype tremor during total movement movement no tremor tremor no tremor tremor wt 7 0 7 wt 8 0 8 Abcd1 ko 6 1 7 Abcd1 ko 8 0 8 Mag ko 1** 8** 9 Mag ko 1** 6** 7 double ko 0** 7** 7 double ko 0** 7** 7

C genotype tremor during total movement no tremor tremor wt 8 0 8 Abcd1 ko 6 0 6 Mag ko 2* 4* 6 double ko 0** 7** 7

Figure 5: Tremor during movement. Number of individuals lacking signs of tremor or exhibiting tremor related to the total number of animals tested. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months. For statistical analysis Fisher’s exact Chi Square test was performed; Statistically significant differences between mutant and wild type: *) p < 0.05; **) p < 0.01

Marked whole body tremor during rest was recorded in most double-knockout mice and in some Mag knockout mice, whereas wild type animals and Abcd1 knockout mice were uniformly unaffected. Chi square test revealed significant impairment of double-knockout mice compared to wild type animals (male and female, 18-19 months: p < 0.01; male, 13-14 months: p < 0.05) and of 18-19 months old double-knockout mice compared to sex- and age-matched Abcd1 knockout mice (male and female: p < 0.01). Though the difference between Mag knockout mice and double-knockout mice was not statistically significant, a more consistent

87 Results occurrence of tremor during rest in double-knockout mice when compared with Mag knockout mice seemed apparent.

A B genotype tremor during rest total genotype tremor during rest total no tremor tremor no tremor tremor wt 7 0 7 wt 8 0 8 Abcd1 ko 7 0 7 Abcd1 ko 8 0 8 Mag ko 4 5 9 Mag ko 4 3 7 double ko 0** 7** 7 double ko 0** 7** 7

C genotype tremor during rest total no tremor tremor wt 8 0 8 Abcd1 ko 6 0 6 Mag ko 5 1 6 double ko 2* 5* 7

Figure 6: Tremor during rest. Number of individuals lacking signs of tremor or exhibiting tremor related to the total number of animals tested. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months. For statistical analysis Fisher’s exact Chi Square test was performed; Statistically significant differences between mutant and wild type: *) p < 0.05; **) p < 0.01

4.1.3 Hind limb reflex

When gently lifted by the tail, a wild type mouse typically extends its hind limbs outwards in a steady 90-120° angle. Upon impairment of the motor system mice tend to retract their hind limbs, holding them close to the body, often with inversion and flexion of paws. Pan et al. recorded an impaired hind limb reflex in 6 months old Mag knockout mice (176). We reformed their scaling system and tested the hind limb reflex of our mutant and wild type animals (figure 7). In accordance with the results of Pan and colleagues we found an impaired hind limb reflex in male and female Mag knockout mice of both age groups. Double-knockout mice also displayed an impaired reflex response, which was quite equal to that of Mag knockout mice. The performance of male Abcd1 knockout mice was indistinguishable from that of wild type mice at the age of 13-14 months, but deteriorated in 18-19 months old male animals.

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Figure 7: Hind limb reflexes. Wild type and mutant animals were lifted by their tail and the position of their hind limbs was scored from 0 (no reflex) to 4 (normal reflex). The best performance value of three trials for each animal was subjected to statistical analysis. Number of analyzed individuals indicated in parenthesis. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months. Boxplot graphics show median value, 1st and 3rd quartiles (boxed, icluding 50 % of values), range (whiskers) between minimum and maximum value, and mild (open circle) and extreme (asterisk) outliers

Kruskal Wallis nonparametric testing showed significance (p < 0.01 for all groups). Wilcoxon rank sum test (Mann Whitney U test) revealed significant differences between Mag knockout and wild type mice in all sex- and age groups (male and female animals, 18-

89 Results

19 months: p < 0.05; male animals, 13-14 months: p < 0.01). The 13-14 months old Mag knockout mice were also significantly impaired compared to sex- and age matched Abcd1 knockout animals. Double-knockout mice showed a significant decrease in hind limb reflex response compared to wild type animals at the age of 18-19 months (male and female: p < 0.05). However, due to a wider dispersion of values no statistical significance was reached in the comparison of 13-14 months old double-knockout and wild type animals. Male Abcd1 knockout mice were significantly impaired compared to wild type animals at the age of 18-19 months.

4.1.4 Motor coordination and balance

Pan et al. performed rotarod testing in Mag knockout mice at the age of 6 months (176). They used a rotarod apparatus in which the cylinder rotates at a constant speed of either 5 rpm or 10 rpm. At 10 rpm they recorded an impaired motor coordination in Mag knockout mice compared to wild type animals. Mice fell of the apparatus in less than 40 seconds. Defects in motor performance were also described in Abcd1 knockout mice (145). They showed severe impairment at the age of 20 months, as they fell off the cylinder (rotating at 10 rpm) in less than 20 seconds. To assess motor coordination and balance of our mice, we performed rotarod testing (figure 8) using an accelerating mode (2-20 rpm). Here, 10 rpm are reached after 150 seconds, 20 rpm after 300 seconds (5 minutes). In accord with the results from Pan et al., we found an impaired motor coordination in 18-19 months old Mag knockout mice. Male Mag knockout mice fell of the rotarod with a median of less than 120 seconds corresponding to a speed < 9.2 rpm), female animals in less than 180 seconds (speed < than 12.8 rpm). Similar results were obtained from double-knockout animals. Male double- knockout mice also showed a median latency to fall smaller than 120 seconds (speed < 9.2 rpm) and female individuals fell off in less than 150 seconds (speed < 11 rpm). Kruskal Wallis nonparametric testing was significant (male: p < 0.001; female p < 0.05). Wilcoxon rank sum test (Mann Whitney U test) revealed significant impairment of Mag knockout mice compared to wild type control animals (male: p < 0.001 and female: p < 0.05) and of double-knockout mice compared to sex- and age-matched wild type animals (male: p < 0.01; female: p < 0.05). However, no significant differences could be found between Abcd1 knockout mice and wild type controls. Though a tendency to a decrease in

90 Results latencies was encountered in male Abcd1 knockout individuals, results were far off from values of 20 months old animals reported by Pujol et al.. Over all female mice displayed a higher performance in the rotarod test, than male individuals. However, also a much wider spread of values was encountered in the female sample groups.

