Generation and analysis of mouse models for Alzheimer's disease

286 Pages·2016·16.5 MB·English

by  Anke Annette Wittmann

Technische Universität München

Lehrstuhl für Entwicklungsgenetik

„Generation and Analysis of Mouse Models
for Alzheimer’s Disease”

Anke Annette Wittmann

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Harald Luksch
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Wolfgang Wurst
2. Univ.-Prof. Dr. Aphrodite Kapurniotu

Die Dissertation wurde am 21.03.2016 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 08.07.2016 angenommen.

Index of contents

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Index of contents
Zusammenfassung ...................................................................................................................................................... 1
Summary ....................................................................................................................................................................... 3
1. Introduction ........................................................................................................................................................ 5
1.1 Alzheimer’s disease .................................................................................................................................. 5
1.1.1 AD facts and numbers – prevalence, FAD and SAD classification ..................................... 5
1.1.2 AD symptoms – four stages of AD progression ...................................................................... 5
1.1.3 AD pathology ................................................................................................................................... 6
1.1.3.1 APP and Amyloid-β ................................................................................................................... 7
1.1.3.2 pTAU tangles .............................................................................................................................. 8
1.1.3.3 Neuronal inflammation and neuronal death ........................................................................ 9
1.1.3.4 Lipid accumulation ................................................................................................................... 10
1.1.4 APP processing – amyloidogenic and non-amyloidogenic pathway ................................ 10
1.1.4.1 ADAM 10 ................................................................................................................................... 11
1.1.4.2 BACE1 ......................................................................................................................................... 12
1.1.4.3 y-secretase .................................................................................................................................. 12
1.1.5 AD genetics – Risk factors for FAD......................................................................................... 13
1.2 Sphingolipid metabolism ...................................................................................................................... 14
1.2.1 Anabolic pathway .......................................................................................................................... 14
1.2.1.1 De novo Synthesis of Ceramide............................................................................................. 14
1.2.1.2 Transport of Ceramide for further processing .................................................................. 15
1.2.1.3 Complex Sphingolipids, Ceramide-1-Phosphate and their synthesizing enzymes ... 15
1.2.2 Catabolic pathways ....................................................................................................................... 16
1.2.2.1 Sphingomyelinases ................................................................................................................... 16
1.2.2.2 Ceramidases................................................................................................................................ 17
1.3 AD and lipids ........................................................................................................................................... 18
1.3.1 How do lipids influence AD associated proteins? ................................................................. 18
1.3.1.1 Lipid rafts ................................................................................................................................... 18
1.3.1.2 Endocytosis and Autophagy .................................................................................................. 20
1.3.1.3 Direct interaction ...................................................................................................................... 20
1.3.2 Observed lipid alterations in AD patients ............................................................................... 20
1.3.2.1 Ceramide ..................................................................................................................................... 21
1.3.2.2 Sphingomyelin ........................................................................................................................... 22
1.3.2.3 Phospholipids – Phosphatidylcholine, phosphatidylethanolamine and plasmalogens
23

