同时抑制NR2B和增强NR2A对AD模型动物神经突触可塑性和学习记忆能力的影响及初步机制探讨

N-methyl-D-aspartate receptors (NMDARs) play an important role in Alzheimer's disease (AD). NMDARs exist as heteromeric complexes, most frequently composed of two NR1 and two NR2 (A, B, C and D) subunits. Lots of evidences indicated that NR2A subunits mediate protective pathway and NR2B subunits makes a major contribution to excitotoxic pathway. Some researches indicate over activation of NMDA receptor induced excitotoxicity is a main pathogenic factor for AD. Contradictory, there are many data support the functions of NMDA receptor were down regulated in AD. In our opinion, the function of NMDA receptor in AD are both over activated and down regulated. Over activation refers to NR2B induced excitotoxicity, down regulation refers to normal physiological function of NR2A. In our previous study, we found that treating hippocampal slices with Aβ[25-35] or NMDA suppressed the induction of long-term potentiation (LTP) significantly. NR2B antagonist ifenprodil could not prevent the disruption of LTP in slices treated with Aβ[25-35] by perfusion. Co-applying ifenprodil and NMDA has protective effect on LTP against Aβ[25-35] perfusion. As the NMDARs are mainly consisting of NR2B and NR2A in the hippocampal tissues of adult rats, co-applying ifenprodil with NMDA means mainly activating NR2A. These results indicated that downregulating the function of NR2A and activating NR2B may be a main factor contributing to Aβ playing its disruptive effect on LTP. So we propose that blocking NR2B and activating NR2A simultaneously might be a potential treatment strategy for AD. In this study, two AD animal models will be used to elucidate the effect of blocking NR2B and activating NR2A simultaneously on cognitive behaviors; then we investigate the role of NR2A and NR2B in Aβ induced LTP impairment; finally drugs used in clinic are used to confirm our proposal. We hope this study will provide valuable data for the R&D of AD treatment drug.

NMDA受体在AD的病理生理过程中起着重要的作用。但现有研究存在矛盾之处，有研究表明AD病人NMDA受体兴奋过度，而另一些研究提示AD病人NMDA受体功能低下。我们认为AD病人同时存在NMDA受体兴奋过度和功能不足：不过兴奋过度是NMDA受体NR2B亚型介导，不足是介导正常生理功能的NR2A功能不足。我们前期研究初步表明，同时激活NR2B和抑制NR2A可能是Aβ介导LTP损伤的机制之一。所以我们认为，同时抑制NR2B和增强NR2A对防治AD可能具有更好的疗效。本研究分三部分（效果考察、机制初探和进一步验证），首先考察同时抑制NR2B和增强NR2A对AD模型动物认知功能的影响；接着系统考察NR2A和NR2B在Aβ介导的LTP损伤中的作用，以便进行机制的初步探讨；最后考察AD治疗药物对AD模型动物认知功能影响的异同，并对我们的假设进一步验证。以期在此基础上为AD药物研发提供科学实验依据。

基于现有研究，我们认为AD病人NMDA受体亚型功能平衡紊乱（介导正常生理功能的NR2A功能不足，而介导兴奋性毒性的NR2B功能增强）。本研究以AD的主要病理损伤因素Aβ作为损伤因素，在整体动物水平、组织水平和信号转导通路水平考察Aβ是否会导致NR2A/B平衡紊乱，以及恢复其平衡是否可改善Aβ致神经突触可塑性和学习记忆行为损伤。结果表明，Aβ可明显损伤离体海马片的LTP；给予非特异性的NMDA受体激动剂（NMDA）、拮抗剂（Memantine）和NR2B特异性拮抗剂（Ifenprodil）均对Aβ所致LTP损伤无明显影响；NR2A特异性激动剂D-serine以及联合给给药方式实现NR2A功能增强（NMDA和Ifenprodil）均可明显改善Aβ所致的LTP损伤；联合给予D-serine和Ifenprodil同时抑制NR2B和激动NR2A也可改善Aβ所致LTP损伤，且其改善强度较单独激活NR2A更强，本部分实验结果表明NR2A功能的降低和NR2B功能的增强可能是Aβ诱导LTP损伤的机制之一。接着在Aβ侧脑室注射模型上我们也得到了类似的结果，Aβ可诱导动物多种认知相关行为的损伤，单独给予memantine、Ifenprodil和D-cycloserine均对认知行为有一定的改善作用，但同时给予Ifenprodil和D-cycloserine实现同时抑制NR2B和激动NR2A的改善效果最佳。信号通路分析也证实，Aβ处理的动物海马组织中NR2A和NR2B的比例降低，NR2A相关的TORC和REK1/2磷酸化水平降低，从而导致其下游信号p-CREB和BDNF水平降低，给予memantine、Ifenprodil和D-cycloserine均可一定程度恢复NR2A和NR2B相关信号通路的紊乱，但同时给予Ifenprodil和D-cycloserine实现同时抑制NR2B和激动NR2A对信号通路的紊乱改善作用最强。本研究表明，抑制NR2A和增强NR2B从而导致NR2A/B功能失衡是Aβ诱导突触可塑性和认知功能损伤的重要途径之一，恢复NR2A/B的功能平衡可改善Aβ所致的突触可塑性和认知功能损伤，提示同时抑制NR2B和增强NR2A可能是AD治疗和相关药物研发的一个有效策略。基于本研究的相关数据，已基本证明了研究计划中提出的相关假说和回答了研究计划中提出的关键问题。