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Figure 8: Motor coordination ability of mutant and wild type animals in a rotarod test at accelerating speed (2-20 rpm). Motor coordination was evaluated by measuring the latency to fall (in seconds). Mean values of three trials per animal were subjected to statistical analysis. Number of analyzed individuals indicated in parentheses. (A) male mice, 18-19 months; (B) female mice, 18-19 months.

4.2. Neuropathological analysis

Previous studies demonstrated that both Mag and Abcd1 knockout mice show subtle axon and myelin pathologies in PNS, as well as in CNS (145, 176, 183). We intended to examine the effects of Mag-loss on the pathology present in Aldp-deficient mice. Thus we performed standard histological staining, immunohistochemistry, confocal microscopy and also electron microscopy to determine the status of tissue damage in double-knockout mice compared to age- and sex-matched Mag, Abcd1 knockout and wild type animals.

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4.2.1 Gross histopathological examination of brain, spinal cord and sciatic nerve sections

Hematoxylin eosin stains of cerebrum, cerebellum, spinal cord and longitudinal sciatic nerve sections of 6-8, 13-14 and 18-19 months old animals failed to reveal any focal or dispersed infiltrates of immune cells. No malformations or gross tissue damage could be detected in any of the mutant or wild type individuals. In areas of corpus callosum, internal capsule and anterior commisure, regions that are typically the first affected in human cALD, no obvious pathological features were encountered in neither group. Also no gross abnormalities were found in spinal cord (figure 9), which is first affected in human AMN. Klüver Barrera-PAS staining for myelin showed a normal distribution of myelinated and unmyelinated CNS areas (figure 10). In particular we did not find signs of generalized or focal demyelination in brain and spinal cord, not in single- and also not in double-knockout mice. No conspicuous vacuolar degeneration could be detected. Also myelin of the peripheral nervous system appeared intact. Bielschowsky silver impregnation revealed a completely normal distribution of axons and no gross axonal loss (figure 11).

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Figure 9: Hematoxylin eosin stained cross-sections of spinal cord. Light microscopic view of paraffine-embedded spinal cords of 18-19 months old animals. Normal appearance of grey and white matter areas, no inflammatory infiltrates. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout. Original magnification, x100

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Figure 10: Luxol fast blue-PAS stained cross-sections of spinal cord. Light microscopic view of paraffine-embedded spinal cords of 18-19 months old animals. Normal distribution of grey and white matter areas, no focal or generalized demyelination. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout. Original magnification, x100.

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Figure 11: Bielschowsky silver stained cross-sections of spinal cord. Light microscopic view of paraffine-embedded spinal cords of 18-19 months old animals. Normal distribution of grey and white matter areas, no gross axonal loss. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout. Original magnification, x100.

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Torpedoes, which are defined as enlarged but otherwise intact axons that develop due to impaired axonal transport, were described by Loers et al. in granular cell layer of cerebellar sections derived from aged Mag knockout mice (188). We also found such structures in double-knockout mice (figure 12). At 18-19 months of age, Mag- and double-knockout mice showed significantly elevated numbers of torpedoes compared to wild type animals (p < 0.05) and compared to Abcd1 knockout mice (p < 0.05).

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Figure 12: Axonal injury (torpedoes) in the cerebellum. Sections were stained with Bielschowsky silver impregnation to evaluate chronic axonal pathology. (A) Torpedoes of Purkinje cells in the cerebellar granule cell layer of an 18-19 months old double-knockout mouse; x400 and x1000; Arrows: torpedoes. (B) Density of axonal spheroids in the cerebellum of 18-19 months old wild type and mutant mice. Number of analyzed individuals indicated in parentheses.

Some axons in spinal cord sections of Mag, double-knockout, but also to some extent those of Abcd1 knockout mice, were surrounded by an enlarged periaxonal collar (figure 13). In Bielschowsky silver impregnation the periaxonal collar did not contain silver reactive material. Lassmann et al. described such profiles in spinal cord white matter of 8 months old Mag knockout mice and found that the periaxonal material was immunoreactive for CNP (183). However, as such structures are hard to distinguish from preparation artefacts, we forgo quantifying them.

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Figure 13: Paraffin-embedded sections of spinal cord white matter of 18-19 months old double knockout mice. The arrows indicate axons, which are surrounded by an enlarged periaxonal collar. (A) Hematoxylin eosin staining, x1000; (B) Luxol fast blue-PAS staining, x400; (C) Bielschowsky silver impregnation, x1000; Axons are stained black; an axon (arrow) is surrounded by an enlarged periaxonal collar; nearby two invading cells (arrowheads).

4.2.2 Microglia activation in spinal cord and cerebellum

As Hematoxylin eosin and Luxol fast blue-PAS stains failed to reveal any infiltrates of immune cells, we decided to perform immunostaining for Mac-3, a marker for activated microglia and macrophages, since these cells are usually good indicators for pathological alterations within the central nervous system. Pujol et al. found signs of microglia activation in spinal cord sections of 22 months old Abcd1 knockout mice (52). Loers et al. described increased numbers of Mac-3 positive cells in spinal cords of aged Mag knockout mice (188). We consistently detected an increased number of activated microglia in spinal cords of double-knockout mice of all age groups (figure 14). Also most Mag knockout mice showed increased numbers of Mac-3 positive cells. Microglia activation in Abcd1 knockout animals was age-dependent, since we did not find signs of activation in 6-8 and 13-14 months old animals, but encountered elevated levels of Mac-3 positive cells in 18-19 months old Abcd1 knockout mice. Kruskal Wallis nonparametric test for comparison of more than two groups was significant in all sex- and age-matched sample groups (p < 0.01). Wilcoxon rank sum test (Mann Whitney U test) revealed a significant increase in activated microglia cells in double- knockout mice compared to wild type controls (male and female, 18-19 and 13-14 months:

