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Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination.

Malik A, Kondratov RV, Jamasbi RJ, Geusz ME - PLoS ONE (2015)

Bottom Line: It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ).This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core.The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Bowling Green State University, Bowling Green, Ohio, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Neuronal commitment is diminished in Bmal1-/- DG neurospheres.In WT DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons expressing BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons expressing NeuN (green) at day 14 and lacking GFAP (red). In contrast, Bmal1-/- neurospheres displayed reduced neuronal differentiation and increased astrocyte proliferation. C: Lack of BetaIII-tubulin expression (green) shown with GFAP (red) in a Bmal1-/- neurosphere at day 7. D: Lack of NeuN (green) shown with GFAP (red) at day 14. All nuclei were stained with Hoechst (blue). Scale bar = 50 μm. E: Percentage of positive cells for DCX (neuroblasts), BetaIII-tubulin, and NeuN at days 4, 7, and 14 after differentiation in B27 medium, respectively.
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pone.0139655.g005: Neuronal commitment is diminished in Bmal1-/- DG neurospheres.In WT DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons expressing BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons expressing NeuN (green) at day 14 and lacking GFAP (red). In contrast, Bmal1-/- neurospheres displayed reduced neuronal differentiation and increased astrocyte proliferation. C: Lack of BetaIII-tubulin expression (green) shown with GFAP (red) in a Bmal1-/- neurosphere at day 7. D: Lack of NeuN (green) shown with GFAP (red) at day 14. All nuclei were stained with Hoechst (blue). Scale bar = 50 μm. E: Percentage of positive cells for DCX (neuroblasts), BetaIII-tubulin, and NeuN at days 4, 7, and 14 after differentiation in B27 medium, respectively.

Mentions: To analyze whether Bmal1, an essential component of the circadian clock, is necessary for neurogenesis in vitro, both secondary DG neurospheres from WT and KO dishes were transferred to new 35mm poly-D-lysine-coated Mattek dishes with neural differentiation medium (B27M). Neurospheres were allowed to differentiate for 4 or 6–7 days in B27M with no added stem cell-maintaining growth factors (bFGF and EGF). Immunocytochemistry was used to determine the percentage of neuroblasts (DCX+), immature neurons (BetaIII-tubulin+), and astrocytes (GFAP+) in the culture. The percentage of DCX+ cells in Bmal1-/- DG spheres was significantly lower relative to WT controls after 4 days of differentiation in B27M (KO: 5.42 ±7.91%, n = 6; WT: 27.86 ±21.53%, n = 7; t = 3.42, p = 0.01). When compared with WT (Fig 5A) the percentage of immature BetaIII+ neuronal cells, after differentiation in B27 medium at day 7, was significantly reduced in Bmal1-/- DG spheres (Fig 5C) (KO: 5.88 ±8.56%, n = 9; WT: 55.79 ±7.38%, n = 8; t = 4.93, p = 0.001). In addition, these differentiated neurospheres exhibited an increased astrocyte proliferation when compared to their WT littermates (KO: 76.01 ±7.09%, n = 9; WT: 5.22 ±4.19%, n = 8; t = 32.13, p<0.001).


Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination.

Malik A, Kondratov RV, Jamasbi RJ, Geusz ME - PLoS ONE (2015)

Neuronal commitment is diminished in Bmal1-/- DG neurospheres.In WT DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons expressing BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons expressing NeuN (green) at day 14 and lacking GFAP (red). In contrast, Bmal1-/- neurospheres displayed reduced neuronal differentiation and increased astrocyte proliferation. C: Lack of BetaIII-tubulin expression (green) shown with GFAP (red) in a Bmal1-/- neurosphere at day 7. D: Lack of NeuN (green) shown with GFAP (red) at day 14. All nuclei were stained with Hoechst (blue). Scale bar = 50 μm. E: Percentage of positive cells for DCX (neuroblasts), BetaIII-tubulin, and NeuN at days 4, 7, and 14 after differentiation in B27 medium, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4595423&req=5

pone.0139655.g005: Neuronal commitment is diminished in Bmal1-/- DG neurospheres.In WT DG neurospheres the sequence of cell types during differentiation in B27 medium parallels events during in situ neurogenesis. A: Immature neurons expressing BetaIII-tubulin (green) at day 7 and lacking GFAP co-localization (red). B: Mature neurons expressing NeuN (green) at day 14 and lacking GFAP (red). In contrast, Bmal1-/- neurospheres displayed reduced neuronal differentiation and increased astrocyte proliferation. C: Lack of BetaIII-tubulin expression (green) shown with GFAP (red) in a Bmal1-/- neurosphere at day 7. D: Lack of NeuN (green) shown with GFAP (red) at day 14. All nuclei were stained with Hoechst (blue). Scale bar = 50 μm. E: Percentage of positive cells for DCX (neuroblasts), BetaIII-tubulin, and NeuN at days 4, 7, and 14 after differentiation in B27 medium, respectively.
Mentions: To analyze whether Bmal1, an essential component of the circadian clock, is necessary for neurogenesis in vitro, both secondary DG neurospheres from WT and KO dishes were transferred to new 35mm poly-D-lysine-coated Mattek dishes with neural differentiation medium (B27M). Neurospheres were allowed to differentiate for 4 or 6–7 days in B27M with no added stem cell-maintaining growth factors (bFGF and EGF). Immunocytochemistry was used to determine the percentage of neuroblasts (DCX+), immature neurons (BetaIII-tubulin+), and astrocytes (GFAP+) in the culture. The percentage of DCX+ cells in Bmal1-/- DG spheres was significantly lower relative to WT controls after 4 days of differentiation in B27M (KO: 5.42 ±7.91%, n = 6; WT: 27.86 ±21.53%, n = 7; t = 3.42, p = 0.01). When compared with WT (Fig 5A) the percentage of immature BetaIII+ neuronal cells, after differentiation in B27 medium at day 7, was significantly reduced in Bmal1-/- DG spheres (Fig 5C) (KO: 5.88 ±8.56%, n = 9; WT: 55.79 ±7.38%, n = 8; t = 4.93, p = 0.001). In addition, these differentiated neurospheres exhibited an increased astrocyte proliferation when compared to their WT littermates (KO: 76.01 ±7.09%, n = 9; WT: 5.22 ±4.19%, n = 8; t = 32.13, p<0.001).

Bottom Line: It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ).This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core.The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Bowling Green State University, Bowling Green, Ohio, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus