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TNF-Induced Chronic Neuroinflammation: Disrupting Human Neural Stem Cell Pool and the Modulatory Effects of AIM2
No full textNeuroinflammation acts as a critical causative and/or exacerbating factor in diseases, physical trauma, stroke and aging, disrupting brain homeostasis. During fetal neurodevelopment, neuroinflammation is particularly concerning as it can interfere with the plasticity of human neural stem cells (hNSCs), which generate the diverse neuronal and macroglial cells of the central nervous system (CNS). This disruption can lead to long-lasting or permanent effects on brain function, highlighting the importance of understanding how neuroinflammation hinders the neurogenic potential of hNSCs.
Chronic exposure to tumor necrosis factor (TNF), a key neuroinflammatory cytokine, has been linked to neurodevelopmental disorders. However, its impact on the plasticity of fetal hNSCs during gestation remains unclear. This study investigated the effects of sustained prenatal neuroinflammation on hNSCs by exposing 3D starPEG-heparin hydrogel cultures of primary human fetal astrocytes (3D pHA) to chronic TNF levels. The study found that chronic TNF exposure triggered significant morphological changes in neural networks, characterized by reduced branching and longer connected paths, along with cells with elongated nuclei. TNF exposure led to suppressed stem cell plasticity and neurogenic potential, evidenced by a dramatic reduction in GFAP+ and GFAP+/SOX2+ cell populations. These alterations were associated with transcriptome remodeling, revealing changes in gene expression related to inflammation, innate immunity, cell structure, adhesion, and motility. Surprisingly, chronic TNF exposure did not affect the total cell number in the 3D pHA cultures, suggesting activation of compensatory mechanisms that promote cell survival or prevent cell death, including senescence. The study also identified the unique upregulation of AIM2, an inflammasome sensor, and hypothesized that inhibiting AIM2 could mitigate TNF-induced inflammation. Treatment with LL-37, an AIM2 inhibitor, effectively attenuated TNF-induced upregulation of various cytokines and chemokines, as well as components of the NFKB pathway. Additionally, replenishing TNF-downregulated proteins showed some efficacy in mitigating TNF's impact, although their overall ability to restore expression of a select set of genes to untreated control levels was limited.
In conclusion, this dissertation provides compelling evidence of the detrimental role of TNF-induced chronic neuroinflammation on hNSC plasticity during fetal brain development, offering insights into the underlying molecular mechanisms. The study's findings also reveal potential therapeutic targets, such as AIM2 and TNF-downregulated proteins, for mitigating the adverse effects of neuroinflammation on brain development and function.:COVER PAGE I
SIGNATURE PAGE II
DEDICATION III
ACKNOWLEDGEMENTS IV
SUMMARY VI
ZUSAMMENFASSUNG VII
TABLE OF CONTENTS IX
LIST OF PUBLICATIONS XII
LIST OF ABBREVIATIONS XIII
LIST OF TABLES XIX
LIST OF FIGURES XX
LIST OF SUPPLEMENTARY FIGURES XXI
1 INTRODUCTION 22
1.1 Human Neural Stem Cells in Neurodevelopment 22
1.1.1 Neurogenesis 22
1.1.2 Gliogenesis 25
1.2 Plasticity in Human Neurodevelopment 26
1.3 Neuroinflammation in The Brain Tissue 27
1.3.1 Neuroinflammation Spectrum: Acute and Chronic States 27
1.4 TNF as a Mediator of Neuroinflammation 29
1.5 Mechanisms of TNF Signaling 31
1.5.1 TNF-TNFR1 Signaling through Complex I, IIa, IIb and IIc 31
1.5.2 TNF-TNFR2 Signaling 33
1.6 TNF in Neurodevelopmental Disorders 33
1.6.1 Effect of TNF-mediated Neuroinflammation on Fetal and Adult Neural Stem Cells 37
1.7 TNF and Inflammasome Link in Neuroinflammation Context 39
2 AIMS AND OBJECTIVES OF THE STUDY 43
3 MATERIALS AND METHODS 44
3.1 Main Materials and Resources 44
3.2 Medium and Solutions 47
3.3 Software 48
3.4 Methods 48
3.4.1 Primary Human Fetal Astrocyte Culture 48
3.4.2 Cell Encapsulation 48
3.4.3 Cell Culture Treatments 49
3.4.3.1 TNF, R9-LL-37 or Combination Treatments 49
3.4.3.2 Pooled Protein Treatments 49
3.4.4 Immunofluorescence 50
3.4.5 Microscope Imaging 51
3.4.5.1 Leica TCS SP5 MP Microscope Imaging 51
3.4.5.2 Zeiss Axio Observer.Z1/7 Spinning Disk Confocal Microscope Imaging 51
3.4.6 Image Processing and Quantification 52
3.4.7 RNAseq and Data Analysis 52
3.4.8 Quantitative RT-PCR (qRT-PCR) 53
3.4.9 Synthesis of R9-LL-37 54
3.4.10 Protein Arrays 56
3.4.10.1 Human Cytokine Proteome Profiling 57
3.4.10.2 Human NFKB Pathway Proteome Profiling 61
3.4.11 Densitometric Analysis 63
4 RESULTS 64
4.1 Transcriptome Remodeling and Morphological Alterations are Triggered in hNSC by Chronic TNF Exposure 64
4.2 Chronic TNF exposure induces a strong inflammatory and innate immune response in hNSC 70
4.3 Plasticity and Neurogenic Potential in hNSC are Suppressed by Chronic TNF Exposure 74
4.4 TNF Effect Can Be Alleviated by Inhibiting AIM2 in hNSC 81
4.4.1 TNF Effects Demonstrate Time- and Dose-Dependent Dynamics 81
4.4.2 AIM2 Inhibition Mitigates TNF-Induced Molecular Alterations 84
4.5 Targeted Protein Pooling Can Mitigate TNF-Induced Gene Downregulation in hNSC 91
5 DISCUSSION 96
6 CONCLUSION 105
7 FUTURE DIRECTIONS 107
8 REFERENCES 110
9 SUPPLEMENTARY INFORMATION 129
9.1 Senescence and Quiescence Genes 129
9.2 Ferroptosis Signaling 130
9.3 Effects of AIM2 Inhibition on Cytokine and Chemokine Signaling 131
9.4 Effects of AIM2 Inhibition on NFKB Signaling 140
9.5 Pooled Protein Treatments 144
10 DECLARATIONS 147
Annex 1 147
Annex 2 148Neuroinflammation ist ein kritischer ursächlicher und/oder verschlimmernder Faktor bei Krankheiten, körperlichen Traumata, Schlaganfällen und Alterung und stört die Homöostase des Gehirns. Während der fötalen Neuroentwicklung ist Neuroinflammation besonders bedenklich da sie die Plastizität menschlicher neuronaler Stammzellen (hNSCs) beeinträchtigen kann, welche die verschiedenen neuronalen und makroglialen Zellen des zentralen Nervensystems (ZNS) erzeugen. Diese Störung kann zu lang anhaltenden oder dauerhaften Auswirkungen auf die Gehirnfunktion führen, was die Bedeutung von Fachwissen unterstreicht, wie Neuroinflammation das neurogene Potenzial von hNSCs behindert.
