早期MVAD对突触可塑性翻译调节机制的影响

Vitamin A deficiency（VAD）is one of the three micronutrient deficiency diseases in the world. Many studies have shown that there are close relationship between vitamin A and neurodevelopment as well as cognition, while the mechanism remains unclear. In our previous study, we have found that marginal vitamin A deficiency (MVAD) from pregnancy might have effect on synaptic plasticity signaling pathway related genes expression in offsprings. The mRNA and protein levels of NMDA receptor subunit NR1 in MVAD rats were significantly lower that those of control rats, and retinoic acid (RA) can promote the mRNA and protein expression of NR1. We also found that RA receptor RARα regulated the expression of NR1 by non-transcription way. Recent research revealed that RARα may have translation regulation role in dendrite. Src family kinase (SFKs) may play key role between RA and NR1. In the present study, we will perform in vitro and in vivo studies to discuss whether RARα located in RNA granule in dendrite in VA normal offspring, and if MVAD from pregnancy have influence on the expression and location of RARα, if RA signaling pathway can regulate the protein expression of SFKs by RARα, if SFKs have effect on expression of NR1. And we will further discuss the influence of MVAD from pregnancy on the regulation of SFKs and NR1. The results of the study will explicit the mechanism of RA on cognition, as well as the new translation regulation role of nuclear transcription factor RARα, as well as the mechanism of MVAD from pregnancy on synaptic plasticity.

维生素A缺乏(VAD)是世界三大微量营养素缺乏性疾病之一，大量研究已证实维生素A营养与神经发育及认知功能有密切关系，而VAD对认知功能影响的机制尚未完全清楚。本课题组前期研究发现孕期开始的边缘型VA缺乏（MVAD）可影响海马NR1表达，体外实验中加入视黄酸（RA）可促进NR1的表达，RA信号可能并不通过直接转录调节影响NR1；国外研究表明RARα可能在树突发挥翻译调节作用；Src家族激酶（SFKs）可能是连接RA和NR1的关键分子。本课题通过体内、外实验，探讨VA正常仔鼠海马神经元中RARα是否定位于树突RNA颗粒；孕期开始的MVAD是否影响RARα的表达及定位；RARα是否翻译调节SFKs，进一步调节NR1的表达；孕期开始的MVAD是否影响RARα对SFKs及NR1表达的调节。研究结果将探讨RARα作为核转录因子的翻译调节新作用，探讨孕期MVAD影响突触可塑性的可能机制。

目的 探讨孕期开始的边缘型维生素A缺乏（MVAD）对仔鼠学习记忆功能影响中，视黄酸受体RARα对NR1的影响是否通过调节Src实现，探讨RARα、Src和NR1之间的联系。方法 构建孕期VA正常（VAN）和MVAD大鼠模型（VAN组和MVAD组）；培养两组新生鼠原代海马神经元；检测新生鼠海马组织及神经元细胞经视黄酸（RA）诱导前后RARα、SFKs和NR1的mRNA、蛋白表达和荧光表达；用RARα沉默和过表达及空载腺病毒转染两组神经元，检测三个指标的mRNA表达；Src家族抑制剂PP2诱导处理神经元后，检测三个指标蛋白表达；免疫共沉淀检测RARα与Src以及NR1是否有相互作用；钙影像仪检测钙兴奋性；水迷宫实验检测7周龄仔鼠学习记忆能力。结果 ①成功构建孕期MVAD和VAN孕鼠模型；②新生鼠原代海马神经元95%表达神经元特异性烯醇化酶；③孕期MVAD新生鼠海马组织及神经元中RARα、SFKs和NR1的mRNA和蛋白表达明显低于VAN组（P＜0.05）；④孕期MVAD组神经元钙兴奋性明显低于VAN组（P＜0.05）；⑤水迷宫实验发现孕期MVAD组仔鼠在生后7W学习记忆功能明显低于孕期VAN组仔鼠（P＜0.01）；⑥予1μmol/L的 RA处理神经元24h后，RARα、SFKs和NR1的mRNA和蛋白表达明显增加（P＜0.001），免疫荧光发现树突明显增长且树突中RARα表达明显升高，且RA处理后的神经元钙兴奋性明显升高（P＜0.01）；⑦予神经元RARα沉默（Si-RARα）和过表达（Over-RARα）干预后，Src的mRNA表达变化同RARα一致，有相应的降低和升高，且变化有统计学差异（P＜0.05），NR1无显著变化；⑧神经元予Src家族激酶抑制剂PP2处理，NR1的蛋白表达明显降低，RARα蛋白无显著变化；⑨两组生后2W仔鼠海马免疫共沉淀发现RARα与Src有相互作用，而RARα与NR1无相互作用；⑩水迷宫训练后，血清VA浓度和RARα、Src和NR1的mRNA水平不变，行为学实验可升高海马组织中RARα、Src和NR1的蛋白表达。结论 孕期MVAD能影响仔鼠学习记忆功能，其机制可能是通过RA信号通路中的RARα转录和非转录作用调节Src的表达，进而影响NR1以及钙内流，从而影响仔鼠海马突触可塑性以及学习记忆功能。