Circadian Clocks in Neural Stem Cells and their Modulation of Adult Neurogenesis, Fate Commitment, and Cell Death
Adult neurogenesis creates new neurons and glial cells from stem cells in the human brain throughout life, and it is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have been identified in the olfactory bulb and the hippocampus, but the role of any endogenous circadian oscillator cells in neurogenesis and their importance in learning and memory remains unclear. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from mPer1::luc mouse SVZ and DG. Circadian bioluminescence rhythms were recorded in neurospheres maintained in culture medium that induces neurogenesis but not in one that maintains the stem cell state. Cell types were also characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Evidence was provided that neural stem progenitor cells (NSPCs) derived from the SVZ and DG of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Extremely rare percentages of mature neuronal cells were observed during ontogeny of rhythms. The bulk of the neurosphere cells were undifferentiated, indicating that they are the circadian clock cells producing timing signals. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To further test whether circadian clocks in NSPCs are necessary for growth, differentiation and cell survival, neurospheres were cultured from Bmal1-/- and Cry1-/-,2-/- knockout mice. Neurosheres from Bmal1-/- knockout mice displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few DCX and BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also had areas visibly devoid of cells and overall higher cell death. Neurospheres from mice lacking Cry1 and Cry2 showed significantly reduced growth. Altered NSPC proliferation and differentiation in these mice may impede memory formation and could provide a way to identify circadian timing effects in neurodegenerative disorders and impaired brain functions.