Die chronische Aussetzung gegenüber Tumornekrosefaktor (TNF), einem entscheidenden neuroinflammatorischen Zytokin, ist mit neurologischen Entwicklungsstörungen in Verbindung gebracht worden. Seine Auswirkungen auf die Plastizität fetaler hNSCs während der Schwangerschaft bleiben jedoch unklar. Diese Studie untersuchte die Auswirkungen einer anhaltenden pränatalen Neuroinflammation auf hNSCs, indem 3D-StarPEG-Heparin-Hydrogelkulturen primärer menschlicher fötaler Astrozyten (3D pHA) chronischen Konzentrationen von TNF ausgesetzt wurden. Die Studie ergab, dass chronische Aussetzung gegenüber TNF signifikante morphologische Veränderungen in neuronalen Netzwerken auslöste, die durch reduzierte Verzweigung und längere verbundene Pfade sowie Zellen mit verlängerten Nuklei gekennzeichnet waren. Die TNF-Exposition führte zu unterdrückter Stammzellplastizität und unterdrücktem neurogenen Potenzial, was durch eine dramatische Verringerung der GFAP+ und GFAP+/SOX2+ Zellpopulationen belegt wurde. Diese Veränderungen waren mit einer Umgestaltung des Transkriptoms verbunden und offenbarten Veränderungen in der Genexpression im Zusammenhang mit Entzündungen, angeborener Immunität, Zellstruktur, Adhäsion und Motilität. Überraschenderweise hatte die chronische TNF-Exposition keinen Einfluss auf die Gesamtzellzahl in den 3D pHA-Kulturen, was auf die Aktivierung von Kompensationsmechanismen hindeutet, die das Zellüberleben fördern oder Zelltod, einschließlich Seneszenz, verhindern. Die Studie identifizierte außerdem die einzigartige Hochregulierung von AIM2, einem Inflammasom-Sensor, und stellte die Hypothese auf, dass die Hemmung von AIM2 die durch TNF induzierte Entzündung lindern könnte. Die Behandlung mit LL-37, einem AIM2-Hemmer, schwächte die durch TNF induzierte Hochregulierung verschiedener Zytokine und Chemokine sowie Komponenten des NFKB-Signalwegs wirksam ab. Darüber hinaus zeigte die Wiederaufstockung herunterregulierter TNF-Proteine eine gewisse Wirksamkeit bei der Abschwächung der Auswirkungen von TNF, obwohl ihre allgemeine Fähigkeit, die Expression einer ausgewählten Gruppe von Genen auf unbehandelte Kontrollmengen wiederherzustellen, begrenzt war.
Zusammenfassend lässt sich sagen, dass diese Dissertation wissenschaftlich signifikante Nachweise für die nachteilige Rolle der durch TNF induzierten chronischen Neuroinflammation auf die Plastizität von hNSC während der fötalen Gehirnentwicklung liefert und Einblicke in die zugrunde liegenden molekularen Mechanismen bietet. Die Ergebnisse der Studie enthüllen außerdem potenzielle therapeutische Targets wie AIM2 und herunterregulierte TNF-Proteine zur Abschwächung der nachteiligen Auswirkungen der Neuroinflammation auf die Entwicklung und Funktion des Gehirns.:COVER PAGE I
SIGNATURE PAGE II
DEDICATION III
ACKNOWLEDGEMENTS IV
SUMMARY VI
ZUSAMMENFASSUNG VII
TABLE OF CONTENTS IX
LIST OF PUBLICATIONS XII
LIST OF ABBREVIATIONS XIII
LIST OF TABLES XIX
LIST OF FIGURES XX
LIST OF SUPPLEMENTARY FIGURES XXI
1 INTRODUCTION 22
1.1 Human Neural Stem Cells in Neurodevelopment 22
1.1.1 Neurogenesis 22
1.1.2 Gliogenesis 25
1.2 Plasticity in Human Neurodevelopment 26
1.3 Neuroinflammation in The Brain Tissue 27
1.3.1 Neuroinflammation Spectrum: Acute and Chronic States 27
1.4 TNF as a Mediator of Neuroinflammation 29
1.5 Mechanisms of TNF Signaling 31
1.5.1 TNF-TNFR1 Signaling through Complex I, IIa, IIb and IIc 31
1.5.2 TNF-TNFR2 Signaling 33
1.6 TNF in Neurodevelopmental Disorders 33
1.6.1 Effect of TNF-mediated Neuroinflammation on Fetal and Adult Neural Stem Cells 37
1.7 TNF and Inflammasome Link in Neuroinflammation Context 39
2 AIMS AND OBJECTIVES OF THE STUDY 43
3 MATERIALS AND METHODS 44
3.1 Main Materials and Resources 44
3.2 Medium and Solutions 47
3.3 Software 48
3.4 Methods 48
3.4.1 Primary Human Fetal Astrocyte Culture 48
3.4.2 Cell Encapsulation 48
3.4.3 Cell Culture Treatments 49
3.4.3.1 TNF, R9-LL-37 or Combination Treatments 49
3.4.3.2 Pooled Protein Treatments 49
3.4.4 Immunofluorescence 50
3.4.5 Microscope Imaging 51
3.4.5.1 Leica TCS SP5 MP Microscope Imaging 51
3.4.5.2 Zeiss Axio Observer.Z1/7 Spinning Disk Confocal Microscope Imaging 51
3.4.6 Image Processing and Quantification 52
3.4.7 RNAseq and Data Analysis 52
3.4.8 Quantitative RT-PCR (qRT-PCR) 53
3.4.9 Synthesis of R9-LL-37 54
3.4.10 Protein Arrays 56
3.4.10.1 Human Cytokine Proteome Profiling 57
3.4.10.2 Human NFKB Pathway Proteome Profiling 61
3.4.11 Densitometric Analysis 63
4 RESULTS 64
4.1 Transcriptome Remodeling and Morphological Alterations are Triggered in hNSC by Chronic TNF Exposure 64
4.2 Chronic TNF exposure induces a strong inflammatory and innate immune response in hNSC 70
4.3 Plasticity and Neurogenic Potential in hNSC are Suppressed by Chronic TNF Exposure 74
4.4 TNF Effect Can Be Alleviated by Inhibiting AIM2 in hNSC 81
4.4.1 TNF Effects Demonstrate Time- and Dose-Dependent Dynamics 81
4.4.2 AIM2 Inhibition Mitigates TNF-Induced Molecular Alterations 84
4.5 Targeted Protein Pooling Can Mitigate TNF-Induced Gene Downregulation in hNSC 91
5 DISCUSSION 96
6 CONCLUSION 105
7 FUTURE DIRECTIONS 107
8 REFERENCES 110
9 SUPPLEMENTARY INFORMATION 129
9.1 Senescence and Quiescence Genes 129
9.2 Ferroptosis Signaling 130
9.3 Effects of AIM2 Inhibition on Cytokine and Chemokine Signaling 131
9.4 Effects of AIM2 Inhibition on NFKB Signaling 140
9.5 Pooled Protein Treatments 144
10 DECLARATIONS 147
Annex 1 147
Annex 2 14
TNF-Induced Chronic Neuroinflammation: Disrupting Human Neural Stem Cell Pool and the Modulatory Effects of AIM2
No full textNeuroinflammation acts as a critical causative and/or exacerbating factor in diseases, physical trauma, stroke and aging, disrupting brain homeostasis. During fetal neurodevelopment, neuroinflammation is particularly concerning as it can interfere with the plasticity of human neural stem cells (hNSCs), which generate the diverse neuronal and macroglial cells of the central nervous system (CNS). This disruption can lead to long-lasting or permanent effects on brain function, highlighting the importance of understanding how neuroinflammation hinders the neurogenic potential of hNSCs.
Chronic exposure to tumor necrosis factor (TNF), a key neuroinflammatory cytokine, has been linked to neurodevelopmental disorders. However, its impact on the plasticity of fetal hNSCs during gestation remains unclear. This study investigated the effects of sustained prenatal neuroinflammation on hNSCs by exposing 3D starPEG-heparin hydrogel cultures of primary human fetal astrocytes (3D pHA) to chronic TNF levels. The study found that chronic TNF exposure triggered significant morphological changes in neural networks, characterized by reduced branching and longer connected paths, along with cells with elongated nuclei. TNF exposure led to suppressed stem cell plasticity and neurogenic potential, evidenced by a dramatic reduction in GFAP+ and GFAP+/SOX2+ cell populations. These alterations were associated with transcriptome remodeling, revealing changes in gene expression related to inflammation, innate immunity, cell structure, adhesion, and motility. Surprisingly, chronic TNF exposure did not affect the total cell number in the 3D pHA cultures, suggesting activation of compensatory mechanisms that promote cell survival or prevent cell death, including senescence. The study also identified the unique upregulation of AIM2, an inflammasome sensor, and hypothesized that inhibiting AIM2 could mitigate TNF-induced inflammation. Treatment with LL-37, an AIM2 inhibitor, effectively attenuated TNF-induced upregulation of various cytokines and chemokines, as well as components of the NFKB pathway. Additionally, replenishing TNF-downregulated proteins showed some efficacy in mitigating TNF's impact, although their overall ability to restore expression of a select set of genes to untreated control levels was limited.