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Index of contents

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1.3.2.4 Cholesterol ................................................................................................................................. 23
1.3.3 Effects of life-style on AD ........................................................................................................... 23
1.3.3.1 Diet and AD ............................................................................................................................... 24
1.3.3.2 Stress and AD ........................................................................................................................... 26
1.3.3.3 Physical exercise and AD ....................................................................................................... 26
1.4 The sphingomyelin synthase family .................................................................................................. 27
1.4.1 Sphingomyelin synthase 1 and 2 ............................................................................................... 27
1.4.1.1 The human SMS1 gene ........................................................................................................... 28
1.4.1.2 The mouse SMS1 gene ............................................................................................................ 28
1.4.1.3 SMS1 protein structure ........................................................................................................... 28
1.4.2 Sphingomyelin synthase related protein ................................................................................. 29
1.5 The Tg2576 or APP Swedish mouse - a common AD model ..................................................... 30
1.5.1 Genetic characteristics ................................................................................................................. 30
1.5.2 Neuropathological characteristics ............................................................................................. 30
1.5.3 Behavior and life expectancy ...................................................................................................... 30
1.5.4 Lipid alterations............................................................................................................................. 31
1.6 Sphingomyelin Synthase 1 knock out mice ...................................................................................... 31
1.7 Aim of the study ..................................................................................................................................... 32
2. Material and Methods .................................................................................................................................... 33
2.1 Material .................................................................................................................................................... 33
2.1.1 Animals ............................................................................................................................................ 33
2.1.1.1 Sms1 mouse line ........................................................................................................................ 33
2.1.1.2 Tg2576 mouse line ................................................................................................................... 33
2.1.1.3 Sms1 x APPswe mouse line ..................................................................................................... 33
2.1.2 Antibodies ....................................................................................................................................... 34
2.1.3 Buffers, Gels and Media............................................................................................................... 36
2.1.4 Chemicals and basic substances ................................................................................................. 40
2.1.5 Consumables and other material ............................................................................................... 41
2.1.6 Enzymes and Vectors ................................................................................................................... 42
2.1.7 Fatty acid diet ................................................................................................................................ 42
2.1.8 Kits .................................................................................................................................................... 42
2.1.9 Machinery ....................................................................................................................................... 43
2.1.10 Primer sets for genotyping, splice variant detection and probe generation ................... 44
2.1.11 Primer probe sets for TaqMan assays ..................................................................................... 45
2.1.12 Software ........................................................................................................................................... 46
2.2 Methods .................................................................................................................................................... 47
2.2.1 Animal housing .................................................................................................................................. 47

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2.2.2 Accession of birth rates .................................................................................................................... 47
2.2.3 Accession of body weight gain ....................................................................................................... 47
2.2.4 Accession of death rates ................................................................................................................... 47
2.2.5 PUFA diet experiments ................................................................................................................... 47
2.2.5.1 Fertility experiment ................................................................................................................. 47
2.2.5.2 Weight gain experiment ......................................................................................................... 48
2.2.6 Behavioral Tests ................................................................................................................................. 48
2.2.6.1 Open Field .................................................................................................................................. 49
2.2.6.2 Y-Maze ........................................................................................................................................ 49
2.2.6.3 Grip strength ............................................................................................................................. 49
2.2.6.4 Rotarod test ................................................................................................................................ 50
2.2.6.5 Stress reactivity test (SRT) .................................................................................................... 50
2.2.7 Animal blood and organ preparation ............................................................................................ 51
2.2.7.1 Blood taking ............................................................................................................................... 51
2.2.7.2 Pathological analysis................................................................................................................ 51
2.2.7.3 Perfusion ..................................................................................................................................... 51
2.2.7.3.1 Organ preparation and perfusion with PBS ................................................................ 51
2.2.7.3.2 Organ preparation and perfusion with PFA ............................................................... 51
2.2.7.4 Tissue embedding ..................................................................................................................... 51
2.2.7.5 Cutting of tissues ...................................................................................................................... 52
2.2.7.5.1 Microtome cutting ............................................................................................................ 52
2.2.7.5.2 Cryostate cutting ............................................................................................................... 52
2.2.7.6 Organ withdrawal for RNA and Protein Isolation .......................................................... 52
2.2.7.7 Dissection of HPA-axis related brain parts and organs for RNA isolation ............... 52
2.2.7.8 Organ withdrawal for Lipid profiling and enzyme activity measurements ............... 53
2.2.8 Accession of organ weight ............................................................................................................... 53
2.2.9 Histological analysis and organ pathology ................................................................................. 53
2.2.10 Nucleic acid based methods ............................................................................................................. 53
2.2.10.1 DNA based methods ............................................................................................................ 53
2.2.10.1.1 DNA Isolation .................................................................................................................. 53
2.2.10.1.2 Genotyping – PCRs ........................................................................................................ 54
2.2.10.1.3 Gel-electrophoresis ......................................................................................................... 54
2.2.10.1.4 Sequencing ........................................................................................................................ 54
2.2.10.2 RNA based methods ............................................................................................................ 54
2.2.10.2.1 RNA Isolation .................................................................................................................. 54
2.2.10.2.2 RNA – cDNA conversion / RT-PCR ........................................................................ 55
2.2.10.2.3 Detection of splice variants ........................................................................................... 55
2.2.10.2.4 Generation of Northern blot probes ........................................................................... 55

