Neuroprotection by a secreted component of the cellular stress response

项目摘要 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。 旁分泌方式值得注意的是，这种蛋白质来源于内质网（ER）的C-末端。 膜结合转录因子CREB 3L 2。蛋白水解裂解释放N-末端转录 因子，并导致ER管腔结构域分泌到细胞外空间。我们的初步数据 表明该C-末端结构域的产生和分泌是由细胞应激触发的， 通过音速刺猬（Shh）信号增加线粒体功能。在这个项目中，我们正在测试 CREB 3L 2-C是适应性神经元应激反应的旁分泌成分的一般假设。 我们将培养原代大鼠皮层神经元，并将其暴露于各种细胞应激触发物，包括氧化应激。 应激、ER应激、蛋白酶体抑制和葡萄糖剥夺。我们将确定是否激活 整合的应激反应途径是上调合成和裂解所必需的， CREB3L2我们将研究CREB 3L 2的管腔C末端结构域如何从细胞分泌， 其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 CREB 3L 2影响神经元，我们将纯化它并将其应用于初级皮质神经元。我们将专注于其 提出了在加强Shh信号传导中的作用，并确定它是否与Shh及其 神经元上的受体Patched-1。我们将确定是否应用的分泌，C-末端结构域的 CREB 3L 2足以增加Shh信号传导，以及增强的Shh信号传导是否是其所必需的。 影响细胞存活和线粒体功能。为了评估分泌的C-末端结构域的作用， CREB 3L 2对线粒体的影响，我们将采用各种测试来量化线粒体质量的变化， 丰富的线粒体蛋白质，和正常条件下神经元中的线粒体功能， 诱导氧化应激、ER应激、蛋白酶体抑制和葡萄糖剥夺。裂解 CREB 3L 2的表达必然激活两种不同的信号分子，即细胞内转录因子和 分泌的C末端。我们将使用染色质免疫沉淀与测序相结合， RNA测序以确定基线下和诱导下神经元中CREB 3L 2的转录靶点 应激条件下，以确定两种信号分子是否在适应性细胞应激中协同作用 反应最后，我们将使用先前产生的条件性CREB 3L 2敲除小鼠来研究CREB 3L 2基因的表达。 细胞自主（即转录因子）和非细胞自主臂的相对作用 CREB 3L 2信号在体外和体内神经元应激反应中的作用总之，该项目的成果将 揭示了一种新的细胞间信号通路，该通路在神经元中响应细胞应激而被激活， 赋予接受神经元增强的弹性和线粒体功能。

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.