基于“D3R/β-arrestin/GSK-3β通路—PD情感/动机障碍关联”新假说探讨新型高选择性D3R激动剂干预PD的分子机制

Parkinson’s disease (PD) is the second most common neurodegenerative disease including motor symptoms and a plethora of neuropsychiatric impairments. The neuropsychiatric symptoms are frequently observed in PD patients before motor disorder and become a comorbidity of a large majority of PD patients. However, no drug is approved to treat patients with PD-related neuropsychiatric deficits. As recent literature reported, the loss of dopaminergic nigrostriatal neurons accounts for the motivational and affective deficits in PD. Additionally, the activation of D3R but not D2R or D1R reversed PD-related motivational and affective deficits. However, the underling biological mechanisms remain shrouded in mystery. Our identified high D3R-biased agonist PU69 exhibited potent anti-parkinsonism activity and deregulated GSK-3β; while GSK-3β (D3R cascade) mediates the core pathological markers of PD: α-synuclein, as well as reverses D3R-related neuropsychiatric symptoms. Accordingly, we proposed a novel hypothesis that D3R/β-arrestin/GSK-3β pathway mediates the PD-related affective/ motivational deficits. In this regard, neurobehavioral, ptoteomics, molecular biological, and stem cell strategies were utilized to investigate the effects of D3R-biased agonist PU69 against PD-related motivational/affective deficits, the regulation of D3R cascade as well as the morphology and function of dopaminergic neurons. Taken together, this project will elucidate the underling pathological mechanism of PD comorbidity (motivational/affective deficits) by our new probe molecule from in vitro, in vivo and molecular perspectives. Furthermore, the project also will pave the theoretical way for the early and objective diagnosis as well as etiological treatment.

帕金森病（PD）包括运动和神经精神症状，后者常早于运动症状发生，并伴随大多数PD患者，但临床缺乏相关药物。最新研究发现PD情感/动机障碍源于黑质纹状体多巴胺神经元损伤，主要靶标为多巴胺D3受体（D3R），但确切分子机制不明。申报人前期筛选出的高选择性D3R激动剂PU69能够改善PD症状，下调GSK-3β；而GSK-3β介导PD核心病理产物α-synuclein聚集，进而调控D3R相关的精神症状。因此，我们提出“D3R/β-arrestin/GSK-3β通路—PD情感/动机障碍关联”新假说。本项目采用神经行为学、蛋白质组学、分子生物学和干细胞等技术探究PU69对PD大鼠情感/动机障碍行为、通路相关蛋白及其磷酸化、多巴胺神经元形态和功能的影响。从整体、细胞及分子水平揭示新型D3R激动剂治疗PD的药效机制，探讨PD情感/动机障碍的分子机理，为PD的早期客观诊断和针对性治疗提供新思路和理论基础。

多巴胺是一种调控运动、认知、情感的神经递质，多巴胺受体通过传导这类递质发挥生物学功能，然而其功能失调会导致几乎所有的神经精神疾病。“不死的癌症”帕金森病病理核心就是多巴胺释放和多巴胺能神经传递受损，临床缺乏治疗PD的相关药物，长期服用左旋多巴会出现异动症；经典抗抑郁药抗焦虑药对PD抑郁焦虑无效；多巴胺能激动剂也因对多巴胺D2类受体的非特异性结合出现多种副作用，这些副作用主要与D2R的结合相关。大多数帕金森病患者在罹受运动障碍的同时也会遭受焦虑抑郁和冷漠等精神症状的折磨。研究表明多巴胺D3R选择性激动剂能够逆转PD情感、动机障碍，因此D3R选择性激动剂可能可以改善帕金森的运动障碍和神经精神症状。.利用药物化学策略设计合成了苯基哌嗪–邻苯二甲酰亚胺和苯基哌嗪–芳基脲类化合物，通过竞争抑制性和功能性实验发现9i和17a是D3R高选择性激动剂，尤其后者选择性高达万倍，特异性的介导D3R下游通路。分子对接提示D3R的别构位点Gly94、Ser182、Ile183可能是其选择性的分子机制。神经行为学研究表明先导化合物及其盐酸盐可显著改善PD症状；细胞活性实验发现其具有多巴胺能神经元保护的作用。免疫共沉淀说明先导化合物介导D3R与β-arrestin2的结合；western提示D3R激动剂抑制Akt 的Ser473磷酸化需要β-Arrestin2参与；免疫共沉淀证明D3R激动剂促进β-arrestin2与Akt的结合，抑制Akt与PDK1的结合；Western提示先导化合物抑制GSK-3β的磷酸化，而Akt又促进GSK-3β的磷酸化。说明先导化合物抗PD的分子机制为选择性激活D3R与β-arrestin2结合，进而募集Akt抑制其Ser473磷酸化，抑制GSK-3β的磷酸化。本研究揭示了先导化合物抗PD分子机制和D3R在PD中的重要作用，为PD的早期客观诊断和针对性治疗提供新思路和新靶点。

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