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2.2.10.2.5 Northern blot ................................................................................................................... 58
2.2.10.2.6 TaqMan assay .................................................................................................................. 58
2.2.10.2.7 Microarray and evaluation ............................................................................................ 59
2.2.11 Protein based methods ..................................................................................................................... 60
2.2.11.1 Protein Isolation standard procedure ............................................................................. 60
2.2.11.2 Protein Isolation for APP profiling ................................................................................. 60
2.2.11.3 Protein quantification / BCA assay ................................................................................. 61
2.2.11.4 Westernblot standard procedure ..................................................................................... 62
2.2.11.5 Westernblot APP profile ................................................................................................... 63
2.2.12 Staining procedures ........................................................................................................................... 65
2.2.12.1 Immunohistochemistry protocols .................................................................................... 66
2.2.12.1.1 IHC / DAB staining ....................................................................................................... 66
2.2.12.1.2 IHC / DAB staining anti-mouse ................................................................................. 66
2.2.12.1.3 APP, Aß40 and Aß42 immunostaining and plaque counting ............................... 67
2.2.12.1.4 IHC / Fluorescent staining .......................................................................................... 68
2.2.12.2 Haematoxylin & Eosin staining ....................................................................................... 68
2.2.12.3 Peridoic acid – Schiff’s reagent staining ......................................................................... 68
2.2.12.4 Klüver-Barrera staining ..................................................................................................... 68
2.2.12.5 TUNEL staining and counting of TUNEL positive cells ......................................... 68
2.2.12.6 Β-glactosidase staining ....................................................................................................... 69
2.2.13 Transmission electron microscopy ................................................................................................ 69
2.2.14 Multispectral optoacoustic tomography ...................................................................................... 70
2.2.15 Lipid profiling and enzyme activity assay ................................................................................... 70
2.2.15.1 Protein Isolation and quantification ................................................................................ 70
2.2.15.1.1 Testes preparation .......................................................................................................... 70
2.2.15.1.2 Protein amount determination ..................................................................................... 70
2.2.15.2 Mass spectrometry .............................................................................................................. 71
2.2.15.2.1 Measurement of diacylphosphatidylcholine (PCaa), phospatidylcholine-
plasmalogen (PCae), lyso-phosphatidylcholine (lysoPC) and sphingomyelin (SM) levels .. 71
2.2.15.2.2 Measurement of ceramide levels ................................................................................. 71
2.2.15.3 Enzyme activity assay ......................................................................................................... 72
2.2.16 Cell culture experiments .................................................................................................................. 72
2.2.16.1 Generation of mouse embryonic fibroblasts .................................................................. 72
2.2.16.2 Rapamycin treatment .......................................................................................................... 72
2.2.16.3 Protein Isolation from MEFs ........................................................................................... 73
2.2.17 Statistical evaluation ......................................................................................................................... 73
3. Results ................................................................................................................................................................ 74
3.1 Sms1 gene trap model ............................................................................................................................ 74

