Restoration of Endoplasmic Reticulum Function in Experimental Stroke

描述(申请人提供)：短暂性脑缺血/中风损害内质网(ER)功能，进而触发不可逆转的翻译停滞 在脆弱的神经元中。当内质网功能被严重的应激不可逆转地破坏时，细胞就不能存活。因此，对于短暂的血液供应中断后神经元的功能恢复，ER功能的恢复与缺血组织的再灌注一样重要。然而，内质网功能障碍在最终导致神经元缺血性细胞死亡的病理过程中的确切作用尚未确定。填补这一知识空白至关重要，因为在我们做到这一点之前，通过恢复内质网功能使神经元对短暂性缺血更具抵抗力的可能性仍未被探索。长期目标是了解内质网功能如何被操纵以达到预防和治疗目的。这里的目的是创造表达短型ATF6的转基因动物，ATF6是一种恢复受损ER功能所需的转录因子。一旦被创造出来，转基因动物将成为阐明内质网功能恢复如何调控神经元从缺血诱导的严重代谢应激中恢复的关键平台。中心假设是内质网功能的恢复是神经元从短暂性脑缺血中恢复的关键。这一假设是基于我们实验室产生的数据而提出的。这项研究的基本原理是，一旦我们确定了内质网功能受损在缺血诱导的细胞死亡中的作用，就很可能开发出新的创新方法来预防或治疗与内质网功能受损相关的各种疾病。在强大的初步数据的指导下，这一假说将通过追求以下特定目标来验证：与Cre转基因动物杂交后创造表达ATF6的转基因小鼠。ATF6xCre小鼠将表达ATF6，ATF6保留在细胞质中，需要他莫昔芬诱导才能进行核转位，从而激活ATF6依赖基因的表达。在他莫昔芬激活后，动物将受到短暂性局灶性脑缺血的影响，以观察ATF6和ATF6依赖基因的诱导表达对梗塞面积的影响。这种方法是创新的，因为它首次专注于为中风的治疗目的操纵神经元的内质网功能。这项拟议的研究意义重大，因为它有望导致新的预防和治疗干预策略的开发，使细胞对短暂的脑缺血/中风具有更强的抵抗力。此外，ATF6转基因动物将为未来的研究提供一个有希望的工具，以调查ER功能障碍在多种具有高度临床相关性的疾病中的作用，在与Cre转基因动物杂交后，在所研究的器官/细胞类型中特异性表达ATF6。

DESCRIPTION (provided by applicant): Transient cerebral ischemia/stroke impairs endoplasmic reticulum (ER) function, which in turn triggers translation arrest that is irreversible in vulnerable neurons. Cells cannot survive when ER function is irreversibly damaged by a severe form of stress. For functional recovery of neurons following a transient interruption of blood supply, restoration of ER function is therefore as crucial as reperfusion of the ischemic tissue. However, the precise role of ER dysfunction in the pathological process that culminates in ischemic cell death of neurons has never been established. It is of key importance to fill this gap in our knowledge because until we do, the possibility of making neurons more resistant to transient ischemia by restoring ER function will remain unexplored. The long-term goal is to understand how ER functioning can be manipulated for preventive and therapeutic purposes. The objective here is to create transgenic animals expressing the short form of ATF6, a transcription factor required to restore impaired ER function. Once created, the transgenic animals will be a pivotal platform to elucidate how restoration of ER function modulates recovery of neurons from the severe metabolic stress induced by ischemia. The central hypothesis is that restoration of ER function is essential for recovery of neurons from transient ischemia. This hypothesis has been formulated on the basis of data generated in our laboratories. The rationale for the proposed research is that once we have established the role of impaired ER function in ischemia-induced cell death, new and innovative approaches can most probably be developed to prevent or treat a variety of diseases associated with impaired ER function. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following specific aim: Create transgenic mice expressing ATF6 after crossbreeding with Cre transgenic animals. The ATF6xCre mice will express ATF6 that is retained in the cytoplasm and requires induction by tamoxifen for nuclear translocation and thus activation of the expression of ATF6-dependent genes. After tamoxifen activation, animals will be subjected to transient focal cerebral ischemia to investigate the effects of induced expression of ATF6 and ATF6-dependent genes on size of infarcts. The approach is innovative, because it focuses for the first time on manipulating ER function of neurons for therapeutic purposes in stroke. The proposed research is significant, because it is expected to lead to the development of new strategies for preventive and therapeutic interventions to render cells more resistant to a transient episode of cerebral ischemia/stroke. Furthermore, the ATF6 transgenic animals will provide a promising tool for future studies to investigate the role of ER dysfunction in a large variety of disorders of high clinical relevance, after cross- breeding with Cre transgenic animals to specifically express ATF6 in the organ/cell type under investigation.