The role of proBDNF-p75NTR signaling in hind limb ischemia.

proBDNF-p75 NTR信号通路在后肢缺血中的作用 脑源性神经营养因子（BDNF）及其前体proBDNF已被很好地表征， 它们分别在中枢神经系统中的神经保护和神经炎症中的对比作用。 最近，BDNF和proBDNF已被证明存在于中枢神经系统以外的组织中，但 它们在这些组织中的作用仍不清楚。在我的研究中，整个肌肉裂解物含有高 与全脑裂解物相比，proBDNF水平和相对低水平的成熟BDNF。该项目旨在 探讨骨骼肌源性脑源性神经营养因子原（proBDNF）在后肢缺血再灌注（IR）中的功能作用 损伤在该模型中，其中C57 BL/6小鼠经受右后肢的结扎，proBDNF及其mRNA表达水平降低。 受体p75 NTR均上调，p75 NTR下游信号通路在细胞中被激活。 缺血肌肉与对侧对照肢体肌肉相比。数据进一步表明，治疗 与对照组相比，IR损伤后使用p75 NTR调节剂LM 11 A-31改善了缺血肢体的灌注。 未处理的IR对照，表明内皮功能改善。此外，骨骼肌特异性敲除 与野生型小鼠相比，BDNF显著改善了缺血肢体的血液灌注。先前 研究表明内皮细胞p75 NTR可能介导血管损伤。基于这些结果，我们 假设IR损伤后骨骼肌中上调的proBDNF可能刺激内皮细胞 p75 NTR，促进细胞间结构域（ICD）的切割和随后的PKA诱导的细胞凋亡的抑制。 eNOS的磷酸化和一氧化氮的产生导致血管功能障碍和炎症。测试 根据这一假设，将使用骨骼肌特异性敲除BDNF的小鼠来确定 肌源性proBDNF在IR损伤中的作用然后将内皮特异性敲除p75 NTR的小鼠用于 检测内皮细胞p75 NTR是否介导IR损伤。最后，将使用体外和体内实验 为了确定p75 NTR的ICD对PKA的抑制以及随后的eNOS功能是导致 红外线损伤。该项目的结果将揭示IR损伤的新机制，并提出新的靶点 用于预防和治疗患者的IR损伤。在上级知识分子的指导下， 在该领域拥有20多年经验的导师的支持，实验设计的功能知识， 数据分析和解释，以及科学交流将得到发展，导致博士学位。和有利 在生物医学研究中取得成功和富有成效的职业生涯。实验将使用最先进的 一流的设施中的研究设备，为学习，成长和支持提供了充足的机会。

The role of proBDNF-p75NTR signaling in hind limb ischemia Brain-derived neurotrophic factor (BDNF) and its precursor proBDNF have been well characterized in their contrasting roles in neuroprotection and neuroinflammation in the central nervous system respectively. Recently, BDNF and proBDNF have been shown to exist in tissues outside of the central nervous system, but their role in these tissues is still unclear. In my study it was shown that whole muscle lysate contains high levels of proBDNF and relatively low levels of mature BDNF compared to whole brain lysate. This project aims to investigate the functional role of skeletal muscle-derived proBDNF in hind limb ischemia-reperfusion (IR) injury. In this model, in which C57BL/6 mice are subjected to ligation of the right hind limb, proBDNF and its receptor p75NTR were both upregulated, and p75NTR downstream signaling pathways were activated in the ischemic muscle compared to the contralateral control limb muscles. The data further showed that treatment with LM11A-31, a p75NTR moderator, following IR injury improved perfusion of the ischemic limb compared to untreated IR controls, indicating improved endothelial function. Moreover, skeletal muscle-specific knockout of BDNF significantly improved the blood perfusion in the ischemic limb compared to wild-type mice. Previous studies have suggested that endothelial p75NTR may mediate vascular injury. Based on these results, we hypothesize that the upregulated proBDNF in skeletal muscle following IR injury may stimulate endothelial p75NTR, promoting cleavage of the intercellular domain (ICD) and subsequent inhibition of PKA-induced phosphorylation of eNOS and nitric oxide production leading to vascular dysfunction and inflammation. To test this hypothesis, mice with skeletal muscle-specific knockout of BDNF will be used to determine the role of muscle-derived proBDNF in IR injury. Then mice with endothelial-specific knockout of p75NTR will be used to examine whether endothelial p75NTR mediates IR injury. Finally, in vitro and in vivo experiments will be used to define the inhibition of PKA by the ICD of p75NTR and subsequent eNOS function being responsible for the IR injury. The results of this project will reveal a novel mechanism underlying IR injury and suggest new targets for the prevention and treatment of IR injury in patients. While under the superior intellectual guidance and support of a mentor with over 20 years of experience in the field, functional knowledge of experimental design, data analysis and interpretation, and scientific communication will be developed, leading to Ph.D. and enabling a successful and productive career in biomedical research. Experiments will be carried out using top-of-the-line research equipment in premier facilities with ample opportunities for learning, growth, and support.