Optogenetic and Biochemical Studies of Novel Roles of beta-Catenin Modulation/Addiction in Neuronal Differentiation and Apoptosis

β-连环蛋白调节/成瘾在神经元分化和细胞凋亡中新作用的光遗传学和生化研究

基本信息

  • 批准号:
    8984020
  • 负责人:
  • 金额:
    $ 5.42万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2015
  • 资助国家:
    美国
  • 起止时间:
    2015-07-01 至 2018-06-30
  • 项目状态:
    已结题

项目摘要

 DESCRIPTION (provided by applicant): Parkinson's and Alzheimer's disease are neurodegenerative diseases that affect 40 million people worldwide. Adult neural stem cells (NSC) have great potential as cell replacement therapies. The adult NSC microenvironment is likely highly dynamic, with signaling molecules presented at modulating intensities and durations. The canonical Wnt signaling pathway, whose activation involves ß-catenin stabilization, is involved in regulating NSC behavior, including neurogenesis and apoptosis. To mimic in vivo dynamic signaling, our lab has developed a tunable optogenetic system to modulate ß-catenin signaling through Cry2 oligomerization of the LRP6 intracellular domain. Canonical Wnt pathway activation via Wnt3a can lead to robust neurogenesis, and I have observed that NSCs undergo neuronal differentiation in a signal dosage-dependent manner in the presence of low level fluctuations of dark and light cycles. In contrast, we observed that brie stabilization of ß- catenin followed by extended signal withdrawal induces apoptosis, a novel outcome that may act to eliminate incompletely differentiated neurons. Specifically, catastrophic signal loss was induced by the total withdrawal of light, and increased apoptosis was observed in cells exposed to light for less than 3 days. Under low intensity and varying light cycle conditions, a decrease in total photons also led to increased apoptosis. However, cells "rescued" with light within 24 hrs did not have increased apoptosis whereas those without light for greater than 24 hrs exhibited increased apoptosis. In addition, I show a loss-of-signal `buffer region, within which the neurogenic fates of NSCs were relatively insensitive to signal fluctuation, whereas, outside of the `buffer', NSC fate is significantly shifted towards apoptosis. Therefore, dynamic ß-catenin signaling likely directs NSCs towards differentiation or apoptosis. In Aim 1, we explore the dynamics of ß-catenin (de-) stabilization, with our unique optogenetic system, through the on/off kinetics, transcriptional activity, and downstream effects on NSC fate. The kinetic response of ß-catenin to signal fluctuations and cell fate decisions will be correlate to the levels of ß-catenin phospho- species and other key proteins to gain insight into the molecular mechanism(s) involved. In Aim 2, we will determine the molecular mechanism(s) involved in the apoptosis cell fate. In biased strategies, we are exploring known protein pathways involved in apoptotic activities. Complementary, unbiased searches with ChIP-Seq and RNA-Seq will identify potential transcriptional regulation of apoptotic processes. Notably, ChIP-seq of ß-catenin throughout differentiation will be broadly useful for studies of Wnt signaling and neurogenesis. The precise control of signal induction offered by our optogenetic system allows for unprecedented exploration of ß-catenin dynamics. The observed apoptotic outcome may represent an important regulatory step for in vivo neurogenesis and determination of downstream signaling effectors will yield more complete molecular mechanisms by which canonical Wnt signaling regulates NSC fate. Thus, this work will advance our understanding of Wnt signaling and the efforts to harness NSCs for neuroregeneration.
 描述(由申请人提供):帕金森病和阿尔茨海默病是影响全球4000万人的神经退行性疾病。成体神经干细胞(NSC)作为细胞替代疗法具有巨大的潜力。成年NSC微环境可能是高度动态的,信号分子以调节强度和持续时间呈现。经典的Wnt信号通路,其激活涉及β-连环蛋白稳定,参与调节NSC行为,包括神经发生和凋亡。为了模拟体内动态信号传导,我们的实验室已经开发了一种可调的光遗传学系统,以通过LRP 6细胞内结构域的Cry 2寡聚化来调节β-连环蛋白信号传导。通过Wnt 3a的典型Wnt途径激活可以导致强大的神经发生,并且我已经观察到,在黑暗和光照周期的低水平波动的存在下,NSC以信号剂量依赖性的方式经历神经元分化。相反,我们观察到β-连环蛋白的布里稳定化随后是延长的信号撤回诱导细胞凋亡,这是一种新的结果,可以消除不完全分化的神经元。具体而言,灾难性的信号损失诱导的光的总撤回,并增加细胞凋亡中观察到暴露于光少于3天。在低强度和变化的光周期条件下,总光子的减少也导致细胞凋亡增加。然而,在24小时内用光“拯救”的细胞没有增加的凋亡,而那些没有光超过24小时的细胞表现出增加的凋亡。此外,我显示了一个信号丢失的“缓冲区”,在该缓冲区内,神经干细胞的神经源性命运对信号波动相对不敏感,而在“缓冲区”之外,神经干细胞的命运显著地向凋亡转移。 因此,动态β-连环蛋白信号传导可能引导NSC朝向分化或凋亡。在目标1中,我们探索了β-连环蛋白(去)稳定的动力学,我们独特的光遗传学系统,通过开/关动力学,转录活性,和下游影响NSC的命运。β-连环蛋白对信号波动和细胞命运决定的动力学响应将与β-连环蛋白磷酸化物质和其他关键蛋白质的水平相关,以深入了解所涉及的分子机制.在目标2中,我们将确定参与凋亡细胞命运的分子机制。在有偏见的策略,我们正在探索已知的蛋白质途径参与凋亡活动。使用ChIP-Seq和RNA-Seq的互补、无偏搜索将鉴定凋亡过程的潜在转录调控。值得注意的是,在整个分化过程中β-连环蛋白的ChIP-seq将广泛用于Wnt信号传导和神经发生的研究。由我们的光遗传学系统提供的信号诱导的精确控制允许对β-连环蛋白动力学进行前所未有的探索。所观察到的凋亡结果可能代表了体内神经发生的重要调控步骤,下游信号效应子的确定将产生更完整的分子机制,通过该机制,经典Wnt信号调节NSC命运。因此,这项工作将促进我们对Wnt信号传导的理解,并努力利用NSC进行神经再生。

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