97 Results p < 0.05) and also compared to Abcd1 knockout mice (male and female, 18-19 and 13-14 months: p < 0.05). Significant enhancement in microglia activation was also encountered in 18-19 and 13-14 months old male Mag knockout mice compared to wild type controls.(both p < 0.05). However, due to generally higher levels of activation in female wild type animals no significance was reached in the comparison of Mag knockout mice and wild type controls in the female sample group. Abcd1 knockout mice showed significantly elevated levels of microglia activation compared to wild type animals in the sample group of 18-19 months old male animals (p < 0.05). We found a similar tendency in 18-19 months old female animals, but significance was not reached due to a higher base line activation in wild type individuals, as mentioned above. The number of Mac-3 positive cells in double-knockout mice significantly exceeded the number of positive cells in Mag knockout mice at the age of 18-19 months (male and female: p < 0.05). Equal levels of microglia activation in spinal cords of 13-14 months old Mag and double-knockout mice may reflect a missing effect of Abcd1 deficiency in double-knockout mice at this age. Due to the small sample size, no statistical analysis could be performed in the group of 6-8 months old animals. However, results from single individuals (figure 14D) show a similar trend as those found in 13-14 months old animals. Microglia activation in spinal cord sections of Abcd1 knockout mice resembles wild type levels. Mag knockout and double- knockout mice show an increased number of Mac-3 positive cells compared to wild type and Abcd1 knockout mice, but are indistinguishable from each other. Overall microglia activation is lower in all animals at this age compared to older individuals.

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Figure 14: Density of Mac-3 positive microglial cells in spinal cord. Number of analyzed individuals indicated in parentheses. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13- 14 months; (D) male (blue) and female (yellow) individuals, 6-8 months.

We also found microglia activation in cerebellar white matter of double-knockout animals (figure 15). Kruskal Wallis nonparametric testing was significant (p >0.01) and Wilcoxon rank sum test (Mann Whitney U test) revealed significantly increased numbers of Mac-3 positive cells in 18-19 months old double-knockout mice compared to wild type animals.(p

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< 0.05) Abcd1 and Mag knockout mice also showed slightly elevated numbers of activated cells, but no significance was reached, when compared to wild type controls.

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Figure 15: Density of Mac-3 positive microglial cells in cerebellar white matter of 18-19 months old male mice. Number of analyzed individuals indicated in parentheses.

4.2.3 Axon pathology in spinal cord

Since immunolabelling for Mac-3 revealed elevated numbers of activated microglia and macrophages in spinal cords of most mutant animals, we assessed apoptotic changes as well as axon- and myelin-integrity to detect the underlying cause(s) of microglia activation. Using proteolytically activated caspase-3 (detected by CM1 antiserum) as a marker for apoptotic cells, no evidence was found for increased apoptosis in spinal cord sections of mutant mice at any age (data not shown). Immunohistochemical detection of neurofilaments (NF 150 kD) revealed no gross abnormalities of axons in spinal cord sections of mutant mice (figure 16). We found neither accumulation nor loss of signals. Thus, we determined acute axonal injury by immunolabelling for amyloid precursor protein (APP). In neurons, APP is normally transported distally by fast axonal transport, but under pathological conditions accumulates focally at sites of acute disturbance of axonal transport or in transected axons. The sites of APP accumulation are called “spheroids”. Accumulation of such APP-positive spheroids have previously been documented by quantitative immunohistochemistry in spinal cord of

100 Results our Abcd1 knockout strain at 22 months of age (S: Forss-Petter, personal communication) and was reported by Pujol et al. in spinal cord sections of 12 months old Abcd1 knockout mice and by Loers et al. in spinal cords of 5-months-old Mag knockout mice. We found elevated numbers of spheroids in spinal cord sections of Mag and double-knockout animals of all age groups (figure 17). Some axonal degeneration was also seen in Abcd1 knockout mice at the age of 13-14 months and thereafter.

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Figure 16: Immunostaining for neurofilament (NF 150 kD); x400; Normal appearance of axons in spinal cord white matter (dorsal columns) of 18-19 months old mice. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double knockout mouse

Kruskal Wallis nonparametric testing was significant (p < 0.01 in all sample groups). Wilcoxon rank sum test (Mann Whitney U test) revealed a significant increase in acute axonal degeneration in Mag and double-knockout mice compared to wild type animals (male and female, 18-19 and 13-14 months: p < 0.05). Abcd1 knockout mice showed elevated numbers of APP-positive spheroids compared to wild type controls at the age of 18-19 months (male and female: p < 0.05), whereas no significance was reached in the

101 Results group of 13-14 months old individuals. Due to the small number of individuals no statistical analysis could be performed in the group of 6-8 months old animals. However, single values resembled the results derived from 13-14 month old animals. Equal numbers of spheroids were found in spinal cord sections of Abcd1 knockout and wild type mice. Mag knockout and double-knockout mice showed slightly increased numbers of APP- positive spheroids, but the two genotype groups were indistinguishable.

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Figure 17: Acute axonal injury in spinal cord of mutant mice. Density of amyloid precursor protein (APP)-immunoreactive axonal spheroids in the spinal cords of different genotypes. Number of analyzed individuals indicated in parentheses. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months; (D) single values of male (blue) and female (yellow) individuals, 6-8 months.

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4.2.4 Myelin pathology in spinal cord

Though no apparent signs for focal hypo-, dys-, or demyelination could be seen in Luxol fast blue-PAS stains of spinal cord sections of our mutant mice, subtle myelin abnormalities detectable by ultrastructural analysis were reported in Mag and Abcd1 knockout mice (145, 176, 183). As expected, neuronal cell bodies in spinal cord grey matter of all mutant and wild type animals were APP positive. Surprisingly we found marked immunoreactivity for APP outside of defined axonal spheroids in white matter areas of Mag and double-knockout mice, in the form of conspicuous, small, APP positive circular structures (figure 18 and 20A) and around nuclei in spinal cord white matter of all mutant and wild type animals. To estimate the density of diffuse APP accumulation in the different genotypes, we performed semiquantitative evaluation of spinal cord sections derived from 18-19 and 13-14 months old male mutant mice. We assessed extra-axonal APP immunoreactivity as virtually absent (0) to heavy diffuse APP accumulation (+++). Enhanced immunoreactivity was found in Mag and in double-knockout mice, whereas spinal cord sections of Abcd1 knockout animals were indistinguishable from sections derived from wild type controls in both age groups (figure 19). Kruskal Wallis nonparametric testing was significant (18-19 and 13-14 months: p < 0.01). Wilcoxon rank sum test (Mann Whitney U test) revealed significant enhancement of APP accumulation in Mag and double-knockout mice compared to wild type animals (18-19 and 13-14 months: p < 0.05) and compared to Abcd1 knockout mice (18-19 and 13-14 months: p < 0.05). A significant difference was also seen between Mag and double-knockout mice at the age of 18-19 months.

