ProNGF对恒河猴脑缺血后认知功能障碍发生和丘脑Aβ代谢的作用及机制研究

Cerebral infarction can induce amyloid β (Aβ) deposition in the ipsilateral thalamus, which considered as a main reason to cause vascular cognitive impairment (VCI). However, the mechanism of such phenomenon is still unclear. In our previous work, we found for the first time, that unprocessed precursor of nerve growth factor (proNGF) was stably increased in the ipsilateral thalamus following focal cerebral infarction and activated neuron death in vivo, but the expression of mature NGF was not significantly change. Moreover, proNGF has been suggested to play a crucial role in the accumulation of Aβ. Based on these evidences, we hypothesize that cognitive impairment following focal cerebral infarction may result from increased proNGF in the ipsilateral thalamus and Aβ deposition may involved in this neurobiological process. We had established a stable acute middle cerebral artery occlusion (MCAO) model in rhesus monkeys in the previous study. In this research, we plan to further our study to determine the contribution of proNGF on the evolution of cognitive impairment and amyloid β deposition in the ipsilateral thalamus following focal cerebral infarction in nonhuman primates. Both proNGF overexpression and proNGF pathway inhibitors will be conducted on a monkey model of MCAO. Then we will use molecular biological techniques such as laser confocal microscope to identify the effect of proNGF on the damage of neurovascular unit in the ipsilateral thalamus, analysis its association to behavioral manifestations and Aβ deposition, and investigate its possible mechanisms. Our result may provide an important basis for a better understanding in the pathophysiology of VCI.

局灶脑缺血会诱发非缺血区丘脑β淀粉样蛋白（Aβ）过度沉积，被认为是导致血管性认知功能障碍（VCI）发病重要原因之一，但其机制尚未阐明。前期工作中我们首次发现局灶脑缺血可引起同侧丘脑神经生长因子前体（proNGF）长时间表达上调，诱发神经元死亡；而近年研究认为proNGF是介导Aβ代谢的关键受体。据此我们推测局灶脑缺血可能通过上调丘脑proNGF表达影响Aβ代谢，进而诱发VCI。本课题拟在前期建立的恒河猴大脑中动脉阻塞（MCAO）模型基础上，结合分子生物学、激光共聚焦等技术，应用慢病毒转染上调proNGF并对其作用于Aβ的多个环节给予抑制剂，从细胞和活体动物两个层面，观察干预proNGF对局灶脑缺血后丘脑神经元、微血管内皮细胞和星形胶质细胞凋亡、增殖的影响，分析与恒河猴认知功能状况相关性和对丘脑Aβ代谢的调控作用，并探讨可能的机制。研究结果将对进一步揭示VCI的发生机制具有非常积极的意义。

脑卒中对脑功能的影响是全面的。不仅局限于运动及感觉，而且会严重影响认知等高级皮质功能，引起血管性认知功能损害（VCI）。局灶脑缺血会诱发非缺血区丘脑β淀粉样肽（Aβ）过度沉积，被认为是VCI发病重要原因之一。近年研究认为神经生长因子前体（proNGF）是介导Aβ代谢的关键受体。本自然基金青年项目以proNGF为干预靶点，考察其与VCI的相关性和对丘脑Aβ代谢的调控作用，探索VCI的病理生理机制。项目组成功建立起神经细胞培养体系，并建立糖氧剥夺（OGD）模型。通过免疫荧光、WB和PCR等多种方法均显示神经细胞经OGD后proNGF和P75NTR、Sortilin等相关通路蛋白表达逐步增高。进一步的，项目组通过大脑中动脉栓塞（MCAO）模型并辅以行为学筛选方式，建立起小鼠VCI模型。在该模型发现随时间推移，其丘脑部位proNGF及P75NTR、Sortilin等相关通路蛋白呈现逐步增高的动态变化，且这一变化同Aβ的动态变化呈现一致。慢病毒转染上调proNGF后，VCI鼠的行为学进一步加重，且P75NTR、Sortilin和Aβ的表达也随之上调，神经细胞死亡随之加剧。而加用P75NTR通道阻断剂GST-pro后，免疫荧光和WB显示proNGF相关蛋白P75NTR、Sortilin和Aβ表达量下降，同时神经细胞死亡减少，神经重塑更加活跃。由此可见，项目组在工作中通过体外和体内两个层面，发现proNGF可介导信号通路增加VCI模型丘脑Aβ的沉积，促进神经细胞凋亡和抑制神经功能恢复，从而抑制VCI后神经轴突的重塑，加重疾病的发生发展。而对proNGF的干预，有可能逆转这一过程。这一发现不仅是对VCI作用机制的新补充，而且对疾病提供了一个非常有价值的潜在干预靶点。