Extracellular signal-regulated kinase-dependent circadian expression of clock-related genes in the neural stem cells

The neural stem cells (NSCs), which possess the ability of proliferation and differentiation into neurons and glial cells, exist in the mammalian adult brain, including the hippocampal dentate gyrus. The proliferation of the NSCs in the dentate gyrus was reported to change along the time of day. However, it is still unclear whether this circadian rhythm in the mitotic activity is caused by the daily change in the microenvironment around the NSCs or by the cell-autonomous circadian rhythm driven by clock-related genes. To address the later possibility, we examined the circadian expression of several clock-related genes and the proliferation of the neural stem cells under in vitro conditions. The NSCs from the ganglionic eminence of embryonic mice were expanded by the neurosphere method and then treated. with epidermal growth factor (EGF) to stimulate their mitotic activity. The time courses of the proliferation were examined by WST-8 assay and bromodeoxyuridine (BrdU) incorporation assay and the expression of clock-related genes was examined by RT-PCR and immunocytochemistry. We have found that EGF treatment elicited the circadian change in the increase in mitotic activity of the NSCs. In addition, the gene expression of mPer2, mBMAL1, NPAS2 and Rev-Erb alpha changed rhythmically with a period of 24 h and correlated negatively with the DNA synthesis activity rhythm. Furthermore, the treatment with an antisense oligonucleotide against mPer2 increased the DNA synthesis activity of the NSCs. We also found that the treatment with EGF transiently activated the extracellular regulated kinase 1/2 (ERK1/2), p38 mitogen activated kinase (P38 MAPK) and Jun kinase (JNK) in a concentration-dependent manner. The pretreatment with U0126, a MEK inhibitor, but not the inhibitors of p38MAPK and JNK, abolished the circadian rhythms of clock-gene expression in the NSCs. These results suggest that the proliferation of NSCs is periodically regulated by the cell-autonomous circadian expression of clock-related genes which involve the ERK1/2.