In conclusion, this dissertation provides compelling evidence of the detrimental role of TNF-induced chronic neuroinflammation on hNSC plasticity during fetal brain development, offering insights into the underlying molecular mechanisms. The study's findings also reveal potential therapeutic targets, such as AIM2 and TNF-downregulated proteins, for mitigating the adverse effects of neuroinflammation on brain development and function.:COVER PAGE I
SIGNATURE PAGE II
DEDICATION III
ACKNOWLEDGEMENTS IV
SUMMARY VI
ZUSAMMENFASSUNG VII
TABLE OF CONTENTS IX
LIST OF PUBLICATIONS XII
LIST OF ABBREVIATIONS XIII
LIST OF TABLES XIX
LIST OF FIGURES XX
LIST OF SUPPLEMENTARY FIGURES XXI
1 INTRODUCTION 22
1.1 Human Neural Stem Cells in Neurodevelopment 22
1.1.1 Neurogenesis 22
1.1.2 Gliogenesis 25
1.2 Plasticity in Human Neurodevelopment 26
1.3 Neuroinflammation in The Brain Tissue 27
1.3.1 Neuroinflammation Spectrum: Acute and Chronic States 27
1.4 TNF as a Mediator of Neuroinflammation 29
1.5 Mechanisms of TNF Signaling 31
1.5.1 TNF-TNFR1 Signaling through Complex I, IIa, IIb and IIc 31
1.5.2 TNF-TNFR2 Signaling 33
1.6 TNF in Neurodevelopmental Disorders 33
1.6.1 Effect of TNF-mediated Neuroinflammation on Fetal and Adult Neural Stem Cells 37
1.7 TNF and Inflammasome Link in Neuroinflammation Context 39
2 AIMS AND OBJECTIVES OF THE STUDY 43
3 MATERIALS AND METHODS 44
3.1 Main Materials and Resources 44
3.2 Medium and Solutions 47
3.3 Software 48
3.4 Methods 48
3.4.1 Primary Human Fetal Astrocyte Culture 48
3.4.2 Cell Encapsulation 48
3.4.3 Cell Culture Treatments 49
3.4.3.1 TNF, R9-LL-37 or Combination Treatments 49
3.4.3.2 Pooled Protein Treatments 49
3.4.4 Immunofluorescence 50
3.4.5 Microscope Imaging 51
3.4.5.1 Leica TCS SP5 MP Microscope Imaging 51
3.4.5.2 Zeiss Axio Observer.Z1/7 Spinning Disk Confocal Microscope Imaging 51
3.4.6 Image Processing and Quantification 52
3.4.7 RNAseq and Data Analysis 52
3.4.8 Quantitative RT-PCR (qRT-PCR) 53
3.4.9 Synthesis of R9-LL-37 54
3.4.10 Protein Arrays 56
3.4.10.1 Human Cytokine Proteome Profiling 57
3.4.10.2 Human NFKB Pathway Proteome Profiling 61
3.4.11 Densitometric Analysis 63
4 RESULTS 64
4.1 Transcriptome Remodeling and Morphological Alterations are Triggered in hNSC by Chronic TNF Exposure 64
4.2 Chronic TNF exposure induces a strong inflammatory and innate immune response in hNSC 70
4.3 Plasticity and Neurogenic Potential in hNSC are Suppressed by Chronic TNF Exposure 74
4.4 TNF Effect Can Be Alleviated by Inhibiting AIM2 in hNSC 81
4.4.1 TNF Effects Demonstrate Time- and Dose-Dependent Dynamics 81
4.4.2 AIM2 Inhibition Mitigates TNF-Induced Molecular Alterations 84
4.5 Targeted Protein Pooling Can Mitigate TNF-Induced Gene Downregulation in hNSC 91
5 DISCUSSION 96
6 CONCLUSION 105
7 FUTURE DIRECTIONS 107
8 REFERENCES 110
9 SUPPLEMENTARY INFORMATION 129
9.1 Senescence and Quiescence Genes 129
9.2 Ferroptosis Signaling 130
9.3 Effects of AIM2 Inhibition on Cytokine and Chemokine Signaling 131
9.4 Effects of AIM2 Inhibition on NFKB Signaling 140
9.5 Pooled Protein Treatments 144
10 DECLARATIONS 147
Annex 1 147
Annex 2 148Neuroinflammation ist ein kritischer ursächlicher und/oder verschlimmernder Faktor bei Krankheiten, körperlichen Traumata, Schlaganfällen und Alterung und stört die Homöostase des Gehirns. Während der fötalen Neuroentwicklung ist Neuroinflammation besonders bedenklich da sie die Plastizität menschlicher neuronaler Stammzellen (hNSCs) beeinträchtigen kann, welche die verschiedenen neuronalen und makroglialen Zellen des zentralen Nervensystems (ZNS) erzeugen. Diese Störung kann zu lang anhaltenden oder dauerhaften Auswirkungen auf die Gehirnfunktion führen, was die Bedeutung von Fachwissen unterstreicht, wie Neuroinflammation das neurogene Potenzial von hNSCs behindert.
Die chronische Aussetzung gegenüber Tumornekrosefaktor (TNF), einem entscheidenden neuroinflammatorischen Zytokin, ist mit neurologischen Entwicklungsstörungen in Verbindung gebracht worden. Seine Auswirkungen auf die Plastizität fetaler hNSCs während der Schwangerschaft bleiben jedoch unklar. Diese Studie untersuchte die Auswirkungen einer anhaltenden pränatalen Neuroinflammation auf hNSCs, indem 3D-StarPEG-Heparin-Hydrogelkulturen primärer menschlicher fötaler Astrozyten (3D pHA) chronischen Konzentrationen von TNF ausgesetzt wurden. Die Studie ergab, dass chronische Aussetzung gegenüber TNF signifikante morphologische Veränderungen in neuronalen Netzwerken auslöste, die durch reduzierte Verzweigung und längere verbundene Pfade sowie Zellen mit verlängerten Nuklei gekennzeichnet waren. Die TNF-Exposition führte zu unterdrückter Stammzellplastizität und unterdrücktem neurogenen Potenzial, was durch eine dramatische Verringerung der GFAP+ und GFAP+/SOX2+ Zellpopulationen belegt wurde. Diese Veränderungen waren mit einer Umgestaltung des Transkriptoms verbunden und offenbarten Veränderungen in der Genexpression im Zusammenhang mit Entzündungen, angeborener Immunität, Zellstruktur, Adhäsion und Motilität. Überraschenderweise hatte die chronische TNF-Exposition keinen Einfluss auf die Gesamtzellzahl in den 3D pHA-Kulturen, was auf die Aktivierung von Kompensationsmechanismen hindeutet, die das Zellüberleben fördern oder Zelltod, einschließlich Seneszenz, verhindern. Die Studie identifizierte außerdem die einzigartige Hochregulierung von AIM2, einem Inflammasom-Sensor, und stellte die Hypothese auf, dass die Hemmung von AIM2 die durch TNF induzierte Entzündung lindern könnte. Die Behandlung mit LL-37, einem AIM2-Hemmer, schwächte die durch TNF induzierte Hochregulierung verschiedener Zytokine und Chemokine sowie Komponenten des NFKB-Signalwegs wirksam ab. Darüber hinaus zeigte die Wiederaufstockung herunterregulierter TNF-Proteine eine gewisse Wirksamkeit bei der Abschwächung der Auswirkungen von TNF, obwohl ihre allgemeine Fähigkeit, die Expression einer ausgewählten Gruppe von Genen auf unbehandelte Kontrollmengen wiederherzustellen, begrenzt war.
