多巴胺D4受体基因多态性变异体通过PSD-95调节NMDA受体功能的研究

Abnormal regulation of glutamate receptors functions by dopamine D4 receptors (D4R) in prefrontal cortex (PFC) is highly implicated in mental disorders, such as attention deficit hyperactivity disorder (ADHD) and schizophrenia. The D4 receptor gene contains extensive polymorphism in its exon 3 in a region which codes for proline-rich domains in the third intracellular loop (IC3) of the receptor. A 48 base pair exists 2-11 folds repeats (2X16 to 11X16 amino acids), and the most common variants are D4.4R and D4.7R which contain 4 and 7 repeats, respectively. ..Previous studies have described that the variant D4.7R of D4 receptors associates with an increased risk for the development of ADHD, however, the cellular and molecular mechanisms remain elusive. In our preliminary studies, we found that both D4.4R and D4.7R bind directly with scaffold protein PSD-95 under physiological conditions, moreover, the binding strength between D4.7R and PSD-95 increases after activation of the receptor itself, whereas the binding between D4.4R and PSD-95 remains unchanged (unpublished data). More interesting is that the interaction binding between NR1 subunit of NMDA receptor (NMDAR) and PSD-95 was differently affected by these two variants activation, namely D4.7R has a stronger effect on decreasing the binding between NR1 and PSD-95 than D4.4R. ..The NMDA receptor is an important type of ionotropic glutamate receptor, it plays a key role in synaptic plasticity. Hypofunction of NMDA receptor in PFC is associated with ADHD. Functions of NMDA receptor rely largely on its protein-protein interaction with PSD-95, by which PSD-95 organizes NMDA receptor and its associated signaling proteins, and stabilizes NMDA receptor in the synaptic membrane. Based on our preliminary studies, our hypothesis is that D4.7R binds more PSD-95 than D4.4R after receptor activation, thus causes a competitive decreased binding between NMDAR and PSD-95, and results in NMDA receptor internalization. This effect of D4.7R may lead to NMDA receptor hypofunction. ..In this study, we use a combination of multiple approaches, such as whole-cell patch-clamp recordings and biochemical techniques, to examine the role of PSD-95 in the signaling pathway of regulation of NMDA receptor currents by the D4 receptor variants, and compare the effects of the variants on regulation of NMDA receptor function. Our study will provide new valuable clues for elucidating the role of D4.7R underlying pathogenesis of ADHD.

多巴胺D4受体对前额叶皮层谷氨酸受体功能调节的异常与几种精神病的发生相关，如注意力缺陷多动症(ADHD)，精神分裂症。人类D4受体基因在其外显子3存在多态性，导致受体的第3个胞内环出现16个氨基酸的串联重复，最常见重复数为4和7(D4.4R, D4.7R)。研究发现D4.7R和ADHD的发病呈相关性，但其机制有待深入阐明。我们的前期研究发现，这两个变异体在生理条件下都能和PSD-95发生直接结合，而且当自身激活后，D4.7R与PSD-95的结合增强，D4.4R与PSD-95的结合基本不变。更有意义的是，D4.7R激活后减少NMDA受体NR1亚基和PSD-95之间的相互结合，而D4.4R对它们结合的影响较小。本课题结合生物化学、膜片钳等技术，研究PSD-95在D4受体调节NMDA受体功能中的作用，比较D4受体变异体对NMDA受体功能影响的区别，为D4.7R与ADHD的相关性提供新的理论依据。

人类多巴胺D4受体（D4R）基因存在多态性，其多态表现型D4.7R与注意力缺陷多动障碍（ADHD）存在相关性，但其作用机理有待阐明。本课题研究了D4.7R与支架蛋白PSD-95的相互作用、D4.7R对NMDA受体（NMDAR）功能的调节作用、以及对小鼠行为活动的影响。结果表明：1）D4受体通过其外显子3编码的脯氨酸重复区和PSD-95的SH3结构域发生相互结合。2）D4.7R激活后显著减少NMDA受体NR1亚基和PSD-95的相互结合，并明显降低NR1在神经细胞膜表面表达；而对照D4.4R对NR1和PSD-95相互结合、以及NR1的膜表面表达的影响都较小。3）D4.7R比D4.4R更大程度降低小鼠前额叶皮层（PFC）神经元NMDA诱发电流、以及NMDAR-EPSC电流幅值。4）D4.7R引起小鼠多动样行为和冒险行为，NMDA受体部分激动剂D-环丝氨酸（D-cycloserine）能够改善D4.7R引起的小鼠多动样行为和冒险行为。5）相对D4.4R而言，D4.7R更大程度抑制兴奋性神经网络簇状放电，而更小程度地减弱抑制性同步神经网络簇状放电。6）小鼠腹腔注射ADHD治疗药物利他林后，能够使D4.7R对小鼠同步神经网络活动水平的调节作用回复到正常水平。本课题的研究结果为D4.7R与ADHD的相关性提供了新的理论依据。