AAV-Mediated Overexpression of Neuroserpin in the Hippocampus Decreases PSD-95 Expression but Does Not Affect Hippocampal-Dependent Learning and Memory

Neuroserpin is a serine protease inhibitor, or serpin, that is expressed in the nervous system and inhibits the protease tissue plasminogen activator (tPA). Neuroserpin has been suggested to play a role in learning and memory but direct evidence for such a role is lacking. Here we have used an adeno-associated virus (AAV) vector expression system to investigate the effect of neuroserpin on hippocampal-dependent learning and memory in the young adult rat. A FLAG-tagged neuroserpin construct was initially characterized by in vitro transcription/translation and transfection into HEK293 cells and shown to interact with tPA and be targeted to the secretory pathway. Targeted injection of a chimeric AAV1/2 vector expressing FLAG-neuroserpin resulted in localized overexpression in the dorsal hippocampus. Neuroserpin overexpression led to the appearance of an unstable neuroserpin:tPA complex in zymographic assays consistent with interaction with endogenous tPA in vivo. Rats overexpressing neuroserpin also showed a significant decrease in the levels of postsynaptic density protein 95, a major postsynaptic scaffolding protein. Three weeks after injection, a range of behavioural tests was performed to measure spatial and associative learning and memory, as well as innate and acquired fear. These tests provided no evidence of a role for neuroserpin in hippocampal-dependent learning and memory. In summary this study does not support a role for neuroserpin in hippocampal-dependent learning and memory in young adult rats but does suggest an involvement of neuroserpin in hippocampal synaptic plasticity.神经丝氨酸蛋白酶抑制蛋白（Neuroserpin）是一类丝氨酸蛋白酶抑制剂家族（serpin）成员，在神经系统中表达，可靶向抑制组织型纤溶酶原激活剂（tissue plasminogen activator, tPA）。既往研究提示该蛋白可能参与学习与记忆过程，但尚未有直接实验证据支持这一功能。本研究借助腺相关病毒（adeno-associated virus, AAV）载体表达系统，探究了神经丝氨酸蛋白酶抑制蛋白对年轻成年大鼠海马依赖性学习记忆的影响。首先，本研究通过体外转录/翻译体系以及转染HEK293细胞，对FLAG标签标记的神经丝氨酸蛋白酶抑制蛋白重组构建体进行了功能鉴定，证实其可与tPA特异性结合，并被定向分选至分泌途径。将携带FLAG-神经丝氨酸蛋白酶抑制蛋白的嵌合型AAV1/2载体定向注射至大鼠背侧海马，成功实现了该脑区的局部过表达。酶谱分析结果显示，神经丝氨酸蛋白酶抑制蛋白过表达后可形成不稳定的神经丝氨酸蛋白酶抑制蛋白:tPA复合物，该现象与体内内源性tPA的结合效应一致。此外，过表达神经丝氨酸蛋白酶抑制蛋白的大鼠，其主要突触后支架蛋白突触后致密蛋白95（postsynaptic density protein 95）的表达水平显著降低。注射三周后，本研究通过一系列行为学实验检测大鼠的空间学习记忆、联合学习记忆能力，以及先天恐惧与获得性恐惧反应。实验结果未发现神经丝氨酸蛋白酶抑制蛋白参与海马依赖性学习记忆的直接证据。综上，本研究不支持年轻成年大鼠中神经丝氨酸蛋白酶抑制蛋白在海马依赖性学习记忆中发挥功能的假说，但提示其可能参与海马突触可塑性过程。