Adult hippocampal neurogenesis entails a continued recruitment of neural precursor cells (NPCs) into active cell cycle and their progressive transition into post-mitotic granule cells. These adult born neurons integrate into the existing circuitry and confer structural plasticity, which aids in key hippocampal functions. For sustained neurogenesis, the cell cycle entry of the NPCs has to be tightly controlled. Environmental cues strongly, and differentially, regulate this checkpoint. Voluntary physical activity represents such an established strong stimulus that results in enhanced proliferation within the neurogenic niche. However, mechanistic insights into the maintenance and regulation of quiescence and the responsiveness of the NPCs to acute physical activity, as a form of adaptive neurogenesis, are yet to be elucidated. In my doctoral studies, we identified redox regulation as a key pathway regulating the cellular state equilibrium. I further explored the role of cellular oxidative stress in the neurogenic course and in adaptive neurogenic responses. Our results show that non-proliferative precursors within the hippocampal dentate gyrus, unlike in other stem cell systems, are marked by high levels of cellular reactive oxygen species (ROS). Using cytometric methodologies, ex vivo bioassays and transcriptional profiling, we revealed that classifying cells based on intracellular ROS content identified functionally defined sub-populations of adult NPCs. We propose that a drop in intracellular ROS content precedes the transition of cellular states, specifically from quiescence to active proliferation. Acute physical activity involves the activation of non- proliferating cells through a transient Nox2-dependent ROS surge in high-ROS, quiescent NPCs. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity- dependent response was abolished. These findings shed new light on the discrete cellular events, which maintain the homeostasis between distinct cellular states of NPCs within the adult murine hippocampus.:Zusammenfassung 3
Summary 4
Acknowledgements 5
Index 8
List of figures 10
List of tables 11
Abbreviations 12
Publications 14
Introduction 15
Adult hippocampal neurogenesis 16
Adult subventricular neurogenesis 21
Methods to study adult neurogenesis 23
Environmental regulation of neurogenesis 26
Redox regulation in a stem cell 29
Working hypothesis 31
Specific aims 31
Materials and methods 32
Mice 34
Physical activity paradigm 35
Thymidine labelling and tissue preparation 35
Fluorescence immunohistochemistry 35
DG and SVZ dissection and dissociation 36
Flow cytometry 36
Gating for ROS classes 36
Neurosphere culture 37
Generation of monolayer culture 37
Inducing quiescence through BMP4 treatment 38
Next Generation sequencing (NGS) 38
RNA extraction 38
Quality control and differential expression 39
Functional enrichment and expression profiles 41
RNA isolation and quantitative RTPCR (qRT-PCR) 43
Ki67 immunochemistry and quantification of in vivo proliferation 45
Quantification and statistical analysis 46
Data and software availability 48
Results 49
Intracellular ROS content functionally delineates subpopulations of neural precursor cells 49
Resolution of ROS profiles of DG and SVZ and neurosphere bioassay 49
Distribution of Nes-GFP cells into different ROS classes 54
Neural precursors of the different ROS classes have distinct molecular profiles 55
Changes in intracellular ROS content precede cell fate changes 65
ROS profiling of other cell types within the DG 70
ROS profiling of Astrocytes and type-1 cells 70
ROS profiling of Doublecortin (Dcx)positive cells of the neurogenic lineage 74
ROS profiling of microglial cells within the DG 77
Resolving the response of Nes-GFP subpopulations to environmental stimulus 78
Nes-GFP+ cells of the hiROS class specifically respond to physical activity 81
Changes in ROS content are not driven by mitochondrial activity 83
In vitro monolayer culture of NPCs as an independent corroboration 86
Discussion 89
The organization of an active stem cell niche with respect to redox content 89
Cytometric classification of cells within the DG 91
Establishing the cellular states of redox defined subsets of Nes-GFP+ adult precursors within the DG 95
Timeline of baseline proliferation within precursors and identifying the subset of precursors responsive to de novo physical activity 97
Monolayer culture to study cellular states and redox regulation 100
Nox2 dependency as a discriminatory feature of adaptive neurogenesis 101
Conclusion 103
References 104
Declarations 122
Anlage 1 122
Anlage 2 124
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:72802 |
| Date | 16 November 2020 |
| Creators | Adusumilli, Vijaya |
| Contributors | Kempermann, Gerd, Karl, Mike O., Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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