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

 描述(申请人提供)：帕金森氏症和阿尔茨海默氏症是影响全球4000万人的神经退行性疾病。成人神经干细胞(NSC)作为细胞替代疗法具有很大的潜力。成年的神经干细胞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的方式出现。典型的Wnt信号通路参与调节神经干细胞的行为，包括神经发生和细胞凋亡，它的激活涉及？连环蛋白的稳定。为了模拟体内的动态信号，我们实验室开发了一种可调的光遗传系统，通过LRP6细胞内结构域的Cry2寡聚来调节？连环蛋白信号。通过Wnt3a激活规范的Wnt通路可以导致强大的神经发生，我观察到NSCs在暗周期和光周期的低水平波动存在的情况下，以信号剂量依赖的方式进行神经元分化。相反，我们观察到，在延长信号撤除之后，çcatenin的Brie稳定化诱导了细胞凋亡，这一新的结果可能有助于消除未完全分化的神经元。具体地说，完全撤光导致灾难性的信号丢失，并观察到暴露在低于3天的光照下的细胞凋亡增加。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的增加。然而，在24小时内被光“拯救”的细胞并没有增加凋亡，而那些没有光超过24小时的细胞则有更多的凋亡。此外，我还显示了一个信号丢失的“缓冲区”，在这个缓冲区内，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，神经干细胞的命运明显转向凋亡。 因此，动态的ç-catenin信号很可能将神经干细胞导向分化或凋亡。在目标1中，我们利用我们独特的光遗传系统，通过开/关动力学、转录活性以及下游对神经干细胞命运的影响，来探索？连环蛋白(De-)稳定的动力学。连接蛋白对信号波动和细胞命运决定的动态响应将与连接蛋白磷酸化物种和其他关键蛋白的水平相关联，以深入了解相关的分子机制(S)。在目标2中，我们将确定参与细胞凋亡命运的分子机制(S)。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通路。用ChIP-Seq和RNA-Seq进行互补的、无偏见的搜索将识别潜在的对细胞凋亡过程的转录调控。值得注意的是，在整个分化过程中，ç-catenin的ChIP-SEQ将广泛用于Wnt信号和神经发生的研究。我们的光遗传系统提供了对信号诱导的精确控制，这使得对ç-catenin动力学的前所未有的探索成为可能。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，对下游信号效应的确定将产生更完整的分子机制，规范的Wnt信号通过这些机制调控NSC的命运。因此，这项工作将促进我们对Wnt信号的理解，以及利用NSCs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.