Neuroprotection by a secreted component of the cellular stress response

细胞应激反应的分泌成分的神经保护作用

基本信息

批准号：
10546480
负责人：
Ulrich Hengst
金额：
$ 52.67万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-15 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10546480
关键词：
Affect Binding C-terminal Caenorhabditis elegans Cell Survival Cell secretion Cells Cellular Stress Central Nervous System ChIP-seq Chondrocytes Cognition Disorders Communication Complex Conditioned Culture Media Data Endoplasmic Reticulum Environment Exhibits Exposure to Extracellular Space Genetic Transcription Glucose Homeostasis In Vitro Knockout Mice Link Malignant Neoplasms Mediating Mediator Membrane Mitochondria Mitochondrial Proteins Modality Molecular N-terminal Nerve Degeneration Nervous System Neurodegenerative Disorders Neurons Oxidative Stress Oxidative Stress Induction Paracrine Communication Pathway interactions Process Production Proteasome Inhibition Protein Fragment Proteins Rattus Reaction Reactive Oxygen Species Reporting Research Project Grants Role SHH gene SOD2 gene Signal Pathway Signal Transduction Signaling Molecule Stimulus Stress Testing Work arm axon growth biological adaptation to stress cell assembly chromatin immunoprecipitation deprivation endoplasmic reticulum stress extracellular improved in vivo intercellular communication nanomolar neuroprotection neurotransmission novel overexpression paracrine programs promote resilience receptor resilience response smoothened signaling pathway stressor transcription factor transcriptome sequencing transmission process

项目摘要

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.

项目概要我们发现神经元分泌一种可溶性蛋白质，可以在细胞应激条件下保护神经元旁分泌方式。值得注意的是，该蛋白质源自内质网 (ER) 的 C 末端膜结合转录因子CREB3L2。蛋白水解裂解释放 N 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 端结构域的产生和分泌是由细胞应激触发的，并且它起作用通过声波刺猬 (Shh) 信号传导增强线粒体功能。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，并将它们暴露于各种细胞应激触发因素，包括氧化应激应激、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整合的应激反应途径对于上调合成和裂解是必要且充分的 CREB3L2 的。我们将研究 CREB3L2 的管腔 C 端结构域如何从细胞中分泌并其分泌途径是否与细胞应激有关。研究分泌的 C 末端结构域如何 CREB3L2影响神经元，我们将纯化它并将其应用于初级皮质神经元。我们将重点关注其提出的在加强Shh信号传导并确定它是否与Shh及其形成复合物中的作用神经元上的受体 Patched-1。我们将确定是否应用分泌的 C 末端结构域 CREB3L2足以增强Shh信号传导，增强Shh信号传导对于其是否是必要的？对细胞存活和线粒体功能的影响。评估分泌的 C 末端结构域的作用 CREB3L2 在线粒体上，我们将采用各种测试来量化线粒体质量的变化，正常条件下神经元中线粒体蛋白的丰度和线粒体功能诱导氧化应激、内质网应激、蛋白酶体抑制和葡萄糖剥夺。乳沟 CREB3L2 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。我们将结合使用染色质免疫沉淀和测序 RNA 测序以确定基线和诱导神经元中 CREB3L2 的转录靶标应激条件，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复。最后，我们将使用先前生成的条件 CREB3L2 敲除小鼠来研究细胞自主臂（即转录因子）和非细胞自主臂的相对作用 CREB3L2 信号在体外和体内神经元应激反应中的作用。总之，该项目的结果将揭示了一种新的细胞间信号传导途径，该途径响应神经元中的细胞应激而被激活，赋予接收神经元增强的弹性和线粒体功能。