GRK-2/DR2/NALCN信号通路调控纹状体投射神经元兴奋性与帕金森病发病的相关性研究

Parkinson’s disease (PD) is a common neurologic disorder that affects approximately 1% of individuals older than 60 years. While extensive researches have been conducted, no current treatments prevent disease progression, and patients become increasingly disabled over time. Recently, a novel pathway governing dopamine-dependent movement has been identified in the model animal system C. elegans. It was shown that the G protein-coupled receptor kinase GRK-2, the D2 type dopamine receptor DOP-3 (D2R), and the sodium (Na+) leak channel (NCA; i.e., NALCN in human) act together to modulate rhythmic locomotion. This model is intriguing because these classes of proteins are highly conserved in humans and have been implicated in various human diseases. Here, we will examine a new hypothesis – the GRK-2/DR2/NALCN pathway regulates the excitability of the medium spiny neurons (MSNs). MSNs are the principal neuron type in the striatum. Induction of PD-like state in mice causes rapid and selective loss of synapses in a subset of MSNs that express D2R, unveiling a role of DR2 MSNs in PD progression. In this study, we will induce dopaminergic damage using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). We will determine the excitability of MSNs in healthy and PD model mice using electrophysiological recordings. We will examine the contribution of NALCN channels using pharmacological approaches. We anticipate that modulation of the excitability of MSNs (e.g., using K+ channel inhibitors) will alter behavioral and pathological features of PD model mice. Results form these studies will elucidate a new mechanism for dopamine-regulated neuronal excitability of MSNs and will ultimately advance the understanding of the pathogenicity of Parkinson’s disease.

帕金森病（PD）有效防治是当前研究热点。研究发现GRK-2/DOP3/NCA信号通路在多巴胺神经元调控节律运动中起到关键作用。NCA突变的秀丽线虫可出现类似PD的运动迟缓和冻结现象，而且NCA离子通道参与神经元的兴奋性调控。在哺乳类，这一信号通路为GRK-2/DR2/NALCN，但尚不清楚DR2对NALCN功能的调控及NALCN功能改变与纹状体投射神经元异常放电间的关系。我们假设：GRK-2/DR2/NALCN通过调控纹状体投射神经元的兴奋性可能参与PD的发病。本课题拟利用CD1来源的E14小鼠纹状体神经元及MPTP诱导的小鼠PD模型，采用神经电生理、免疫荧光、蛋白印迹等技术，拟证实DR2通过GRK-2/DR2/NALCN信号通路参与哺乳动物纹状体投射神经元兴奋性的调控；并通过干预NALCN离子通道观察纹状体投射神经元的兴奋性变化与PD小鼠模型行为学改变的相关性，为治疗PD提供新的策略。

帕金森病（PD）有效防治是当前研究热点。PD患者存在明显的节律性活动异常（包括节律性运动、睡眠障碍等），但其潜在发病机制并不清楚。前期在秀丽隐杆线虫节律运动研究中发现GRK-2/DOP3/NCA信号通路在多巴胺神经元调控节律运动早期的启动过程中起到关键作用。NCA突变的秀丽线虫可出现类似PD的运动迟缓和冻结现象，而且NCA离子通道参与神经元的兴奋性调控。在哺乳类，这一信号通路为GRK-2/DR2/NALCN，但尚不清楚DR2对NALCN功能的调控及NALCN功能改变与纹状体投射神经元异常放电间的关系。我们假设：GRK-2/DR2/NALCN通过调控纹状体投射神经元的兴奋性可能参与PD的发病。本课题利用MPTP制备PD小鼠模型，利用WB和IHC技术研究了GRK-2/DR2/NALCN信号通路蛋白在PD小鼠纹状体内的改变，利用慢病毒转染构建过表达GRK-2蛋白SH-SY5细胞系，并利用细胞膜片钳技术检测SH-SY5Y细胞系电生理改变，探讨GRK-2对SH-SY5Y细胞系电生理的作用。同时我们还通过与美国Fred Hutchinson Cancer Research Center白继红实验室合作进一步探索了节律运动后期维持的调控机制。上述研究发现（1）PD小鼠纹状体内GRK-2/DR2/NALCN信号通路蛋白发生变化（2）过表达GRK-2的SH-SY5Y细胞系的钠离子电流受到抑制，从而导致自发电位减少（3）DA可以通过DOP-1/FLP-11信号通路参与秀丽隐杆线虫节律性运动的调控（4）DOP-3/NALCN通路对于游泳运动的起始至关重要，DOP-1/FLP-11通路则特异性地参与了游泳运动的后期维持（5）DOP-3/NALCN通路主要在运动前中间神经元里发挥调控作用，而DOP-1/FLP-11通路则在RIS睡眠神经元里起作用。我们的研究结果提示（1）DOP-1/FLP-11和DOP-3/NALCN两条信号通路在时间上和空间上的相互协作是节律性运动持续性的重要调控机制，体现了多巴胺系统与睡眠调控系统在节律性运动调控中的相互关系。（2）在PD小鼠模型确实存在GRK-2/DR2/NALCN信号通路异常，GRK-2可以抑制神经元电活动，可能参与帕金森病的发病，为进一步研究提供理论基础。