KINASE-MEDIATED SIGNALING PATHWAYS IN NEURONAL APOPTOSIS

神经元凋亡中激酶介导的信号通路

• 批准号: 6540349
• 负责人: MARY LOU VALLANO
• 金额: $ 26.48万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540349
• 关键词: apoptosis; biological signal transduction; calcium flux; genetically modified animals; granule cell; laboratory mouse; neurotrophic factors; phosphorylation; protein kinase; tissue /cell culture; transcription factor

DESCRIPTION (From the applicant's abstract): We are studying the kinase-mediated signaling pathways that promote neuronal survival and prevent apoptosis in primary cultures of granule neurons. Relevant neurotrophins are insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF). Glutamate is the predominant neurotransmitter that regulates neuronal activity through its interaction with different glutamate receptor-channels, referred to herein as N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Binding of these agents to their respective receptors in the neuronal plasma membrane initiates activation of intracellular signaling cascades that regulate the transcription of pro-apoptotic or anti-apoptotic genes. The balance between these gene products ultimately determines cell fate. Early in granule cell development, before neurons have formed synaptic connections, they rely exclusively on neurotrophic factors, present in the serum that bathes them, for survival. We have novel evidence that particular isoforms of the family of phospholipid-sensitive protein kinases (PKCs), designated as atypical PKCs (aPKCs), are critical components of the neurotrophin-dependent survival pathway. We are quite enthusiastic about these data since almost nothing is currently known about the neuronal functions of aPKCs. Moreover, preliminary data indicate that inhibition of aPKCs reduces phosphorylation on serine-133 of the nuclear transcription factor CREB. One goal of this application is to extend our studies examining the linkage between neurotrophin-dependent activation of aPKCs. phosphorvlation of CREB. and survival. In many neuronal populations, inadequate electrical stimulation by excitatory neurotransmitters or inadequate trophic factor make cell death more likely. A "Ca2+ set-point hypothesis" postulates that the bulk cytosolic Ca2+ activity determines the degree to which immature neurons require trophic factor to suppress the mechanism responsible for apoptosis (Koike et al., 1989). Accordingly, many types of neurons grown in dissociated culture can be rescued from death, induced by neurotrophin deprivation, by inclusion of agents in the media that produce a sustained elevation in Ca2+. These agents include elevated KCI acting through voltage-sensitive Ca2+ channels (VSCCs) and NMDA acting directly upon NMDA receptor channels (NRs). We have novel evidence that a Ca2+ and calmodulin-dependent kinase, designated as CaM KIV, and its substrate CREB are critical components of this Ca2+-dependent signaling pathway. Immunocytochemical evidence also indicated that these treatments induce the redistribution of CaM KIV from the nucleus to the cytosol. A second goal of this application is to extend our studies examining the linkage between calcium, CaM KIV phosphorvlation of CREB, and survival. Phosphorylation and activation of CREB may mediate induction of anti-apoptotic genes, such as bc1-2, or repression of pro-apoptotic genes, such as bax. A third goal in this application is to compare the expression levels of bc1-2 and bax rnRNAs under culture conditions promoting survival or apoptosis.

描述（来自申请人的摘要）：我们正在研究 激酶介导的信号通路可促进神经元存活并预防 颗粒神经元原代培养物中的细胞凋亡。相关的神经营养素是 胰岛素样生长因子（IGF-1）和脑源性神经营养因子 （BDNF）。谷氨酸是调节神经元的主要神经递质 通过与不同谷氨酸受体通道相互作用的活性， 本文中称为N-甲基-D-天冬氨酸(NMDA)和非NMDA受体。 这些药物与神经元血浆中各自的受体结合 膜启动细胞内信号级联的激活，调节 促凋亡或抗凋亡基因的转录。之间的平衡 这些基因产物最终决定细胞的命运。颗粒细胞早期 在神经元形成突触连接之前，它们依赖 专门针对神经营养因子，存在于沐浴它们的血清中， 生存。我们有新的证据表明该家族的特定亚型 磷脂敏感蛋白激酶 (PKC)，称为非典型 PKC (aPKC)，是神经营养蛋白依赖性生存的关键组成部分 途径。我们对这些数据非常感兴趣，因为几乎没有什么是 目前已知 aPKC 的神经元功能。此外，初步 数据表明，抑制 aPKC 会降低丝氨酸 133 的磷酸化 核转录因子CREB。该应用程序的目标之一是 扩展我们的研究，检查神经营养素依赖性之间的联系 aPKC 的激活。 CREB ​​的磷酸化。和生存。在许多神经元 人群，兴奋性神经递质的电刺激不足 或营养因子不足使细胞更有可能死亡。 “Ca2+ 设定点 假说”假设大量胞质 Ca2+ 活性决定了 未成熟神经元需要营养因子来抑制的程度 细胞凋亡的机制（Koike et al., 1989）。据此，许多 在分离培养物中生长的神经元类型可以免于死亡， 由神经营养蛋白剥夺引起，通过在介质中包含试剂来 导致 Ca2+ 持续升高。这些药物包括升高 KCI 作用 通过电压敏感 Ca2+ 通道 (VSCC) 和 NMDA 直接作用于 NMDA 受体通道 (NR)。我们有新的证据表明 Ca2+ 和 钙调蛋白依赖性激酶，称为 CaM KIV，及其底物 CREB ​​是 该 Ca2+ 依赖性信号通路的关键组成部分。 免疫细胞化学证据还表明，这些治疗可诱导 CaM KIV 从细胞核重新分布到细胞质。第二个目标是 这个应用程序是为了扩展我们的研究，检查之间的联系 钙、CREB ​​的 CaM KIV 磷酸化和存活率。磷酸化和 CREB ​​的激活可能介导抗凋亡基因的诱导，例如 bc1-2，或促凋亡基因的抑制，例如bax。本次比赛的第三个进球 应用是比较 bc1-2 和 bax mRNA 在条件下的表达水平 培养条件促进存活或凋亡。