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

 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。成人 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 Wnt 信号通路的激活涉及 β-连环蛋白的稳定，参与调节 NSC 行为，包括神经发生和细胞凋亡。为了模拟体内动态信号传导，我们的实验室开发了一种可调光遗传学系统，通过 LRP6 胞内结构域的 Cry2 寡聚化来调节 ß-catenin 信号传导。通过 Wnt3a 激活典型的 Wnt 通路可以导致强大的神经发生，并且我观察到 NSC 在暗周期和光周期低水平波动的情况下以信号剂量依赖性方式经历神经元分化。相比之下，我们观察到β-连环蛋白的布里稳定性随后延长信号撤回会诱导细胞凋亡，这是一种可能消除不完全分化神经元的新结果。具体来说，光的完全消失会导致灾难性的信号丢失，并且在暴露于光不足3天的细胞中观察到细胞凋亡增加。在低强度和不同的光周期条件下，总光子的减少也会导致细胞凋亡增加。然而，在24小时内用光“拯救”的细胞并没有增加细胞凋亡，而那些没有光超过24小时的细胞则表现出细胞凋亡增加。此外，我还显示了一个信号丢失的“缓冲区”区域，在该区域内，NSC 的神经源性命运对信号波动相对不敏​​感，而在“缓冲区”之外，NSC 的命运显着转向细胞凋亡。 因此，动态β-连环蛋白信号传导可能引导 NSC 分化或凋亡。在目标 1 中，我们利用我们独特的光遗传学系统，通过开/关动力学、转录活性和对 NSC 命运的下游影响来探索 β-连环蛋白（去）稳定性的动态。 β-连环蛋白对信号波动和细胞命运决定的动力学反应将与 β-连环蛋白磷酸化物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。在有偏见的策略中，我们正在探索参与细胞凋亡活动的已知蛋白质途径。利用 ChIP-Seq 和 RNA-Seq 进行互补、公正的搜索将鉴定细胞凋亡过程的潜在转录调控。值得注意的是，β-连环蛋白整个分化过程的 ChIP-seq 将广泛用于 Wnt 信号传导和神经发生的研究。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。观察到的细胞凋亡结果可能代表体内神经发生的重要调节步骤，下游信号传导效应器的确定将产生更完整的分子机制，规范的 Wnt 信号传导通过该机制调节 NSC 命运。因此，这项工作将增进我们对 Wnt 信号传导的理解以及利用 NSC 进行神经再生的努力。

 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.