Zusammenfassend lässt sich sagen, dass diese Dissertation wissenschaftlich signifikante Nachweise für die nachteilige Rolle der durch TNF induzierten chronischen Neuroinflammation auf die Plastizität von hNSC während der fötalen Gehirnentwicklung liefert und Einblicke in die zugrunde liegenden molekularen Mechanismen bietet. Die Ergebnisse der Studie enthüllen außerdem potenzielle therapeutische Targets wie AIM2 und herunterregulierte TNF-Proteine zur Abschwächung der nachteiligen Auswirkungen der Neuroinflammation auf die Entwicklung und Funktion des Gehirns.:COVER PAGE I
SIGNATURE PAGE II
DEDICATION III
ACKNOWLEDGEMENTS IV
SUMMARY VI
ZUSAMMENFASSUNG VII
TABLE OF CONTENTS IX
LIST OF PUBLICATIONS XII
LIST OF ABBREVIATIONS XIII
LIST OF TABLES XIX
LIST OF FIGURES XX
LIST OF SUPPLEMENTARY FIGURES XXI
1 INTRODUCTION 22
1.1 Human Neural Stem Cells in Neurodevelopment 22
1.1.1 Neurogenesis 22
1.1.2 Gliogenesis 25
1.2 Plasticity in Human Neurodevelopment 26
1.3 Neuroinflammation in The Brain Tissue 27
1.3.1 Neuroinflammation Spectrum: Acute and Chronic States 27
1.4 TNF as a Mediator of Neuroinflammation 29
1.5 Mechanisms of TNF Signaling 31
1.5.1 TNF-TNFR1 Signaling through Complex I, IIa, IIb and IIc 31
1.5.2 TNF-TNFR2 Signaling 33
1.6 TNF in Neurodevelopmental Disorders 33
1.6.1 Effect of TNF-mediated Neuroinflammation on Fetal and Adult Neural Stem Cells 37
1.7 TNF and Inflammasome Link in Neuroinflammation Context 39
2 AIMS AND OBJECTIVES OF THE STUDY 43
3 MATERIALS AND METHODS 44
3.1 Main Materials and Resources 44
3.2 Medium and Solutions 47
3.3 Software 48
3.4 Methods 48
3.4.1 Primary Human Fetal Astrocyte Culture 48
3.4.2 Cell Encapsulation 48
3.4.3 Cell Culture Treatments 49
3.4.3.1 TNF, R9-LL-37 or Combination Treatments 49
3.4.3.2 Pooled Protein Treatments 49
3.4.4 Immunofluorescence 50
3.4.5 Microscope Imaging 51
3.4.5.1 Leica TCS SP5 MP Microscope Imaging 51
3.4.5.2 Zeiss Axio Observer.Z1/7 Spinning Disk Confocal Microscope Imaging 51
3.4.6 Image Processing and Quantification 52
3.4.7 RNAseq and Data Analysis 52
3.4.8 Quantitative RT-PCR (qRT-PCR) 53
3.4.9 Synthesis of R9-LL-37 54
3.4.10 Protein Arrays 56
3.4.10.1 Human Cytokine Proteome Profiling 57
3.4.10.2 Human NFKB Pathway Proteome Profiling 61
3.4.11 Densitometric Analysis 63
4 RESULTS 64
4.1 Transcriptome Remodeling and Morphological Alterations are Triggered in hNSC by Chronic TNF Exposure 64
4.2 Chronic TNF exposure induces a strong inflammatory and innate immune response in hNSC 70
4.3 Plasticity and Neurogenic Potential in hNSC are Suppressed by Chronic TNF Exposure 74
4.4 TNF Effect Can Be Alleviated by Inhibiting AIM2 in hNSC 81
4.4.1 TNF Effects Demonstrate Time- and Dose-Dependent Dynamics 81
4.4.2 AIM2 Inhibition Mitigates TNF-Induced Molecular Alterations 84
4.5 Targeted Protein Pooling Can Mitigate TNF-Induced Gene Downregulation in hNSC 91
5 DISCUSSION 96
6 CONCLUSION 105
7 FUTURE DIRECTIONS 107
8 REFERENCES 110
9 SUPPLEMENTARY INFORMATION 129
9.1 Senescence and Quiescence Genes 129
9.2 Ferroptosis Signaling 130
9.3 Effects of AIM2 Inhibition on Cytokine and Chemokine Signaling 131
9.4 Effects of AIM2 Inhibition on NFKB Signaling 140
9.5 Pooled Protein Treatments 144
10 DECLARATIONS 147
Annex 1 147
Annex 2 14
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Effects of stress hormones on multimodal measures of brain and cognition in older adults
No full textBackground
As a consequence of demographic change, rising incidences of neurodegenerative diseases such as Alzheimer’s disease pose a major challenge for modern medicine. While struggling to find disease-preventing therapies, the possibility of preventing or delaying the onset of dementia through modifiable lifestyle factors shifts into focus of applied biomedical research. Chronic stress is one such proposed risk factor and indeed, dysregulation of stress-related pathways have been linked to progression of Alzheimer’s disease dementia (for review see Machado et al. 2014).
Research questions
The first aim of this thesis was to assess links between blood-based cortisol, hippocampal volume, and risk of clinical progression to Alzheimer’s disease over time.
The second aim was to cross-sectionally investigate the effects of circulating, blood-based stress hormone levels, namely norepinephrine, epinephrine, cortisol, and dehydroepiandrosterone sulfate, on multimodal measures of brain health, including brain region volumes, glucose metabolism, and perfusion, as well as cognitive function.
Methods
For the longitudinal study, 304 older adults with amnestic mild cognitive impairment were selected from the database of the Alzheimer’s disease neuroimaging initiative, based on availability of plasma cortisol at baseline and hippocampal volume measures, assessed at baseline and during follow-up visits over a study period of up to 100 months. The effect of baseline plasma cortisol, stratified into low- or high-level groups, on change in hippocampal volume over time (i.e. hippocampal atrophy) was evaluated using linear mixed effects models. The effects of baseline hippocampal volume (stratified into large or small) and plasma cortisol (high/low) on the risk of clinical progression to Alzheimer’s disease dementia was evaluated through survival analyses using Cox proportional hazards models and the
Kaplan-Meier-estimator. Risk groups were defined for all four possible combinations of hippocampal volume (large/small) and cortisol levels (high/low) and the low-risk group large hippocampal volume/low cortisol level was defined as reference group.
For the cross-sectional study, data from 132 cognitively unimpaired older adults from the AgeWell randomized controlled trial were analysed. Stress hormone levels were obtained from overnight fasting morning blood serum (cortisol, dehydroepiandrosterone sulfate) and blood plasma (epinephrine, norepinephrine) samples. Brain regions vulnerable to AD were investigated using multimodal imaging measures: T1 magnetic resonance imaging was used to extract brain region volumes (anterior cingulate cortex, insula, hippocampus),
Fluoro-deoxyglucose positron emission tomography was used to measure glucose metabolism (anterior cingulate cortex, Insula, posterior cingulate cortex, Precuneus), and Florbetapir positron emission tomography was used to measure perfusion (anterior cingulate cortex, Insula, posterior cingulate cortex, Precuneus) and neocortical amyloid deposition. Cognitive function was evaluated using composite scores for verbal episodic memory, short-term memory, and executive function, as well as the Preclinical Alzheimer’s Cognitive Composite 5. Associations between stress biomarkers and indicators for brain health and cognition were assessed in the total sample, in the interaction with sex, and in sex-stratified subsamples.
Results
The longitudinal data analyses within the cohort selected from the Alzheimer’s disease neuroimaging initiative yielded the following results:
Faster decline in hippocampal volume was observed in the high cortisol group, when compared to the low cortisol group. This interaction between baseline plasma cortisol and time on decline in hippocampal volume over time was significant in both unadjusted and adjusted models. Baseline hippocampal volume alone did not predict hippocampal atrophy. When reducing the sample to those participants with a positive cerebrospinal fluid signature for Alzheimer’s disease (amyloid β+/t-tau+), the interaction effect of cortisol and time on hippocampal atrophy was maintained. There were no significant effects of cortisol observed on the volumes of fusiform, lateral and medial orbitofrontal, and middle temporal brain regions. When visualizing the data using the Kaplan-Meier-estimator and forest plots it could be observed that compared to the low risk reference group, all other combination groups of hippocampal volume and cortisol levels showed elevated risks of clinical progression to Alzheimer’s disease dementia. Cox proportional hazards models revealed that small baseline hippocampal volume predicted a higher risk of clinical progression. A similar effect was not found for baseline plasma cortisol alone. Also, the interaction between hippocampal volume and plasma cortisol showed no significant effect on clinical progression to Alzheimer’s disease dementia.