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Index of contents

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3.1.1 Validation of insert localization ................................................................................................. 74
3.1.2 The Sms1 gene trap mouse model showed remaining expression of Sms1 and brain
specific splice variants .................................................................................................................................... 75
3.1.3 Sms1MUT animals appear normal in most instances .............................................................. 77
3.1.4 Sms1MUT animals show slight alterations in heart an spleen .............................................. 78
3.1.5 Lipid profile..................................................................................................................................... 79
3.1.5.1 Sphingomyelin species ............................................................................................................. 79
3.1.5.2 Ceramide ..................................................................................................................................... 80
3.1.5.3 Phosphatidylcholines and Plasmalogens ............................................................................ 80
3.1.5.4 Lyso-Phosphatidylcholine species ........................................................................................ 87
3.1.5.5 Cholesterol and Phosphatidylethanolamine ....................................................................... 88
3.1.6 Enzymes of the Sphingolipid pathway ..................................................................................... 89
3.1.6.1 Alterations in expression levels of catabolic enzymes ..................................................... 91
3.1.6.2 Alterations in expression levels of anabolic enzymes ...................................................... 92
3.1.7 Myelination deficits in the brain and the spinal cord of Sms1MUT ..................................... 94
3.1.8 Strength, coordination and nociception changes in Sms1MUT ............................................ 96
3.1.9 Blood-Brain-Barrier alterations ................................................................................................. 97
3.1.10 HPA axis alterations .................................................................................................................... 99
3.1.10.1 Basic results related to stress-coping and anxiety-like behavior ............................. 99
3.1.10.2 Stress reactivity tests ........................................................................................................ 101
3.1.10.2.1 Stress reactivity test with 15 min restraint stress ................................................ 101
3.1.10.2.2 Stress reactivity test with 2h restraint stress ........................................................ 104
3.1.10.3 Analysis of the major HPA axis components .............................................................. 108
3.1.10.3.1 Pituitary and Adrenal morphology ........................................................................... 108
3.1.10.3.2 Expression level of genes involved in HPA axis signaling................................. 108
3.1.11 Aged Sms1MUT animals show splenomegaly ......................................................................... 110
3.1.12 Sms1MUT showed altered testes histology and partially comprised fertility ................. 114
3.1.12.1 Testes specific transcripts and protein levels of Sms1 were strongly reduced in
Sms1MUT animals ....................................................................................................................................... 114
3.1.12.2 Testes of Sms1MUT showed specific reduction in lipid levels, which differed from
changes observed in brain tissue ........................................................................................................... 115
3.1.12.3 Sms1MUT testes showed strongly changed histology and enhanced apoptosis ... 118
3.1.12.4 Sloughing of immature germ cell takes place between pachytene and round
spermatid stage .......................................................................................................................................... 120
3.1.12.5 Sms1MUT testes showed alterations in junction protein expression and localization
and in blood-testes-barrier functionality............................................................................................. 122
3.1.12.6 Sms1MUT showed partial reduction in fertility, which could be ameliorated by
DHA/EPA diet .......................................................................................................................................... 126
3.2 Sms1 x APPswe mouse model ............................................................................................................ 129

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3.2.1 General characterization of the Sms1 x APPswe mouse model........................................ 129
3.2.1.1 Birth rates ................................................................................................................................. 129
3.2.1.2 Survival rates ........................................................................................................................... 130
3.2.1.3 Body and organ weight ......................................................................................................... 132
3.2.2 Lipid profile .................................................................................................................................. 135
3.2.2.1 Sphingomyelin ......................................................................................................................... 135
3.2.2.2 Diacyl-phosphatidylcholine species ................................................................................... 138
3.2.2.3 Acyl-alkyl-phosphatidylcholine (plasmalogene) species ............................................... 140
3.2.2.4 Lyso-PC species ...................................................................................................................... 142
3.2.2.5 Carnitines ................................................................................................................................. 144
3.2.3 DHA/EPA diet reduced hampered weight gain, but did not affect death rates.......... 145
3.2.3.1 Weight gain ............................................................................................................................. 146
3.2.3.2 Life expectancy ........................................................................................................................ 146
3.2.4 APP processing............................................................................................................................ 146
3.2.4.1 APP cleavage products.......................................................................................................... 146
3.2.4.2 Activity of APP processing enzymes ................................................................................. 150
3.2.4.2.1 BACE1 activity ................................................................................................................ 151
3.2.4.2.2 Γ-secretase complex activit ........................................................................................... 152
3.2.5 pTAU/TAU ratio ....................................................................................................................... 152
3.2.6 Brain histology of Sms1 x APPswe mice ................................................................................ 153
3.2.7 Studies on general behavior and memory performance ..................................................... 155
3.2.8 APP Expression pattern of APPswe in different organs .................................................. 157
3.2.9 Myelination was altered between Sms1WT x APPsweTg and Sms1MUT x APPsweTg
animals depending on the brain region .................................................................................................... 160
3.2.10 Inflammatory hints ..................................................................................................................... 162
3.2.11 Autophagy alterations in brain and MEFs of Sms1 x APPswe nimals........................... 165
4. Discussion........................................................................................................................................................ 170
4.1 Sms1 has tissue specific transcripts in mice ................................................................................... 170
4.2 The intronic gene trap cassette insertion leads to a tissue-specific reduction in SMS1 ..... 170
4.3 The tissue-specific SMS1 reduction is not fully compensated by SMS2 ................................ 171
4.4 Sms1 disruption affects birth rates and survival depending on expression of hAPPsweTg . 172
4.5 Sms1 disruption leads to reduced body weight or weight gain ................................................. 173
4.6 Disruption of Sms1 triggers further changes in SL-metabolism, which are magnified on a
hAPPsweTg background .................................................................................................................................... 173
4.6.1 Maintenance of Cer homeostasis as an important aspect in the brain of Sms1MUT ..... 173
4.6.2 The disruption of Sms1 affects PC species, with specific saturation and elongation
states in Sms1MUT animals ........................................................................................................................... 175

