Malik Astha, Kondratov Roman V, Jamasbi Roudabeh J, Geusz Michael E
Department of Biology, Bowling Green State University, Bowling Green, Ohio, United States of America.
Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, Ohio, United States of America.
PLoS One. 2015 Oct 6;10(10):e0139655. doi: 10.1371/journal.pone.0139655. eCollection 2015.
Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.
成体神经发生在人的一生中由脑内的干细胞产生新的神经元和神经胶质细胞。这一过程在海马体的齿状回（DG）和脑室下区（SVZ）中最易理解。海马体中已发现昼夜节律，但任何内源性昼夜节律振荡器细胞在海马体神经发生中的作用及其在学习或记忆中的重要性仍不清楚。由于大脑其他部位的昼夜节律时钟的调节作用，对DG内固有昼夜节律对干细胞调节的任何研究都变得复杂。为了更孤立地研究昼夜节律振荡器，从两种缺乏功能性昼夜节律时钟的基因敲除小鼠品系的DG以及mPer1::luc小鼠中制备神经球培养物，以确定基因表达中的昼夜节律振荡。在诱导神经发生的培养基中培养的神经球中记录到了昼夜节律性的mPer1基因活性节律，但在维持干细胞状态的培养基中未记录到。尽管神经球中正在分化的神经干细胞祖细胞具有节律性，但成熟神经元的证据极为稀少。昼夜节律定时信号起源于神经球内未分化的细胞。这一结论得到了mPER1蛋白免疫细胞化学的支持，该蛋白定位于内部、更像干细胞的神经球核心。为了测试昼夜节律时钟对神经发生的影响，改变培养基条件以诱导BMAL1基因敲除小鼠的神经球分化。根据GFAP和NeuN的表达，这些培养物分别显示出向神经胶质细胞而非神经元的异常高分化，并且观察到极少的βIII微管蛋白阳性未成熟神经元。基因敲除神经球还显示出明显无细胞的区域，并且总体细胞死亡更高。缺乏另外两个核心时钟基因Cry1和Cry2的无节律小鼠的神经球在分化过程中生长显著减少且星形胶质细胞增殖增加，但它们产生的神经元细胞百分比正常。因此，神经元命运的决定似乎是通过BMAL1的非时钟功能来控制的。这项研究为细胞自主昼夜节律时钟和时钟基因如何调节成体神经干细胞提供了见解，这对于通过操纵神经发生来治疗神经退行性疾病和受损脑功能具有重要意义。