Analyses of the cross-sectional data from the AgeWell randomized controlled trial yielded the following results:
In the total sample, higher stress biomarker levels were not associated with volumes of any of the selected brain regions of interest, after adjusting for covariates. The interaction between epinephrine and sex showed an effect on the anterior cingulate cortex volume, while the interaction between norepinephrine and sex showed an effect on hippocampal volume.
Post-hoc within-group analysis confirmed that higher epinephrine levels were related to larger anterior cingulate cortex volume in women, but not men. In the total sample, higher epinephrine levels were significantly associated with lower glucose metabolism in the anterior and posterior cingulate cortex and the Precuneus. In the total sample, both higher epinephrine levels, as well as higher DHEAS/cortisol ratios were associated with lower cerebral perfusion in the posterior cingulate cortex. The interaction between norepinephrine and sex showed an effect on perfusion in the Precuneus. There were no significant associations found between stress biomarkers and neocortical amyloid deposition in neither the total sample, nor the subgroups. In the total sample, levels of stress biomarkers were not significantly associated with any of the cognitive composite scores. The interaction between cortisol and sex showed effects on verbal episodic memory and post-hoc within-group analysis confirmed that higher cortisol levels were associated with lower verbal episodic memory, as well as with lower Preclinical Alzheimer’s Cognitive Composite 5 scores in men, but not women.
Conclusions
The findings of this study suggest that regulation of the sympathetic-adrenal-medullary and hypothalamic-pituitary-adrenal axes as the two main stress-related pathways, may increase resilience in healthy older adults and might be protective against neural degradation and clinical progression in prodromal stages of Alzheimer’s disease. Also, stress-related reactions vary between men and women. For a possible decrease in vulnerability to adverse effects of stress reactions, regulation through risk-modifying lifestyle interventions need to be targeted and happen as early as possible before the onset of cognitive decline.:TABLE OF CONTENTS
Acknowledgements 4
Personal acknowledgements 4
Study-related acknowledgements 5
Preliminary remarks 7
Data availability statements 8
List of Abbreviations 9
List of Figures 10
List of Tables 12
1 Introduction 13
1.1 Motivation, identification of research gap, and outline 13
1.2 Current state of knowledge 15
1.2.1 Alzheimer’s disease 15
The phase of mild cognitive impairment 16
Risk factors for the development of Alzheimer’s disease 17
1.2.2 ”Fight or Flight” – the stress response system 18
Sympathetic adrenal medullary axis 18
Hypothalamic pituitary adrenal axis 20
1.2.3 What stress can do to brain and cognition 22
Stress and brain health 23
Stress and cognition 24
1.2.4 Sex specificities to stress reactions 25
1.3 The present study 26
1.3.1 Alzheimer’s disease neuroimaging initiative (ADNI) 27
1.3.2 Age-Well (Medit-Ageing) 27
1.3.3 Aims and Hypotheses 28
2 Methods 30
2.1 Study data 30
2.1.1 Criteria for inclusion/exclusion 30
2.1.2 Evaluations 31
2.1.3 Study participants 32
2.1.4 Consent statements and standard protocol approvals 35
2.2 Data Acquisition and Assessment 35
2.2.1 Acquisition and analysis of stress biomarkers 35
2.2.2 Acquisition and analysis of neuroimaging data 36
2.2.3 Acquisition and analysis of CSF Aβ1−42 and t-tau levels 37
2.2.4 Assessment of cognitive scores 38
2.2.5 Assessment of additional variables 39
2.3 Statistical analyses 40
2.3.1 Sample characteristics 41
2.3.2 Subsample with AD-positive CFS signature 41
2.3.3 Linear mixed effects analysis 42
2.3.4 Survival analysis 42
2.3.5 Mediation and moderation effect analysis 43
2.3.6 Multiple linear regression analysis 44
2.3.7 Adjustment for covariates 44
3 Results 45
3.1 Effects of stress hormones in mild cognitive impairment 45
3.1.1 ADNI sample baseline characteristics 45
3.1.2 Plasma cortisol and brain region volumes over time 48
3.1.3 Plasma cortisol, hippocampal volume, and clinical progression 52
3.1.4 Mediation and moderation effects 55
3.2 Effects of stress hormones in healthy older adults 56
3.2.1 Age-Well sample characteristics 56
3.2.2 Associations between stress biomarkers and brain health 58
Brain region volumes 58
Cerebral glucose metabolism 61
Cerebral perfusion 64
Amyloid deposition 67
3.2.3 Associations between stress biomarkers and cognitive function 69
4 Discussion 73
4.1 Introductory summary 73
4.2 Stress in mild cognitive impairment 74
4.2.1 Cortisol and brain region atrophies 74
4.2.2 Cortisol, hippocampal volume and disease progression 75
4.2.3 Summarising remarks 76
4.3 Stress in healthy older adults 76
4.3.1 Stress and brain health 76
4.3.2 Sex specificities in stress-related associations 78
4.3.3 Null results 81
4.3.4 Summarising remarks 82
4.4 Strengths and limitations 82
4.5 Conclusions 84
5 Summary 85
6 Zusammenfassung 88
Bibliography 92
Appendix 121
Declaration
Altersabhängige Neurogenese im Gyrus Dentatus unter dem Einfluss des Chemokinrezeptors CXCR4
No full textWährend der adulten Neurogenese entstehen Körnerzellprogenitoren, welche in die Subgranularzone des Gyrus dentatus (GD) einwandern und sich daraufhin im inneren Drittel der Körnerzellschicht (KZS) differenzieren. Im Hilus gealterter Mäuse, sowie nach experimentellem Schlaganfall und Epilepsie, konnte beobachtet werden, dass dieser Prozess nicht ordnungsgemäß funktioniert, sondern mit einer ektopen Platzierung neu gebildeter Granularzellen einhergeht. Die Signalwege und Faktoren, welche die Integration neu gebildeter Granularzellen unter physiologischen Bedingungen regulieren, sind jedoch weitestgehend unerforscht. Aufgrund der hohen Expression des Chemokins SDF-1 in der KZS des adulten GD und dessen führende Rolle bei der neuronalen Migration im embryonalen Gehirn, wurde in der vorliegenden Arbeit die Funktion des Chemokins SDF-1 und dessen Rezeptor CXCR4 in der adulten Neurogenese analysiert. Um die Bedeutung des CXCR4-Rezeptors für die postnatale und adulte Neurogenese zu untersuchen, wurden mausgenetischen Modelle verwendet, bei denen Cxcr4 konditional durch Glast-CreER ausgeschaltet wurde. Insbesondere zeigten Mutanten 50 Tage nach Cxcr4-Ablation im reifen Gyrus dentatus eine reduzierte Dichte von DCX+-Zellkörpern und Dendriten, sowie eine reduzierte Dichte von Sox2+- und NeuroD+-neuronalen Vorläuferzellen. Viele unreife Neurone wurden ektop im Hilus und der inneren Molekularschicht nachgewiesen und zeigten zum Teil einen anomal entwickelten Dendritenbaum. Die Beobachtung, dass sich in adulten Mutanten gebildete Neuro-blasten stark zerstreut im GD ansiedeln, legt nahe, dass die Migration der Zellen in Richtung des inneren Teils der KZS durch die SDF-1/CXCR4-Achse gesteuert wird. Nur wenige fehlplatzierte Zellen überlebten dauerhaft als ektope Neurone. Diese Ergebnisse zeigen, dass das CXCR4-signaling den Stammzellpool im GD aufrecht erhält und das innere Drittel der KZS als Differenzierungsbereich für unreife Körnerzellen vorgibt. Des Weiteren zeigten Ergebnisse mit Veränderung der Haltungsbedingungen, dass die Haltung in einer reizreichen Umgebung die Anzahl der Vorläuferzellen im Hippokampus von adulten Mäusen erhöht, wodurch die Neurogenesereduktion nach Cxcr4-Deletion kompensiert werden kann und es zu einer besseren Differenzierung und Integration postmitotischer Zellen kommt. Alles in allem stellen die gewonnen Erkenntnisse einen weiteren Schritt zum Verständnis der Faktoren und Regulationsmechanismen, welche die adulte Neurogenese in vivo kontrollieren, dar
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Neural Precursor Cells in Culture: Taking a Closer Look
