Project description:Transcriptome analysis of murine foetal NSCs (E14) after short-term (48 hours) and long-term (13 days) hypoxic (3% oxygen) culture compared to normoxic culture (21% oxygen) We focused on whole-transcriptome analyses using gene chip microarrays to compare expression profiles of NSCs cultured at hypoxic conditions to those of normoxic cells. Therefore, we used NSCs derived from the mesencephalon and the cortex and cultured them for short- and long-term at hypoxia/normoxia.
Project description:Hypoxic pre-conditioning has been demonstrated to increase the resistance of neural stem cells (NSCs) to hypoxic conditions, as well as to improve their capacity for differentiation and neuro-genesis. Extracellular vesicles (EVs) have recently emerged as critical mediators of cell-cell com-munication, but their role in this hypoxic conditioning is presently unknown. Here, we demon-strated that three hours of hypoxic pre-conditioning triggers significant neural stem cell EV release. Proteomic profiling of EVs from normal and hypoxic preconditioned neural stem cells identified 20 proteins that were up-regulated and 22 proteins that were down-regulated after hypoxic precon-ditioning. We also found an upregulation of some of these proteins by qPCR, thus indicating dif-ferences also at the transcript level within the EVs. Among the up-regulated proteins are CNP, Cyfip1, CASK, and TUBB5, which are well known to exhibit significant beneficial effects to neural stem cells. Thus, our results not only show a significant difference of protein cargo in EVs conse-quent to hypoxic exposure, but identify several candidate proteins that might play a pivotal role in the cell-to-cell mediated communication underlying neuronal differentiation, protection, matura-tion and survival following exposure to hypoxic conditions.
Project description:Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to enhance stem cell function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term (ST-NSC) and long-term NSCs (LT-NSCs). ST-NSC divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months, but are pushed out of homeostasis by lengthening quiescence. Single cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC pro-aging factor. Treatment with the Abl-inhibitor Imatinib increased NSC proliferation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable to cellular aging, yet NSC function can be partially restored.
Project description:The differentiation from neural stem cells (NSCs) to oligodendrocyte precursor cells (OPCs) is only incompletely understood. In the current study, we used the so-called neurosphere assay to study this process. We performed in vitro differentiation from neurospheres (which predominantly consist of NSCs) to oligospheres (consisting mainly of OPCs) and investigated their proteome by 6plex TMT-labeling and their phosphoproteome by 3plex dimethyl labeling. Among others, we identified a continuous upregulation of double cortin like kinase 1 (Dclk1) as well as its phosphorylation sites in the so-called SP-rich region. Using western blotting and qPCR, we show that two different Dclk1 isoforms (long and short) are present in NSCs and OPCs which gradually interchange during the investigated differentiation process. We further demonstrate that Dclk1 long is proteolytically processed into Dclk1 short and that this process is regulated via phosphorylation in the SP-rich region. Finally, we generated different BioID constructs fused to individual Dclk1 domains and investigate their interactome as well as potential substrates of Dclk1.
Project description:Neural stem cells (NSCs) generate new neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used chronic in vivo 2-photon microscopy and followed single NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI Family Zinc Finger 1 (Gli1) or Achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they becoming exhausted. Using protein expression profiling and single-cell RNA sequencing (scRNA-seq), we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here provide the cellular framework for how functional diversity of NSCs enables the generation of new neurons in the adult hippocampus.
Project description:Other than in the development of the brain, SOX2 is essential for the long-term self-renewal of neural stem cells (NSCs). The mechanisms of how SOX2 maintains the stemness of NSCs is not yet understood. We have identified Fos as a downstream target of SOX2, and therefore used CUT&RUN to investigate where these transcription factors - and the c-FOS partner c-JUN - interact with the genome. By comparing binding patterns of c-FOS, c-JUN and SOX2, we find that they co-occupate the promoter of the SOCS3 locus, which we also have identified as a gene that rescues SOX2 deletion induced senescence when overexpressed in neurospheres grown from Sox2-deleted mouse NSCs. Taken together, our data provide a basis for elucidating a gene regulatory network necessary for the maintenance of self-renewal in post-embryonic neural stem cells.
Project description:Neural stem cells (NSCs) in the adult mammalian subependymal zone maintain a glial identity and the developmental potential to generate neurons during the lifetime. Production of neurons from these NSCs is not direct but follows an orderly pattern of cell progression which allows the gradual increase along the neurogenic lineage in the expression of pro-neural factors needed for neuronal specification. In this context, tightly regulated translation of existing transcriptional programs represents a potential mechanism to avoid the critical challenge posed by genes that encode proteins with conflicting functions, i.e. self-renew or differentiate. Here, we identify RNA-binding protein MEX3A as a post-transcriptional regulator of a set of stemness-associated transcripts at critical transitions in the subependymal neurogenic lineage. MEX3A binding to a set of quiescence-related RNAs in activated NSCs is needed for their return to quiescence, playing a role in the long-term maintenance of the NSC pool. Furthermore, it is required for the repression of the same program at the onset of neuronal differentiation. Our data indicate that MEX3A is a pivotal regulator of adult mammalian neurogenesis acting as a translational remodeller.
Project description:In mammals, dosage compensation for the sex chromosomes is achieved by transcriptional silencing of one of the two X chromosomes in females. The inactive X adopts a particular epigenetic state, characterised by specific histones, histone marks, DNA methylation and 3D chromatin structure. As allelic resolution with short-read sequencing is limited, we do not yet have chromosome-wide phased methylomes of the active and inactive X. In this study, we obtained such complete X methylomes in mouse placenta and neural stem cells (NSCs) via long-read nanopore sequencing. This accession corresponds to the RNA-seq for the NSCs.
Project description:Using long-read nanopore sequencing, we obtained chromosome-wide phased methylomes of the active and inactive X in mouse placenta and neural stem cells (NSCs), overcoming the limitations if short-read bisulfite sequencing in allelic resolution. We also conducted quantitative analysis of methylation properties like symmetry and entropy, providing a more comprehensive view of epigenetic silencing in X chromosome inactivation. We also resolved the allele-specific genetics and epigenetics of structural macrosatellite Dxz4 and other repeats.
Project description:To investigate why dipeptides accumulate in immature CML cells, we examined upstream gene expression patterns. We isolated the most primitive long-term stem cells, short-term stem cells, and KLS- progenitor cells from healthy littermate control and CML-affected mice and performed gene expression profiling using next-generation RNA-sequencing. Gene expression profiles of the most primitive long-term (LT) stem cells (CD150+CD48-CD135-KLS+ cells), short-term (ST) stem cells (CD150-CD48-CD135- KLS+ cells), and KLS- progenitor cells from healthy littermate control and CML-affected mice