Neuroprotection by a secreted component of the cellular stress response

项目总结 我们发现，神经元分泌一种可溶性蛋白，在细胞应激条件下保护神经元 旁分泌的方式。值得注意的是，这种蛋白来自内质网(ER)的C端。 膜结合转录因子CREB3L2。蛋白水解性切割释放N-端转录 并导致内质网管腔结构域分泌到细胞外空间。我们的初步数据 表明这种C-末端结构域的产生和分泌是由细胞应激触发的，它起作用 通过Sonic Hedgehog(Shh)信号增强线粒体功能。在这个项目中，我们正在测试 一般假设CREB3L2-C是适应性神经元应激反应的旁分泌成分。 我们将培养原代大鼠皮质神经元，并将它们暴露在各种细胞应激触发因素下，包括氧化 应激、内质网应激、蛋白酶体抑制和葡萄糖剥夺。我们将确定是否激活 完整的应激反应途径是上调合成和裂解的必要条件和充分条件。 CREB3L2。我们将研究CREB3L2的管腔C-末端结构域是如何从细胞和 它的分泌途径是否与细胞应激有关。为了研究分泌的C-末端结构域是如何 CREB3L2影响神经元，我们将对其进行纯化，并将其应用于原代皮质神经元。我们将重点关注其 建议在加强Shh信号转导中的作用并确定其是否与Shh及其受体形成复合体 神经元上的受体补丁-1。我们将确定分泌的C-末端结构域的应用是否 CREB3L2足以增加Shh信号，以及增强的Shh信号是否对ITS是必要的 对细胞存活和线粒体功能的影响。为了评价分泌的C-末端结构域的作用 在线粒体上的CREB3L2，我们将使用各种测试来量化线粒体质量的变化， 正常和正常情况下神经元线粒体蛋白的丰度和线粒体的功能 在氧化应激、内质网应激、蛋白酶体抑制和葡萄糖剥夺的诱导下。乳沟 CREB3L2必须激活两个不同的信号分子，细胞内转录因子和 分泌的C末端。我们将使用染色质免疫沉淀和测序相结合的方法 RNA测序确定基线和诱导条件下神经元中CREB3L2的转录靶点 应激条件，以确定两个信号分子是否在适应性细胞应激中协同作用 回应。最后，我们将使用之前生成的条件CREB3L2基因敲除小鼠来研究 细胞自主(即转录因子)和非细胞自主臂的相对作用 CREB3L2信号在体内外神经元应激反应中的作用总而言之，该项目的结果将 揭示一种新的细胞间信号通路，该通路在神经元和细胞应激反应中被激活 赋予接受神经元更强的韧性和线粒体功能。

PROJECT SUMMARY We found that neurons secrete a soluble protein that protects neurons under cell stress conditions in a paracrine manner. Remarkably, this protein is derived from the C-terminus of the endoplasmic reticulum (ER) membrane bound transcription factor CREB3L2. Proteolytic cleavage liberates the N-terminal transcription factor and leads to the secretion of the ER luminal domain into the extracellular space. Our preliminary data indicate that the production and secretion of this C-terminal domain is triggered by cell stress and that it acts by increasing mitochondrial function via sonic hedgehog (Shh) signaling. In this project we are testing the general hypothesis that CREB3L2-C is a paracrine component of the adaptive neuronal stress response. We will culture primary rat cortical neurons and expose them to various cell stress triggers, including oxidative stress, ER stress, proteasome inhibition, and glucose-deprivation. We will determine whether the activation of the integrated stress response pathway is necessary and sufficient to upregulate the synthesis and cleavage of CREB3L2. We will investigate how the luminal C-terminal domain of CREB3L2 is secreted from cells and whether its secretory pathway is linked to cell stress. To investigate how the secreted C-terminal domain of CREB3L2 affects neurons, we will purify it and apply it to primary cortical neurons. We will focus on its proposed role in strengthening Shh signaling and determine whether it forms complexes with Shh and its receptor Patched-1 on neurons. We will determine whether application of the secreted, C-terminal domain of CREB3L2 is sufficient to increase Shh signaling and whether enhanced Shh signaling is necessary for its effects on cell survival and mitochondrial function. To assess the effect of the secreted C-terminal domain of CREB3L2 on mitochondria, we will employ a variety of tests to quantify changes to mitochondrial mass, abundance of mitochondrial proteins, and mitochondrial function in neurons under normal conditions and upon induction of oxidative stress, ER stress, proteasome inhibition, and glucose-deprivation. The cleavage of CREB3L2 activates necessarily two distinct signaling molecules, the intracellular transcription factor and the secreted C-terminus. We will use chromatin immunoprecipitation with sequencing in combination with RNA sequencing to determine the transcriptional targets of CREB3L2 in neurons under baseline and induced stress conditions, to determine whether both signaling molecule act cooperatively in the adaptive cell stress response. Lastly, we will use a previously generated conditional CREB3L2 knockout mouse to study the relative roles of the cell autonomous (i.e. the transcription factor) and the non-cell autonomous arm of CREB3L2 signaling in neuronal stress response in vitro and in vivo. Together, the results from this project will uncover a novel intercellular signaling pathway that is activated in response to cell stress in neurons and confers enhanced resilience and mitochondrial function to receiving neurons.