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Dissection and proteome analysis to characterize the adult neural stem cell niche
Dissection and proteome analysis to characterize the adult neural stem cell niche
Stem cell niches in the adult mammalian brain are decisively shaped by their microenvironment. In these niches, extracellular signals modulate stem cell quiescence, proliferation, migration, and differentiation. Conversely, the microenvironment in the remainder of the brain merely permits gliogenesis, restricts neuronal plasticity, and limits the neurogenic potential of neural precursors. Elucidating the mechanisms rendering neurogenic niches permissive for neurogenesis might foster the improvement of cell replacement therapies for neurological disorders involving neural cell loss. To better understand the molecular composition, the architecture, and the physical properties responsible for the neurogenic nature of the microenvironment in neural stem cell niches, this study pursued a characterization of the subependymal zone of the lateral ventricle (SEZ), which is the largest stem cell niche of the murine brain. To investigate the microenvironment of the SEZ, a bottom-up proteomic approach using mass spectrometry was employed. The analysis of the extracellular microenvironment of this region requires a precise dissection method with minimal tissue perturbation, applicable to unfixed tissue. For this purpose, a novel dissection method, termed Cryo-section Dissection (CSD), was developed. In the first step of the CSD protocol the cortex and the corpus callosum covering the lateral ventricles are removed from the unfixed murine brain. Then, after freezing the tissue on dry ice, the brain is sectioned coronally. Finally, the SEZ is manually isolated from each section using a pre-cooled scalpel. The SEZ as adult neural stem cell niche was compared to the non-neurogenic somatosensory cortex, the olfactory bulb as site of neuronal integration, and the structurally similar, but mostly non-neurogenic medial wall of the lateral ventricle, termed medial ependymal zone (MEZ). A library-matched single shot mass spectrometry analysis employing a label-free quantification algorithm was applied to generate the proteome data of the SEZ, the somatosensory cortex, the olfactory bulb, and the MEZ. This proteome data was used to investigate niche specific protein clusters and filtered for individual candidate proteins. Promising candidates were subjected to immunohistochemical staining. This analysis enabled the detection of the candidate proteins C1ql3, Kininogen 1, and S100a6 potentially involved in neurogenesis. Additionally, the influence of niche stiffness on neural stem cell physiology was investigated, and the extracellular neurogenesis regulator Transglutaminase 2 could be identified.
Dissection, Proteome, Adult Neural Stem Cell Niche, Neurogenesis, Extracellular Matrix, Extracellular Microenvironment
Frieß, Christian
2023
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Frieß, Christian (2023): Dissection and proteome analysis to characterize the adult neural stem cell niche. Dissertation, LMU München: Faculty of Medicine
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Abstract

Stem cell niches in the adult mammalian brain are decisively shaped by their microenvironment. In these niches, extracellular signals modulate stem cell quiescence, proliferation, migration, and differentiation. Conversely, the microenvironment in the remainder of the brain merely permits gliogenesis, restricts neuronal plasticity, and limits the neurogenic potential of neural precursors. Elucidating the mechanisms rendering neurogenic niches permissive for neurogenesis might foster the improvement of cell replacement therapies for neurological disorders involving neural cell loss. To better understand the molecular composition, the architecture, and the physical properties responsible for the neurogenic nature of the microenvironment in neural stem cell niches, this study pursued a characterization of the subependymal zone of the lateral ventricle (SEZ), which is the largest stem cell niche of the murine brain. To investigate the microenvironment of the SEZ, a bottom-up proteomic approach using mass spectrometry was employed. The analysis of the extracellular microenvironment of this region requires a precise dissection method with minimal tissue perturbation, applicable to unfixed tissue. For this purpose, a novel dissection method, termed Cryo-section Dissection (CSD), was developed. In the first step of the CSD protocol the cortex and the corpus callosum covering the lateral ventricles are removed from the unfixed murine brain. Then, after freezing the tissue on dry ice, the brain is sectioned coronally. Finally, the SEZ is manually isolated from each section using a pre-cooled scalpel. The SEZ as adult neural stem cell niche was compared to the non-neurogenic somatosensory cortex, the olfactory bulb as site of neuronal integration, and the structurally similar, but mostly non-neurogenic medial wall of the lateral ventricle, termed medial ependymal zone (MEZ). A library-matched single shot mass spectrometry analysis employing a label-free quantification algorithm was applied to generate the proteome data of the SEZ, the somatosensory cortex, the olfactory bulb, and the MEZ. This proteome data was used to investigate niche specific protein clusters and filtered for individual candidate proteins. Promising candidates were subjected to immunohistochemical staining. This analysis enabled the detection of the candidate proteins C1ql3, Kininogen 1, and S100a6 potentially involved in neurogenesis. Additionally, the influence of niche stiffness on neural stem cell physiology was investigated, and the extracellular neurogenesis regulator Transglutaminase 2 could be identified.