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Figure 18: Extra-axonal APP accumulation. Spinal cord sections were immunostained for APP, dorsal funiculus of wild type and mutant mice are shown; x400 and inserts x1000. Arrowheads: APP negative cells; asterisks: APP positive cells; arrows: APP positive circular (myelin-like) structures. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout mouse

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Figure 19: Semiquantitative analysis of extra-axonal APP accumulation. Spinal cord sections from wild type and mutant animals were immunolabelled for amyloid precursor protein (APP). Diffuse APP immunoreactivity in white matter areas outside of APP-postive spheroids was scored from 0 (no accumulation) to 3 (heavy accumulation). Number of analyzed individuals indicated in parentheses. (A) male mice, 18-19 months; (B) male mice, 13-14 months.

Though APP is widely used as a marker for acute, ongoing axonal injury or degeneration, immunocytochemical studies have shown that APP is not exclusively expressed in neurons, but also in some astrocytes, oligodendrocytes and ependymal cells (193, 194). Consistent with this information, we found APP not only in neurons and axonal endings/swellings but also in some cells of spinal cord white matter. A similar localisation of APP immuno-reactivity around cell nuclei was seen in all mutant mice and also in wild type animals. However, APP-positive, myelin-like ring structures were almost exclusively encountered in Mag and double-knockout animals. To quantify this observation, using a morphometric grid, we counted myelin-like structures in spinal cord white matter of 18-19 months male mice (figure 20). Results were similar to those found in the semiquantitative analysis of APP accumulation in the same age group. Mag and double-knockout mice showed significantly elevated numbers of myelin-like ring structures compared to wild type controls and compared to Abcd1 knockout animals (p < 0.05). Also the difference between Mag and double-knockout mice was statistically significant (p < 0.01). Spinal

105 Results cord white matter of Abcd1 knockout mice was indistinguishable from wild type tissue. AAlmost no myelin-like structures were encountered.

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Figure 20: APP positive myelin-like ring structures. Spinal cord sections of mutant and wild type mice were iiwere mmunostained for APP. (A) spinal cord white matter of a double-knockout mouse; x1000. APP ppositive myelin-like ring structures (arrows) are clearly distinguishable from APP positive axonal spheroids (arrowheads) and APP positive cell bodies (asterisks). (B) 18-19 months old male mice. Quantification of APP positive myelin-like structures in spinal cord white matter. Number of analyzed individuals indicated in parentheses.

To reassess our results more specifically and to broaden them to other sample groups, we also performed computer-aided analysis of APP accumulation in spinal cord sections of mutant and wild type mice. Dorsal columns of lumbar spinal cord cross sections were photographed and APP immunolabelling was subjected to digital assessment (cf. Method section). Findings in 18-19 months old male mice resembled results gained from semiquantitative analysis (figure 19) and from counting myelin-like ring structures (figure 20). Thus we were able to expand our investigations to other sample groups, again using computer-aided analysis. All double-knockout mice and all Mag knockout mice showed elevated ratios of APP-positive areas to measured white matter areas independent of age (figure 21). Ratios of Abcd1 knockout mice remained at wild type levels in all age groups.

106 Results

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Figure 21: Computer-aided analysis of APP accumulation in spinal cord white matter areas of mutant and wild type mice. Number of analyzed individuals indicated in parentheses. (A) male mice, 18-19 months; (B) female mice, 18-19 months; (C) male mice, 13-14 months; (D) single values of male (blue) and female (yellow) individuals, 6-8 months.

Kruskal Wallis nonparametric testing was significant in all groups (male and female, 18-19 months: p < 0.001, male 13-14 months: p < 0.005). Wilcoxon rank sum test (Mann Whitney U test) revealed a significant increase in the ratio of APP-positive areas to the measured white matter area in double-knockout mice of all age groups compared to wild type controls (male and female, 18-19 and 13-14 months: p < 0.05). Also all Mag knockout

107 Results mice showed elevated ratios compared to age- and sex-matched wild type animals (male, 18-19 months: p < 0.01; male 13-14 months and female 18-19 months: p < 0.05). Since no enhanced ratios were found in Abcd1 knockout mice compared to wild type controls, significance was furthermore reached in the comparison of Mag and Abcd1 knockout mice (male and female, 18-19 and 13-14 months: p < 0.05) and of double-knockout and Abcd1 knockout mice (male and female, 18-19 and 13-14 months: p < 0.05). Moreover significant differences were encountered between double-knockout individuals and age-/sex-matched Mag knockout mice (male, 18-19 months: p < 0.01, female, 18-19 months and male 13-14 months: p < 0.05). Due to the small number of individuals no statistical analysis could be performed in the group of 6-8 months old individuals. However, single values are similar to the results gained from 13-14 months old animals. Abcd1 knockout mice showed no elevation of ratios above wild type levels. Increased ratios were seen in Mag and double-knockout mice, but only 2 out of 4 double-knockout individuals displayed higher ratios than those found in Mag knockout mice. Upon the supposition that the detected accumulation of APP is localized in oligodendroglia cells and possibly also in myelin, we immunostained spinal cord sections of mutant mice for proteolipid protein (PLP), which is normally produced in oligodendroglia cell bodies and transported into compact myelin regions. Transgenic rats overexpressing PLP accumulate PLP, MAG, MOG and APP in oligodendroglia cell bodies as a result of impeded protein transport (195). However, assenssment of our mutant mice did not reveal enhanced accumulation of PLP, neither in oligodendroglia cell bodies nor in compact myelin (figure 22). This indicates that compact myelin proteins are either not affected or accumulation is not visible due to the normal localisation of PLP in compact myelin.