Full text linkGene mit gerigem Einfluss auf einen untersuchten Phänotyp können durch den Ein- schluss einer genetischen Variation im Tierversuch untersucht werden. Adulte Neuro- genese, der Prozess der Neubildung und Integration von funktionellen Neuronen in das existierende neurale Netzwerk, wird von vielen solchen Genen mit geringem Effekt beeinflusst. All diese Gene im lebenden Tier zu untersuchen wäre mit einem hohen Arbeitsaufwand verbunden, und würde hohe Tierzahlen erfordern. Bereits publizierte Ergebnisse zeigen, dass diese Gene auch in der Zellkultur unter Verwendung von Zelllinien genetisch rekombinanter Tiere untersucht werden können (Kannan et al., 2016). Die hier verwendeten, ingezüchteten Mausstämme des so genannten BXD Panels stellen die Nachkommen der Kreuzung der beiden Mausstämme C57BL/6J und DBA/2J dar (Peirce et al., 2004), die sich in der Ausprägung von unterschiedlichen Neurogenese bezogenen Phänotypen bereits deutlich unterscheiden (Kempermann et al., 2006). Durch die Verwendung der BXD Tiere wird hierbei die Aussagekraft der genetischen Variation mit dem Zellkultursystem verbunden. Die Aussagekraft dieser Studie ist jedoch darin limitiert, dass aufgrund des verwendeten Protokolls nur eine Zelllinie pro Mausstamm generiert werden konnte. Daher präsentiere ich hier ein neues Protokoll welches es erlaubt eine Zelllinie aus nur einem einzelnen Tier zu generieren. Diese Methode kombiniert zwei bestehende Zellkultursysteme, die Neurosphärenkultur und die Monolayerkultur. Es stellte sich heraus, dass die Überlebensrate der einzelnen Zelllinien vom biologischen Hintergrund der Zellen beeinflusst wird. So ist die Überlebensrate von Zellen der DBA/2J Mäuse deutlich schlechter als die der C57BL/6J oder die der F1 Generation aus der Verpaarung der beiden Stämme. Es zeigte sich allerdings, dass diese Überlebensrate nicht ausschließlich von der vorhandenen Anzahl proliferierender Zellen abhängt, da B6D2F1 (F1 Generation mit einem C57BL/6J Muttertier) signifikant weniger proliferierende (Ki67 positive) Zellen in vivo aufweisen, jedoch keine geringere Überlebensrate der Zelllinien haben. Eine hoch standardisierte, umfangreiche Analyse der Zelllinien aller vier Mausstämme (C57BL/6J, DBA/2J, und die zwei reziproken F1 Nachkommen BDF1 und DBF1) zeigte eine hohe Varianz innerhalb genetisch identischer Linien, was die Be- stimmung eines Effektes, der durch den genetischen Hintergrund der Linien verursacht wird, beeinträchtigte. Die Zelllinien werden signifikant von äußeren Faktoren beeinflusst, wie z.B. durch das Einfrieren der Zellen. Dies gibt Hinweise darauf, dass Untersuchungen in der Zellkultur genau geplant, kritisch hinterfragt, sowie möglichst alle potentiellen Einflussfaktoren gleich gehalten werden müssen. Nur so können valide, aussagekräftige Ergebnisse mit der Zellkultur gewonnen werden. Automatische Zellkultursysteme, neue Mikroskopieverfahren, sowie besser definierte Langzeitstudien werden unser Verständnis von Zellen in der Zellkultur deutlich verbessern und dabei ihren Wert, sowie bestehende Limitationen, endgültig klären.:List of Figures I
List of Tables II
List of Abbreviations III
List of Publications V
1. Introduction 1
1.1 Genetic variation in animal research 2
Recombinant inbred strains 3
The BXD panel 4
The Gene Network 5
Genetic modifications 5 \u2028
1.2 Adult hippocampal neurogenesis 6
History 7
Clinical relevance 8
The BXD panel and adult hippocampal neurogenesis 9 \u2028
1.3 Developmental stages of neural precursor cells 9 \u2028
1.4 Studying adult neurogenesis in vitro 11
Culturing hippocampal precursor cells 11
A mouse cell culture genetic reference panel 13 \u2028
1.5 Tracking 13 \u2028
1.6 Objectives 15 \u2028
2. Materials and Methods 16
2.1 Components and equipment 16 \u2028
2.2 Antibodies 20 \u2028
2.3 Recipes 21 \u2028
General buffers and solutions 21
Cell culture solutions 21
Immunocytochemistry solutions 23
Immunohistochemistry solutions 24 \u2028
2.4 Experimental animals 25 \u2028
2.5 Cell culture 25
Coating of cell culture vessels 25
Fire-polished pipettes 25
Dentate gyrus isolation 26
Neurosphere assay 26
Monolayer culture 27 \u2028
2.6 Immunocytochemistry 29
BrdU staining preparations 29
Staining protocol 30
Imaging and counting 30 \u2028
2.7 Immunohistochemistry 30
Sample preparation 30
Staining protocol 31
Cell counting 31 \u2028
2.8 Tracking 32
Cell preparation and imaging setup 34
Image processing 35
Data analysis 35 \u2028
2.9 Generation of CRISPR/Cas mediated knock-out lines 36
Construct design and cloning 36
E. coli Top10 transformation and plasmid isolation 37
Transfection of neural precursor cells and expansion of knock-out lines 38
Genotyping of the generated cell lines 39
Agarose gel electrophoresis 40 \u2028
2.10 Statistical analysis 40
2.11 Data visualization 40
3. Results 41
3.1 Single animal monolayer cultures41
The three phenotypes of the neurosphere assay 44
Neurosphere assay phenotypes could not predict the survival of a cell line 45
The genetic background had an influence on all three phenotypes of the neurosphere assay 46
Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48
BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 \u2028
3.2 A new cell line phenotyping standard operation procedure and its application 50
Line generation data 52
Marker staining 54
Cell tracking 55 \u2028
3.3 Cell culture – a system with limitations 59
Freezing effect 60
Cell culture data - technical variance hinders the analysis of small effects 62
3.4 Migration speed and GFAP 63
The strength of the BXD panel – cumulative data 65 \u2028
3.5 Other applications of the tracking procedure 68 \u2028
Tracking labeled cells in an embryonic zebrafish xenograft model 68
Cell tracking in mouse retina explants 68
4. Discussion 70
4.1 Single animal monolayer cultures – a new protocol 70 \u2028
4.2 A new phenotyping pipeline 74 \u2028
4.3 Semi-automated (user-supervised) cell tracking 77 \u2028
4.4 A possible correlation between migration speed and differentiation 79
4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82
4.6 The problem of the validity of cell culture experiments - a comment 83
4.7 Conclusion 84 \u2028
Bibliography 88
A Single animal cell line generation protocol 106 \u2028
B Cell line characterization SOP 112 \u2028
C R Scripts 117 \u2028Uncovering gene loci that assert only small effects onto a phenotype of interest, can be achieved by including genetic variation in animal research. Adult hippocampal neurogenesis, the process of the formation of new neurons and their functional integration into existing circuitry, is influenced by a broad range of such small effect genes. Analyzing all of these genes in vivo would be laborious and require a high number of animals. Previously published data merged the power of genetic variation with a cell culture system by using cell lines generated from the BXD recombinant inbred mouse strains (Kannan et al., 2016). These strains are inbred progeny of F2 crosses originating from the two mouse strains C57BL/6J and DBA/2J (Peirce et al., 2004), which already differ quite extensively in neurogenesis related phenotypes (Kempermann et al., 2006). As previous studies were limited by the number of strains that could be generated due to the demand for high numbers of animals, I developed a new method that allows the generation of a cell line from one single animal. For this new method, I combined the neurosphere culture with a subsequent monolayer culture. The survival of the resulting cell lines, is thereby greatly influenced by the genetic background. The survival rate of cell lines derived from DBA/2J animals is much lower as compared to C57BL/6J-derived lines or lines from the F1 generation of crossing the two strains. Whether or not a cell line survived did not seem to be solely influenced by the number of proliferating cells in vivo, as B6D2F1 (F1 progeny with a C57BL/6J mother) showed significantly less proliferative (Ki67 positive) cells in vivo while exhibiting a survival rate that exceeded both parental strains. An extensive study of the cell lines gained from all four mouse strains (C57BL/6J, DBA/2J, and the two reciprocal F1 progeny B6D2F1 and D2B6F1) in a highly standardized manner showed that the individual difference between single cell lines was rather high, hampering the successful detection of in-between strain differences. The standardized characterization of the generated cell lines, further allowed the identification of external factors, influencing the cells, as for example the freezing of the cells. This indicates that cell culture experiments need to be thoroughly planned and critically scrutinized, while all external factors should be kept as constant as possible to ensure the validity of the resulting data. Automated cell handling, new imaging technologies, as well as more defined long-term studies will greatly improve the understanding of cells in culture and thereby show their true values and limitations.:List of Figures I