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4.6.3 Sms1MUT x APPsweTg accumulate PCaa and PCae species which could influence
inflammation and acetylcholine availability ............................................................................................ 176
4.7 EPA/DHA supplemented diet improves weight gain, without influencing life span of
Sms1MUT x APPsweTg animals ......................................................................................................................... 178
4.8 Changes in lipid composition lead to altered myelination patterns in CNS and PNS ........ 178
4.9 Is the disruption of Sms1 protective concerning AD pathology of the brain? ...................... 181
4.9.1 The APP level seems to be influenced by Sms1 ................................................................... 181
4.9.2 hAPPswe expression shifts APP processing of towards the β-secretory pathway...... 182
4.9.3 Is there an impact of Sms1 disruption on APP processing? ............................................. 182
4.9.4 Sms1 x APPsweTg animals develop diffuse plaques early in life and a reduced number
of Aß-40 positive plaques with the disruption of Sms1 ........................................................................ 184
4.9.5 Memory performance is not altered between the different genotypes of the Sms1 x
APPswe mouse line ........................................................................................................................................ 185
4.10 Do Sms1MUT and Sms1MUT x APPswe animals show indications related to a “healthier life
style” in humans with respect to AD risk? .................................................................................................. 185
4.10.1 Sms1MUT and Sms1MUT x APPsweTg animals show a higher physical activity rate ...... 185
4.10.2 Sms1MUT show altered stress-coping behavior ..................................................................... 186
4.11 hAPPswe is also expressed in lung and heart of APPsweTg mice .............................................. 190
4.12 Sms1 seems to be involved in regulation of the immune system and the autophagic flux . 192
4.12.1 Sms1MUT animals suffer from age-dependent splenomegaly ............................................. 192
4.12.2 Does the BBB provide sufficient barrier function? ............................................................. 193
4.12.3 Neuro-inflammatory processes might be alterd in Sms1MUT x APPsweTg animals ..... 195
4.13 Autophagy alterations in CNS, connections between LSDs and AD ...................................... 197
4.14 Sms1 is crucial for male fertility ........................................................................................................ 199
4.14.1 The Sms1 mutation altered the lipid profile of mutant testis ........................................... 200
4.14.2 BTB functionality was compromised in Sms1 mutants...................................................... 200
4.14.3 DHA/EPA diet rescued the sloughing phenotype of infertile Sms1 mutants .............. 201
5. Conclusion ....................................................................................................................................................... 203
6. List of Figures ................................................................................................................................................ 205
7. List of Tables .................................................................................................................................................. 207
8. List of abbreviations ...................................................................................................................................... 209
9. References ........................................................................................................................................................ 214
10. Supplementary data .................................................................................................................................. 246
Acknowledgments .................................................................................................................................................. 276

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