108 Results

A B

C D

Figure 22: Immunostaining for proteolipid protein (PLP) in spinal cord; x1000. Normal distribution of PLP (brown) in white matter areas of mutant and wild type mice. No apparent accumulation in oligodendroglia cells or myelin. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double knockout mouse

4.2.5 APP accumulation in compact myelin

To further assess the localisation of extra-axonal APP accumulation found in spinal cord white matter areas of Mag and double-knockout mice, we performed a series of double staining and confocal fluorescent laser microscopy analyses. Various CNS-specific cells and structures, including astrocytes, microglia, oligodendroglia, myelin, neurons and axons, were labelled with specific antibodies and co-stained with APP. In accordance with literature (194), oligodendrocytes (marked with either carbonic anhydrase, CAII, or 2’,3’-cyclic nucleotide 3’-phosphodiesterase, CNP) in spinal cord white matter of wild type mice, and also of our mutant mice, revealed immunoreactivity for APP in their cell bodies (figures 24 and 26 G-I). Also neuronal cell bodies in spinal cord grey matter and axonal spheroids in spinal cord white matter (both immunostained with SMI 31+32 antibodies that recognize neurofilament) were APP-positive (figure 26 A-

109 Results

C). In contrast no APP immunoreactivity was found within microglia cells (immunostained for ionized calcium binding adaptor molecule I, IBA-1; figure 23) and within astrocytes (immunostained for glial fibrillary acidic protein, GFAP; figures 25 and 26 D-F). APP- positive, myelin-like ring structures detected in spinal cord sections of Mag and double- knockout mice turned out to co-localise with PLP (figure 26 J-L) and myelin basic protein, MBP (data not shown), two markers of compact myelin. As APP is normally absent in compact myelin, pathological accumulation points to a disturbance of myelin integrity in Mag knockout mice, which, according to our quantitative analysis, seems to be enhanced in double-knockout animals.

A B *

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Figure 23: Immunohistochemical detection of microglia cells and APP in dorsal funiculus of 18-19 months old wild type and mutant mice. Spinal cord sections were immunostained for IBA-1 (red) and APP (brown). Double-labelling indicates no co-localisation of APP and IBA-1 in wild type and mutant animals. Arrowheads: microglia cells; asterisks: APP-positive cells; arrows: APP-positive ring-structures. (A) wild type control; (B) Abcd1 knockout; (C) Mag knockout; (D) double-knockout mouse.

110 Results

A B

C D

Figure 24: Immunohistochemical detection of oligodendrocytes and APP in dorsal funiculus of 18-19 months old wild type and mutant mice. Double-labelling for CAII (red) and APP (brown) indicates co- localisation in wild type and mutant mice. Arrowheads: APP positive oligodendrocytes, arrows: APP positive ring structures. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout mouse

A B

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Figure 25: Immunohistochemical detection of astocytes and APP in dorsal funiculus of 18-19 months old wild type and mutant mice. Double-labelling for GFAP (red) and APP (brown) indicates no co- localisation in wild type and mutant mice. Arrowheads: APP positive astrocytes, arrows: APP positive ring structures. (A) wild type control, (B) Abcd1 knockout, (C) Mag knockout, (D) double-knockout mouse

111 Results

A B C

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Figure 26: Confocal fluorescent laser microscopy of spinal cord white matter of 18-19 months old double-knockout mice. The last column represents the overlay of the two fluorescence signals shown in the first and second column. (A) labelling for SMI 31+32 (blue); (B, E, H, K) labelling for APP (red); (C) co- localisation of APP in axonal spheroids; (D) labelling for GFAP (green); (F) no accumulation of APP in astrocytes; (G) labelling for CAII (green); (I) co-localisation of APP in oligodendrocytes; (J) labelling for PLP (green); (L) co-localisation of APP in compact myelin

112 Results

As mentioned above PLP-overexpressing rats accumulate APP as well as various myelin proteins in oligodendrocytes (195). Ultrastructural analysis revealed swelling of the endoplasmic reticulum (ER) in these cells as well as a strong expression of the ER proteins: protein disulfide isomerase (PDI) and BiP-chaparone. PDI is an enzyme that catalyzes protein-folding reactions, whereas BiP maintains proteins in a folding-competent state (205). As such PDI and BiP are involved in ER quality control. Upregulation of these proteins is commonly seen during ER stress, as a response to the accumulation of unfolded or misfolded proteins (205). However, no increased immunoreactivity for PDI or BiP was found in spinal cord of any of our mutant mice at the age of 18-19 months (data not shown). Thus, it appears unlikely, that ER stress is the reason for the encountered accumulation of APP in myelin of Abcd1/Mag double-knockout mice.

4.2.6 Light microscopy of semithin sections of spinal cord white matter Detailed analysis of resin-embedded spinal cord sections of 18-19 months old double- knockout mice at high magnification (figure 27) revealed similar alterations as described for Mag and Abcd1 knockout mice (145, 174, 183). In spinal cord white matter of all mutant animals, we found several fibers undergoing axonal degeneration (figure 27 B, C), while others were surrounded by redundant myelin sheaths. Especially in Mag and double- knockout mice some fibers showed inclusions with partly membranous material located between axons and myelin sheaths (figure 27 C, D). Many fibers were surrounded by disrupted compact myelin lamellae. Also axons with abnormally thin myelin sheaths were sometimes encountered (figure 27E, F). However, as such abnormalities are occasionally seen in spinal cord sections of wild type animals as well, fixation artefacts cannot be excluded. Oligodendroglia cells appeared largely normal in all mutant animals.

113 Results

A B

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Figure 27: Pathological features in spinal cord white matter of mutant mice. Toluidine blue-stained semithin spinal cord cross-sections of 18-19 months old mutant mice show fibers with redundant myelin, undergoing axonal degeneration (small arrows), fibers containing vesicular and amorphous inclusions (big arrows) and axons surrounded by abnormally thin or split myelin sheaths (arrowheads). Oligodendrocytes appear normal (asterisks). (A) wild type control (B, C, E) double-knockout; (D, F) Mag knockout mouse.