List of Tables II
List of Abbreviations III
List of Publications V
1. Introduction 1
1.1 Genetic variation in animal research 2
Recombinant inbred strains 3
The BXD panel 4
The Gene Network 5
Genetic modifications 5 \u2028
1.2 Adult hippocampal neurogenesis 6
History 7
Clinical relevance 8
The BXD panel and adult hippocampal neurogenesis 9 \u2028
1.3 Developmental stages of neural precursor cells 9 \u2028
1.4 Studying adult neurogenesis in vitro 11
Culturing hippocampal precursor cells 11
A mouse cell culture genetic reference panel 13 \u2028
1.5 Tracking 13 \u2028
1.6 Objectives 15 \u2028
2. Materials and Methods 16
2.1 Components and equipment 16 \u2028
2.2 Antibodies 20 \u2028
2.3 Recipes 21 \u2028
General buffers and solutions 21
Cell culture solutions 21
Immunocytochemistry solutions 23
Immunohistochemistry solutions 24 \u2028
2.4 Experimental animals 25 \u2028
2.5 Cell culture 25
Coating of cell culture vessels 25
Fire-polished pipettes 25
Dentate gyrus isolation 26
Neurosphere assay 26
Monolayer culture 27 \u2028
2.6 Immunocytochemistry 29
BrdU staining preparations 29
Staining protocol 30
Imaging and counting 30 \u2028
2.7 Immunohistochemistry 30
Sample preparation 30
Staining protocol 31
Cell counting 31 \u2028
2.8 Tracking 32
Cell preparation and imaging setup 34
Image processing 35
Data analysis 35 \u2028
2.9 Generation of CRISPR/Cas mediated knock-out lines 36
Construct design and cloning 36
E. coli Top10 transformation and plasmid isolation 37
Transfection of neural precursor cells and expansion of knock-out lines 38
Genotyping of the generated cell lines 39
Agarose gel electrophoresis 40 \u2028
2.10 Statistical analysis 40
2.11 Data visualization 40
3. Results 41
3.1 Single animal monolayer cultures41
The three phenotypes of the neurosphere assay 44
Neurosphere assay phenotypes could not predict the survival of a cell line 45
The genetic background had an influence on all three phenotypes of the neurosphere assay 46
Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48
BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 \u2028
3.2 A new cell line phenotyping standard operation procedure and its application 50
Line generation data 52
Marker staining 54
Cell tracking 55 \u2028
3.3 Cell culture – a system with limitations 59
Freezing effect 60
Cell culture data - technical variance hinders the analysis of small effects 62
3.4 Migration speed and GFAP 63
The strength of the BXD panel – cumulative data 65 \u2028
3.5 Other applications of the tracking procedure 68 \u2028
Tracking labeled cells in an embryonic zebrafish xenograft model 68
Cell tracking in mouse retina explants 68
4. Discussion 70
4.1 Single animal monolayer cultures – a new protocol 70 \u2028
4.2 A new phenotyping pipeline 74 \u2028
4.3 Semi-automated (user-supervised) cell tracking 77 \u2028
4.4 A possible correlation between migration speed and differentiation 79
4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82
4.6 The problem of the validity of cell culture experiments - a comment 83
4.7 Conclusion 84 \u2028
Bibliography 88
A Single animal cell line generation protocol 106 \u2028
B Cell line characterization SOP 112 \u2028
C R Scripts 117 \u202
Analysis of zebrafish Lrrk2 loss-of-function during brain development and adult brain regeneration
No full textThe neurodegenerative disorder Parkinson's Disease (PD) represents both a major socioeco-nomic challenge and an individual burden for many patients. Despite major efforts, neither satisfactory explanations of the pathogenesis of PD, nor disease-modifying drugs have been developed to date. Mutations of the multidomain kinase LRRK2 represent the overall most common cause of he-reditary PD. Furthermore, LRRK2 mutations have been linked to dysregulations of the immune system such as inflammatory bowel disease, cancer, or the susceptibility towards mycobacte-rial infections. Several pathogenic point mutations have been identified that – directly or indi-rectly – lead to a pathological gain-of-function of the protein’s kinase domain. Despite recent advances, the physiological functions of LRRK2, as well as the underlying processes of LRRK2-mediated pathologies, remain largely unknown. Much research effort has aimed at generating reliable animal models for the study of LRRK2. Nevertheless, neither loss-of-function of the gene, nor overexpression of normal or mutant LRRK2, has yielded definitive results. Previous work from our research group on zebrafish (Danio rerio) has generated two genetic lrrk2 knock-out lines using different mutagenesis strat-egies: lrrk2TILLING and lrrk2CRISPR (Ahrendt, 2011; Suzzi, 2017). These studies’ results were par-tially contradictory, and the described phenotypes were not stable. Whilst this previous work investigated lrrk2 loss-of-function, so far, no genetic knock-in line carrying one of the multiple known pathogenic lrrk2 mutations, has been reported in zebrafish. Therefore, this work aimed to further investigate the effects of Lrrk2-deficiency in zebrafish and to establish a genetic knock-in of the common pathogenic G2019S substitution to model the genotype of PD patients more accurately. A variety of methods was applied to achieve these aims. Immunohistochemistry and conventional histology studies were performed on zebrafish brains and kidneys at different developmental stages, both under physiological conditions and following the induction of brain regeneration. Since the zebrafish’s neuroregenerative capabil-ity is closely linked to an initial neuroinflammation, and previous studies on lrrk2 knock-out zebrafish have suggested an impaired immune response and reduced brain regeneration, the neuroinflammation and neuroregeneration of adult lrrk2TILLING zebrafish were investigated by inducing a telencephalic stab-lesion and a subsequent BrdU-pulse-chase analysis. To investi-gate functional effects of the gene knock-out, a set of behavioral experiments was performed. Using CRISPR/Cas9 genome editing, the basis for a knock-in of the G2019S substitution was established. Immunohistochemistry analyses of larval and adult zebrafish brains were performed in a set of experiments. By quantifying all mitotic cells in larval brains at different time points, the basal brain proliferation levels during development were analyzed, as well as the levels of constitutive neurogenesis during adulthood. Under physiological conditions, basal brain prolif-eration was found unimpaired in Lrrk2-deficient zebrafish. Similarly, the number of microglia found in the telencephalon of Lrrk2-deficient zebrafish was not reduced under physiological conditions, although one experimental group showed signs of neuroinflammation. Upon induc-tion of a traumatic brain injury in adult fish, neither the trauma-induced proliferation of leuko-cytes, nor the number of regenerated neurons were altered in Lrrk2-deficient animals. A multi-dimensional behavioral analysis of Lrrk2-deficient zebrafish revealed no significant constraints. The total swimming distance, average velocity and ratio of mobility states were unimpaired upon lrrk2-knock-out, as was the fish’s