Regulation of Cellular Responsiveness to BDNF

描述(申请人提供)：神经营养因子，脑源性神经营养因子(BDNF)，通过调节神经元存活和突触可塑性，在大脑发育和功能中发挥关键作用。BDNF信号的缺陷可能导致发育、神经退行性和精神障碍。细胞对BDNF的反应是由酪氨酸受体激酶TrkB介导的，其大小由活性的全长TrkB和截短的TrkB亚型的相对水平决定，它们是BDNF信号的显性-负性抑制因子。 对皮质神经元的初步研究表明，全长TrkB的表达，而不是截短的TrkB的表达，是通过增加细胞内钙离子水平而被膜去极化刺激的。钙离子对TrkB基因的两个启动子(TrkB)具有相反的作用，提示启动子的使用调节TrkB亚型的表达。这些观察结果导致了一个可行的假设：TrkB亚型的表达，因此，神经元对BDNF的反应，是由钙离子依赖的TrkB转录调节的。 利用重组DNA技术研究钙离子对TrkB启动子的差异调控机制，以确定TrkB中的钙依赖调控元件以及与其相互作用的转录因子。通过检测带有启动子特异的5‘-非翻译序列的全长和截短TrkB的去极化诱导表达，研究钙离子介导的启动子利用在调节全长和截短TrkB差异表达中的作用。 为了将钙依赖的TrkB调控置于更具生理学意义的背景下，我们将研究模拟体内神经元活动的电诱导钙瞬变模式，以差异激活TrkB启动子和差异驱动全长和截短TrkB亚型的表达的能力。 本研究的长期目标是阐明钙离子调节TrkB亚型表达的机制，从而调节发育中的、成人的和患病的大脑中神经元对BDNF的反应。

DESCRIPTION (provided by applicant): The neurotrophin, brain derived neurotrophic factor (BDNF), plays a critical role in brain development and function by regulating neuron survival and synaptic plasticity. Defects in BDNF signaling may lead to developmental, neurodegenerative and psychiatric disorders. Cellular responses to BDNF are mediated by the tyrosine receptor kinase, trkB, and their magnitude is determined by the relative levels of active, full-length trkB, and of truncated trkB isoforms, which act as dominant-negative inhibitors of BDNF signaling. Preliminary studies in cortical neurons revealed that expression of full-length trkB, but not that of truncated trkB, is stimulated by membrane depolarization via increased levels of intracellular Ca2+. Ca2+ was also found to exert opposing effects on the two promoters of the trkB gene (TRKB), suggesting that promoter usage regulates trkB isoform expression. These observations have led to the working hypothesis: TrkB isoform expression and consequently, neuronal responsiveness to BDNF, is regulated by Ca2+-dependent TRKB transcription. The mechanism by which Ca2+ differentially regulates the TRKB promoters will be studied using recombinant DNA technology to identify the Ca2+-dependent regulatory elements in TRKB and the transcription factors hat interact with them. The role of Ca2+-mediated promoter utilization in regulating the differential expression f full-length and truncated trkB will be studied by measuring the depolarization-induced expression f full-length and truncated trkB mRNA with promoter-specific 5'-untranslated sequences. In order to place Ca2+-dependent TRKB regulation in a more physiological context, we will investigate the ability of patterns of electrically evoked Ca2+ transients, which mimic in vivo neuronal activity, to differentially activate the TRKB promoters and differentially drive expression of full-length and truncated trkB isoforms. The long-term goal of this research is to elucidate the mechanisms by which Ca2+ regulates expression of trkB isoforms and, thus, modulates neuronal responsiveness to BDNF in the developing, adult and diseased brain.