114 Results

4.2.7 Electron microscopy of spinal cord white matter

Electron microscopic studies of spinal cord white matter corroborated the pathological alterations seen in toluidine blue stained semithin sections (figure 28). Double-knockout mice assimilate the morphological defects of both single-knockout mice. In spinal cord white matter sections of 18-19 months old Mag and double-knockout animals, some myelin sheaths were split and contained loops of oligodendrocyte cytoplasm in compact myelin (figure 28 A and B), features, which have already been described in optic nerve cross sections of Mag mutants (175). In many myelinated fibers the cytoplasm of the periaxonal collar was disorganized. Intramyelinic oligodendroglia processes were enlarged and contained partly vesicular, partly amorphous and granular osmiophilic material (figure 28 B and E). Such changes have been described as “dying-back oligodendropathy” in some demyelinative diseases, where they are associated with acute loss of myelin sheaths and clinical deficits (184-186). However, although occasionally axons with abnormally thin myelin sheaths were encountered in Mag and double-knockout animals, we found no signs of acute demyelination, neither in single- nor in double-knockout mice. “Onion bulb”-like structures, which have been described in PNS of Mag knockout mice as Schwann cell processes forming centrically arranged, crescent-shaped wrappings around axons (180), could also be detected in spinal cord white matter of Mag and double-knockout animals (figure 28 C). Most oligodendrocyte cell bodies in sections of mutant mice appeared normal. Just few showed an enlarged cytoplasmic space, where normal cell organelles were hardly detectable. Others contained depots of densely packed mitochondria and lysosomes (figure 28 D). However, qualitatively and quantitatively similar abnormalities were seen in Mag and double-knockout mice, indicating no enhancement of structural myelin/oligodendrocyte pathology in double-knockout animals. Axon degeneration has been described in spinal cords of Mag and Abcd1 knockout mice (52, 145, 176, 188). We found ultrastructural aberrations of axons in all mutant mice (figure 28 C), but no aggravation was evident in double-knockout mice. Occasionally macrophages containing deposits of lipidic materials and paracrystalline, spicular needle-like inclusions were encountered (figure 28 F).

115 Results

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Figure 28: Electron microscopy images of spinal cord white matter of 18-19 months old mutant mice. (A) Mag knockout mouse; disrupted compact myelin, intramyelinic cytoplasmic loops (arrow); (B) double- knockout mouse; split myelin sheath with intramyelinic cytoplasmic loops (arrow); osmiophilic inclusions (arrowheads); (C) double-knockout mouse; degenerating axon (asterisk), axon surrounded by an abnormally thin myelin sheath (arrow) and “onion bulb” (arrowhead); (D) double-knockout mouse; oligodendrocyte containing depots of densely packed mitochondria and lysosomes; (E) double-knockout mouse; axon with enlarged periaxonal space containing vesicular structures and osmiophilic inclusions; (F) double-knockout mouse; macrophage containing lipidic material and paracrystalline, spicular needle-like inclusions.

116 Results

4.2.8 Light microscopy of semithin sections of sciatic nerves

Although hematoxylin-eosin and Luxol fast blue-PAS staining of longitudinal sciatic nerve sections failed to reveal abnormalities, ultrastructural alterations in peripheral nerves have been described for both Abcd1 and Mag knockout mice (145, 174, 179, 180). In order to assess the status of tissue damage in sciatic nerves of our double-knockout mice we produced semithin cross sections of 18-19 months old male animals and compared them to sections derived from age- and sex-matched Abcd1, Mag and wild type animals (figure 29). Pujol et al. described abnormally thick and disorganized myelin sheaths suggesting focal hypermyelination in sciatic nerve sections of 16 months old Abcd1 knockout mice (145). Such “myelin tomacula” have also been reported in peripheral nerve fibers of one month old Mag knockout mice. However, sciatic nerve cross sections of our mutant mice appeared surprisingly normal. Myelin thickenings and so-called “myelin-figures” were occasionally encountered in double-knockout mice, but did not exceed alterations found in Mag and Abcd1 knockout mice. We scarcely found axonal degeneration in mutant mice. However, similar abnormalities occasionally appeared in wild type animals at this age. Fixation and preparation artefacts cannot be excluded.

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Figure 28: Toluidine-blue stained semithin sections of sciatic nerve of mutant and wild type mice. Arrows: myelin tomacula. (A) wild type control; (B) Abcd1 knockout; (C) Mag knockout; (D) double- knockout mouse.

117 Discussion

CHAPTER 5

DISCUSSION

118 Discussion

5. DISCUSSION

X-ALD is a clinically very heterogeneous disorder. Though the inborn error of metabolism is the same in all patients, some develop the severe, cerebral, inflammatory form of the disease, whereas others do not. The cause of this clinical heterogeneity is unknown; in particular the factors that trigger or prevent fulminant inflammation remain elusive. Moreover, Aldp deficient mice, which have been generated as a model for X-ALD and which accumulate VLCFA in brain (63-65), do not show the phenotypic variability of human patients. Late in life they display a mild phenotype involving the spinal cord with modest axonal degeneration and subtle myelin abnormalities without cerebral inflammation and demyelination (145). Testing the hypothesis, that dysmyelination and “spontaneous” myelin breakdown could initiate an inflammatory response in human patients, but in the mouse the extent of myelin damage induced by VLCFA accumulation does not reach a critical threshold, we accelerated the destabilization of myelin in a genetically modified X-ALD mouse model with combined Abcd1 and Mag deficiencies. As also Abcd1/Mag double-knockout aged without any indication of a severe inflammatory, demyelinating phenotype, we designed this study to cover the time window from 13 to 19 months when phenotypic alterations and neurological deficits have been reported to start in Abcd1 knockout mice (145). Although the basic defects within the Abcd1 gene in our knockout mice and in Aldp deficient mice generated by Lu and coworkers (65) are functional null mutations with similar biochemical features of X-ALD, the two strains display different levels of impairment. Pujol and co-workers reported severe motor incoordination in 20 months old Abcd1 knockout mice (145), which is not reproduced in our Abcd1 knockout strain. In the Rotarod test, our 18-19 months old Abcd1 mutant mice did not differ significantly from age- and sex-matched wild type littermates. These conflicting result could be due to differences in genetic background (although in both cases a major contribution of C57BL/6 is expected), in animal housing conditions, or in the test procedure. Phenotypic alterations and clinical deficits have been described in detail for Mag null mice. As this work was in progress, it became apparent that on the C57BL/6 genetic background, body tremor and motor impairment becomes more severe than in the original report and