exploratory behavior in an anxiety model using a light-dark-box. In a test for social preference, Lrrk2-deficient and wild-type zebrafish showed the same tendency to join a group of conspecific animals, suggesting no major deficits in overall social interaction. In contrast to these preserved functions, adult Lrrk2-deficient kidneys revealed a pronounced accumulation of vacuole-like particles in the proximal renal tubules, a finding that may indicate disruptions in the endolysosomal pathway and that is in line with phenotypes described in LRRK2-deficient rodents as well as with the side effects induced by pharmacological LRRK2 inhibitors. These findings represent a promising lead for future exploration. During this work a CRISPR/Cas9 target site with high cleavage efficiency was established within the Lrrk2 kinase domain of freshly spawned zebrafish eggs. In combination with recent advances in CRISPR methodology, these results provide an opportunity for the generation of a genetic Lrrk2-G2019S knock-in line in zebrafish. In summary, this work found Lrrk2-deficient zebrafish unimpaired regarding various physiolog-ical functions. While in line with previously reported results, a satisfactory explanation for Lrrk2-mediatied pathogenesis is still lacking. Morphological alterations of Lrrk2-deficient kidneys hint towards perturbations in the lysosomal homeostasis, and a promising target for future re-search. Modelling human LRRK2 genotypes more precisely will hopefully provide further in-sights into the enigma of LRRK2 and its link to neurodegeneration.Die neurodegenerative Erkrankung Morbus Parkinson (Idiopathisches Parkinson-Syndrom, IPS) stellt sowohl eine individuelle Belastung für betroffene Menschen als auch eine große sozio-ökonomische Herausforderung für die Gesellschaft dar. Trotz großer Anstrengungen konnten bisher weder zufriedenstellende pathophysiologische Erklärungen des IPS, noch krankheits-modulierende Medikamente entwickelt werden. Mutationen der Kinase LRRK2 sind die insgesamt häufigste Ursache für erbliche Parkinson-Syndrome. Darüber hinaus wurden LRRK2-Mutationen mit immundysregulatorischen Syndro-men wie chronisch-entzündlichen Darmerkrankungen, Malignomen oder der Anfälligkeit ge-genüber Mykobakterien-Infektionen in Verbindung gebracht. Verschiedene pathogene Punktmutationen von LRRK2 sind bekannt. Diese führen – direkt oder indirekt – zu einer pa-thologischen Überaktivierung seiner Kinasedomäne. Trotz jüngster Fortschritte in der For-schung sind die Funktionen von LRRK2 und die Prozesse, die zu den LRRK2-vermittelten Pathologien führen, weiterhin weitgehend unbekannt. Viele Studien haben sich um die Entwicklung zuverlässiger Tiermodelle für die Untersuchung von LRRK2 bemüht. Dennoch haben weder die Untersuchung eines Gen-Funktionsverlusts noch die Überexpression von normalem oder mutiertem LRRK2 bislang zu eindeutigen Ergeb-nissen geführt. Frühere Arbeiten unserer Arbeitsgruppe haben in Zebrafischen (Zebrabärbling, Danio rerio) zwei genetische lrrk2-Knockout-Linien mit unterschiedlichen Mutagenesestrate-gien erzeugt: lrrk2TILLING und lrrk2CRISPR (Ahrendt, 2011; Suzzi, 2017). Die Ergebnisse dieser Studien widersprachen sich teilweise, und die beschriebenen Phänotypen waren nicht stabil reproduzierbar. Während alle bisherigen Arbeiten einen Funktionsverlust von Lrrk2 untersuch-ten, wurde bisher noch keine genetische Knock-in-Linie im Zebrafisch publiziert, die eine der zahlreichen bekannten pathogen-überaktivierenden LRRK2-Mutationen trägt. Ziel dieser Arbeit war es daher zum einen, die Auswirkungen eines Lrrk2-Funktionsverlusts in Zebrafischen weiter zu untersuchen, und zum anderen eine genetische Knock-in-Linie der häufigen pathogenen G2019S-Mutation zu etablieren, um den Genotyp menschlicher Parkin-son-Patienten präziser zu modellieren. Um diese Ziele zu erreichen, wurde eine Vielzahl von Methoden angewandt. Es wurden im-munhistochemische und konventionelle histologische Untersuchungen an Gehirnen und Nie-ren von Zebrafischen in verschiedenen Entwicklungsstadien durchgeführt, sowohl unter phy-siologischen Bedingungen als auch nach der Induktion einer Gehirnregeneration. Da die Fä-higkeit des Zebrafischs zur umfassenden Neuroregeneration durch eine initiale Neuroinflam-mation vermittelt wird und frühere Studien an lrrk2-Knockout-Zebrafischen in Folge traumati-scher Hirnverletzungen eine beeinträchtigte Immunreaktion und eine verringerte Neurorege-neration feststellen konnten, wurden die posttraumatische Neuroinflammation und die Neuroregeneration von adulten lrrk2TILLING-Zebrafischen untersucht, indem eine Stichverlet-zung des Großhirns induziert und eine anschließende BrdU-Pulse-Chase-Analyse durchge-führt wurde. Um die funktionellen Auswirkungen des Gen-Knockouts zu untersuchen, wurde eine Reihe von Verhaltensexperimenten durchgeführt. Mit Hilfe von CRISPR/Cas9-Genom-Editierung wurde die Grundlage für den Knock-in der G2019S-Mutation geschaffen. In einer ersten Reihe von Experimenten wurden larvale und adulte Zebrafischgehirne immun-histochemisch analysiert. Durch die Quantifizierung aller zerebraler mitotischer Zellen zu ver-schiedenen Zeitpunkten wurden die basale Hirnproliferation während der larvalen Entwicklung sowie die konstitutive Neurogenese im Erwachsenenalter analysiert. Unter physiologischen Bedingungen war die basale Hirnproliferation bei Lrrk2-defizienten Zebrafischen nicht beein-trächtigt. Auch die Anzahl der Mikroglia im Telenzephalon der Lrrk2-defizienten Zebrafische war unter physiologischen Bedingungen nicht verringert, obwohl eine Versuchsgruppe Anzei-chen einer Neuroinflammation zeigte. Infolge einer gezielten Verletzung einer Großhirnhemi-sphäre waren bei Lrrk2-defizienten Tieren weder die traumabedingte Proliferation von Leuko-zyten noch die Anzahl der anschließend regenerierten Neuronen verändert. Eine Verhaltensanalyse von Lrrk2-defizienten Zebrafischen ergab keine signifikanten Ein-schränkungen. Die Gesamtschwimmdistanz, die Durchschnittsgeschwindigkeit und das Ver-hältnis verschiedener Mobilitätszustände waren durch den Lrrk2-Knock-out unbeeinträchtigt, ebenso wie das Erkundungsverhalten der Fische in einem Angstmodell mit einer Hell-Dunkel-Kammer. In einem Test auf soziale Präferenz zeigten Lrrk2-defiziente und Wildtyp-Zebrafische die gleiche Tendenz, sich einer Gruppe von Artgenossen anzuschließen, was auf keine größeren Defizite in der allgemeinen sozialen Interaktion hindeutet. Im Gegensatz zu diesen unauffälligen Ergebnissen zeigten erwachsene Lrrk2-defiziente Nie-ren eine ausgeprägte Anhäufung vakuolenartiger Partikel in den proximalen Tubuli. Dieser Befund könnte auf Störungen im endolysosomalen Weg hinweisen und ist konsistent zu den bei LRRK2-defizienten Nagetieren beschriebenen Phänotypen, sowie den durch pharmakolo-gische LRRK2-Inhibitoren hervorgerufenen Nebenwirkungen. Diese Ergebnisse sind ein viel-versprechender Ansatzpunkt für künftige Experimente. Im Rahmen dieser Arbeit wurde eine CRISPR/Cas9-target-site mit hoher Schnitteffizienz in-nerhalb der LRRK2-Kinasedomäne von Zebrafisch-Embryonen etabliert. In Kombination mit Fortschritten in der CRISPR-Methodik bilden diese Ergebnisse eine Grundlage zur Erzeugung einer lrrk2-G2019S Knock-in-Linie. Zusammenfassend zeigt sich in dieser Arbeit, dass Lrrk2-defiziente Zebrafische in Hinblick auf verschiedene physiologische Funktionen nicht beeinträchtigt zu sein scheinen. Obwohl dies im Einklang mit früher berichteten Ergebnissen steht, bleibt eine zufriedenstellende Erklärung für die Lrrk2-vermittelte Pathogenese weiterhin aus. Morphologische Veränderungen in Lrrk2-defizienten Nieren deuten auf Störungen in der Homöostase des Lysosoms hin und bieten ein vielversprechendes Forschungsziel. Eine präzisere Modellierung des menschlichen LRRK2-Genotyps in fortschrittlichen Tiermodellen könnte zukünftig mehr Einblick in das Rätsel von LRRK2 und seiner Rolle in der Neurodegeneration bieten
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