Adachi Naoki, Suzuki Shingo, Matsuoka Hidetada, Fushimi Satoko, Ono Junichiro, Ohta Ken-Ichi, Hirai Yohei, Miki Takanori, Koshimizu Hisatsugu
Department of Biomedical Chemistry, School of Science and Technology, Kwansei Gakuin University, Sanda, Japan.
Faculty of Medicine, Department of Anatomy and Neurobiology, Kagawa University, Kagawa, Japan.
J Neurochem. 2018 Jan 22. doi: 10.1111/jnc.14310.
A recent study revealed that corticotropin-releasing hormone (CRH) in the cerebral cortex (CTX) plays a regulatory role in emotional behaviors in rodents. Given the functional interaction between brain-derived neurotrophic factor (BDNF) and the CRH-signaling pathway in the hypothalamic-pituitary-adrenal axis, we hypothesized that BDNF may regulate gene expression of CRH and its related molecules in the CTX. Findings of real-time quantitative PCR (RT-qPCR) indicated that stimulation of cultured rat cortical neurons with BDNF led to marked elevations in the mRNA levels of CRH and CRH-binding protein (CRH-BP). The BDNF-induced up-regulation of CRH-BP mRNA was attenuated by inhibitors of tropomyosin related kinase (Trk) and MEK, but not by an inhibitor for PI3K and Phospholipase C gamma (PLCγ). The up-regulation was partially blocked by an inhibitor of lysine-specific demethylase (KDM) 6B. Fluorescent imaging identified the vesicular pattern of pH-sensitive green fluorescent protein-fused CRH-BP (CRH-BP-pHluorin), which co-localized with mCherry-tagged BDNF in cortical neurons. In addition, live-cell imaging detected drastic increases of pHluorin fluorescence in neurites upon membrane depolarization. Finally, we confirmed that tetrodotoxin partially attenuated the BDNF-induced up-regulation of CRH-BP mRNA, but not that of the protein. These observations indicate the following: In cortical neurons, BDNF led to gene expression of CRH-BP and CRH. TrkB, MEK, presumably ERK, and KDM6B are involved in the BDNF-induced gene expression of CRH-BP, and BDNF is able to induce the up-regulation in a neuronal activity-independent manner. It is suggested that CRH-BP is stored into BDNF-containing secretory granules in cortical neurons, and is secreted in response to membrane depolarization.
最近的一项研究表明，大脑皮质（CTX）中的促肾上腺皮质激素释放激素（CRH）在啮齿动物的情绪行为中起调节作用。鉴于脑源性神经营养因子（BDNF）与下丘脑-垂体-肾上腺轴中的CRH信号通路之间存在功能相互作用，我们推测BDNF可能调节CTX中CRH及其相关分子的基因表达。实时定量PCR（RT-qPCR）结果表明，用BDNF刺激培养的大鼠皮质神经元可导致CRH和CRH结合蛋白（CRH-BP）的mRNA水平显著升高。BDNF诱导的CRH-BP mRNA上调被原肌球蛋白相关激酶（Trk）和MEK的抑制剂减弱，但未被PI3K和磷脂酶Cγ（PLCγ）的抑制剂减弱。赖氨酸特异性去甲基化酶（KDM）6B的抑制剂部分阻断了这种上调。荧光成像确定了pH敏感的绿色荧光蛋白融合的CRH-BP（CRH-BP-pHluorin）的囊泡模式，其在皮质神经元中与mCherry标记的BDNF共定位。此外，活细胞成像检测到膜去极化时神经突中pHluorin荧光急剧增加。最后，我们证实河豚毒素部分减弱了BDNF诱导的CRH-BP mRNA上调，但未减弱蛋白质的上调。这些观察结果表明：在皮质神经元中，BDNF导致CRH-BP和CRH的基因表达。TrkB、MEK、可能还有ERK和KDM6B参与BDNF诱导的CRH-BP基因表达，并且BDNF能够以不依赖神经元活动的方式诱导上调。提示CRH-BP储存在皮质神经元中含BDNF的分泌颗粒中，并在膜去极化时分泌。