119 Discussion could be documented already at the age of 6 months (174, 176). In accordance with the results of Pan and coworkers (176), we found whole body tremor, deficits in hind limb reflex and motor coordination also in our Mag knockout strain. Similar results were obtained from Abcd1/Mag double-knockout animals. Thus we failed to observe any contribution from Aldp deficiency to the motor deficits in double-knockout mice. Standard staining of CNS and PNS sections of 18-19 months old and younger Abcd1/Mag double-knockout mice failed to reveal any inflammatory infiltrates. Also myelination was normal in Luxol fast blue stains. Apparently even this additional destabilization of VLCFA-enriched myelin sheaths is not sufficient to trigger neuroinflammation in Aldp deficient mice. This indicates that additional, so far unknown triggers are needed to set off a fulminant immune response. Possibly, species differences in some component that modulates susceptibility for a demyelinating immunologic attack contribute to the limited phenotypic repertoire in mice. Recently, allelic differences in the human CD1 locus, which encodes lipid antigen presenting molecules and which is strikingly different in mice, have been suggested as a possible determinant for presence or absence of inflammation (190). Moreover, signs of oxidative stress that have been reported in brain sections of human cALD patients were absent in Abcd1 knockout mice (113). This further indicates species differences in the cellular susceptibility and the intrinsic predisposition to develop the cerebral form of X-ALD. Although double-knockout mice failed to develop cerebral inflammation, microglia/macrophage activation was present in spinal cord and cerebellar sections of all mutant mice. In accordance with previous publications (188, 52), the numbers of activated microglia were slightly elevated in spinal cord sections of 18-19 months old Abcd1 knockout mice and clearly elevated in Mag knockout mice of all age groups. At the age of 18-19 months, the number of activated cells in spinal cord sections of double-knockout animals exceeded the number of activated cells in Mag knockout mice, thus pointing to an additional effect of Aldp deficiency in double-knockout mice at this age, which is not yet present in 13-14 months old animals. In cerebellar sections of 18-19 months old animals, we found significantly increased microglia activation in double-knockout mice, whereas the number of activated cells in single-knockout mice was only slightly elevated and did not reach significance. However, since the levels of microglia activation in single-knockout mice were only slightly

120 Discussion surpassed in double-knockout animals, a simple summation of pathologies, caused by loss of Mag and Aldp, in double-knockout mice can thus be assumed. Analysis of apoptotic changes, axon- and myelin integrity should disclose the basic cause(s) of microglia activation found in mutant mice. While apoptosis seems to be no primary feature in any of the mutant mice, standard staining of CNS sections revealed conspicuous profiles of deformed axons surrounded by an enlarged periaxonal collar. These structures are indicative of axon and/or myelin disturbances. Immunohistochemical staining for neurofilaments, as a measure of cumulative axonal degeneration, did not indicate any gross abnormalities or severe loss of axons in any of the mutant mice. However, further detailed histopathological analysis demonstrated acute axonal damaged throughout all segments of the spinal cord in all 18-19-months-old mutant mice. While Mag and double-knockout mice of both age groups showed similarly and clearly enhanced numbers of APP positive axonal spheroids, ongoing axonal damage in Abcd1 knockout mice was less pronounced and increased with age. These results are in agreement with the development of a late-onset, mild AMN-like phenotype in X-ALD mice. No noticeable contribution from Aldp deficiency to the axonopathy in the double- knockout mice could be detected. “Torpedoes” are, per definition, enlarged axonal segments, where axonal transport is impaired (196). In our study we concentrated on torpedoes in Purkinje cell axons present in the cerebellar granular cell layer. We encountered elevated numbers of torpedoes in cerebellar sections of Mag knockout mice, which is in accordance with results from Loers and coworkers (188). However, analysis of cerebellar sections of double-knockout mice did not demonstrate further enhancement of torpedoes above the levels found in Mag knockout mice. An influence of Aldp-deficiency on axonal degeneration in cerebellar granular cell layers could thus be rejected. Nevertheless, Ferrer et al. reported atrophy and death of Purkinje cells in aged Abcd1 knockout mice (147). So it seems that not axons get impaired and degenerate, but whole neurons are primarily lost in the cerebellum of X-ALD mice. Most interestingly immunohistochemical staining and confocal laser fluorescence microscopy revealed accumulation of APP in compact myelin of spinal cord white matter of Mag and double-knockout mice. This was quite surprising, as previously done immunocytochemical and in situ hybridisation studies in wild type mice have reported APP exclusively in neurons, axons, some astrocytes, ependymal cells, choroid plexus cells,

121 Discussion microglia and oligodendrocyte cell bodies (193, 197). Ikeda and Tomonaga reported immunoreactivity for APP also in entire myelin of rat CNS (198). By Western blot analysis Sapirstein and coworkers (199) confirmed that APP is present in axolemma and periaxolemmal-myelin fractions from rat, bovine and human brains but found it absent from compact myelin. They concluded that within the myelin complex APP is produced in oligodendrocyte cell bodies and afterwards targeted and transported to the periaxolemmal myelin domain. Since we detected APP accumulation in myelin almost exclusively in spinal cord sections of Mag and double-knockout mice but only rarely in Abcd1 knockout and wild type control mice, it appears that APP is likely present in compact myelin, but usually below the immunohistochemically detectable amount. Structural or metabolic defects in myelin sheaths, as well as impaired protein expression and transport may lead to the accumulation of APP, and most likely also of other proteins, in compact myelin lamellae. However, it should be noted that the antibody (cf. Materials & Methods) that was used here (and in many previous studies of APP) is directed against the extracellular, amino-terminal domain of APP (pre-A4) but has also been described to cross react with the widely expressed Amyloid beta A4 Precursor-Like Protein 2 (APLP2) (204). Thus, a contribution from APLP2 to the increased APP reactivity is possible. Analysis of semithin and ultrathin sections of spinal cord white matter derived from Mag and double-knockout animals revealed that some fibres were surrounded by split, disrupted myelin sheaths and cytoplasmic loops within compact myelin that could, at least to some extent, account for the accumulation of APP found in these regions. Although some redundant myelin sheaths were also found in spinal cord white matter of Abcd1 knockout mice, they lack, just like wild type animals, cytoplasmic loops within compact myelin. This could explain the absence of APP accumulation in these mice. However, APP accumulation in compact myelin of double-knockout mice is enhanced compared with Mag knockout mice, although the ultrastructural abnormalities appear qualitatively and quantitatively similar in both groups. This indicates that Aldp deficiency contributes to disturbances other than structural defects in compact myelin of double-ko mice. At the ultrastructural level APP has been found in intracellular organelles, endocytotic vesicles and in plasma membranes (193, 200). In neurons it is transported anterogradely via fast axonal transport (201). However, little is known about trafficking and metabolism of APP in glia cells. It may be possible that elevated cholesterol levels described in membrane, raft and myelin fractions of adult Mag knockout mice (188) may alter APP transport and/or

122 Discussion metabolism, via modification of membrane fluidity or by influencing signalling pathways in oligodendrocytes. Recently it has been shown that X-ALD mice also display some abnormalities in cholesterol metabolism including elevated plasma cholesterol levels and dysregulated hepatic expression of cholesterol sensitive genes (202). The effects in the nervous system remain to be established but nevertheless may a further aggravated disturbance of cholesterol metabolism contribute to the enhanced APP reactivity in compact myelin of double-knockout mice. Under pathologic conditions, in transgenic PLP-overexpressing rats, oligodendrocytes suffer from stress in the endoplasmic reticulum (ER), which was detected histologically as increased immunoreactivity for the ER chaperone molecules, protein disulfide isomerase (PDI) and BiP/GRP78, and accumulation of APP in the ER of oligodendrocytes (195). However, no increase in immunoreactivity for PDI or BiP was found in spinal cord of any of the mutant mice at 18 months of age (data not shown). Thus, ER stress appears unlikely as the reason for accumulation of APP in myelin in the Abcd1/Mag double-ko mice. Still a matter of controversy in X-ALD and AMN is the question, whether axonal degeneration precedes or is secondary to myelinopathy and, whether one accounts for the other. Histopathological analysis of the CNS in cALD shows marked loss of myelin and oligodendrocytes, while axons are initially spared In AMN it seems to be vice versa. Results from electrophysiological studies in AMN patients are conflicting; some found signs of primary axonal degeneration (203), other studies favoured the predominance of demyelinating patterns in presymptomatic and early AMN or a mixture of axonal loss and multifocal demyelination (101). The X-ALD mouse model also furnished contradicting results. Analysis of compound muscle action potentials latencies and amplitudes suggested primary myelin abnormalities in 15-month-old animals (145). Histopathological analysis in 12 and 22-month-old animals, on the other hand, suggested that axonal degeneration precedes myelin degeneration (52). Based on these results, Pujol and coworkers proposed that the fundamental lesion in X-ALD mice is an axonopathy. However, it cannot be ruled out, that metabolic defects other than VLCFA load or subtle structural abnormalities, that are not detectable by light- and standard electron microscopy, are present in human and murine myelin sheaths ab initio. Small changes in membranes or cytoplasm of terminal loops of myelin sheaths in paranodal regions or Schmidt-Lanterman incisures, for instance, could escape histological depiction. In addition, different scenarios may be possible at different lesion sites (i.e. cerebrum, spinal cord and peripheral nerves).

123 Discussion

However, the results obtained in the present study indicate that some, presumably metabolic, deficits develop in myelin of Abcd1 knockout mice, which are not obvious in histopathological analysis of spinal cord sections from the Abcd1 single-knockout mouse. In the context of Abcd1/Mag double-deficiency, however, aggravation of some Mag- associated pathological features could be utilized as a more sensitive indicator of an X- ALD related metabolic disturbance that preferentially affects myelin/oligodendrocytes. As elaborated above, double-knockout animals do not show other, more severe or more numerous structural alterations in myelin compared to Mag-knockout mice. Nevertheless APP accumulation in compact myelin is enhanced in double-knockout mice. This enhancement is already visible in some of the 6-8 months old double-knockout mice and is clearly present at the age of 13-14 months. On the other hand, significant acute axonal degeneration does not become apparent in Abcd1 knockout mice until the age of 18-19 months. Taken together this suggests an early presence of metabolic stress in myelin, which precedes axonal degeneration in spinal cords of X-ALD mice. Though a connection of tomacula-formation and axon degeneration has been proposed in Mag knockout mice, Loers and coworkers did not find a clear spatial correlation between dysmyelination and axonal alterations (188), indicating the autarkic occurrence of both pathologies. The same seems to be true for Abcd1 knockout mice. Moreover, taking into account that metabolic alterations in myelin were enhanced in double-knockout mice compared to Mag-knockout mice, whereas axonal degeneration was not, makes a direct correlation between both pathologies in Abcd1 knockout mice unlikely. In summary, we conclude that an accelerated destabilization of myelin sheaths per se is not sufficient to initiate demyelination and cerebral inflammation in X-ALD mice and that some additional, so far unknown, trigger or genetic predisposition is needed. Phenotypic impairment of Abcd1/Mag double-knockout mice was comparable with Mag single- knockout mice, as was the extent of axonal degeneration. However, through the detailed analysis of double-ko animals, which bring along subtle alterations in myelin sheaths ab initio, we were able to demonstrate that Abcd1 deficiency contributes to rather early metabolic disturbances that promote dysregulation of protein expression and/or trafficking in the oligodendrocyte/myelin compartment without accelerated damage to the structural integrity of myelin and axons. Furthermore, these deficits manifested themselves in the double-knockout many months before increased axonal degeneration and microglia activation could be detected in Abcd1 deficiency.

124 References

CHAPTER 6

REFERENCES

125 References

6. REFERENCES

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138 Curriculum Vitae

CURRICULUM VITAE

PERSONAL INFORMATION

ƒ Citizenship: Austria

ƒ Born: 7th of October 1979 in Vienna, Austria

EDUCATION

ƒ Since 2007 Residency in pathology at Pathologisch-Bakteriologisches Institut, Krankenanstalt Rudolfstiftung, Vienna

ƒ 2004-2006 Study of Medical Science (N090) at the Medical University of Vienna Dissertation at the Center for Brain Research, Division of Neuroimmunology Supervisors: Ao. Univ. Prof. Dr. Johannes Berger, Univ. Prof. Dr. Hans Lassmann and Univ. Prof. Dr. Klaus-Armin Nave

ƒ 2001-2006 Study of Molekular Biology (A490) at the University of Vienna (without Graduation)

ƒ 1998-2004 Study of Human Medicin (N201) at the Medicial Universitiy of Vienna Graduation with special training in genetics and medical computer sciences

ƒ 1990-1998 AHS Realgymnasium Alt Erlaa, Vienna

ƒ 1986-1990 Volksschule Rodaun, Vienna

139 Outcome of the Thesis

OUTCOME OF THE THESIS

Dumser, M., Bauer, J., Lassmann, H., Berger, J. and Forss-Petter, S., Lack of Adrenoleukodystrophy protein enhances oligodendrocyte disturbance and microglia activation in mice with combined Abcd1/Mag deficiency.

Manuscript submitted to Acta neuropathologica in July 2007.

140