转录因子Sp1调节MAO-B活性对帕金森病的神经保护作用研究

Parkinson's disease (PD) is characterized as a series of abnormal behaviors arised by the selective demise of dopaminergic neurons in the substantial nigra pars compacta (SNc). Till now, the cause of cell death remains largely unknown which may relate to the apoptosis mechanism induced by oxidative stress. Monoamine oxidase B (MAO-B) inhibitor is approved and widely used in clinical treatment of PD, which could inhibit dopamine decomposition by antagonizing the MAO-B activity in neurons, while reducing the oxidative stress level. But the mechanism of the abnormal increasing MAO-B activity in PD is unclear yet. Transcription factor Sp1 directly regulates the MAO-B expression by binding repeatable Sp1 binding sequence in MAO-B promoter. In this study, using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) and in vitro PD model of neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced mesencephalic cells, we will detect the Sp1 level and function by analyzing the mRNA、protein expression and the Sp1 DNA binding and transcription activity at the first. Then, we will investigate the neuroprotective effect and its mechanisms of Sp1. In PD models interfered by over expression or down regulation of Sp1 activity via lentivirus, we will count the dopaminergic neurons in SNc, detect the MPP+/dopamine levels by HPLC method in striatal and the oxidative stress level in cells and brain tissue, when combined with the behavioral tests. Finally, we will detect the MAO-B expression and activity changes in the same PD models mentioned above. Collectedly, our studies will identify Sp1 as a potent neuroprotective factor whose effectiveness relies on its ability to effectively regulate the MAO-B activity against toxic stress in models of PD in vitro and in vivo, which is also a new therapeutic target or procedure for PD therapy.

帕金森病（PD）以黒质致密部多巴胺能神经元丢失引起的运动及行为学异常为特点。氧化应激引起的凋亡可能是这种选择性神经元死亡的原因之一。虽然PD中单胺氧化酶B(MAO-B)活性异常增高，但其机制仍不清楚。转录因子Sp1能结合MAO-B启动子上相应结合序列直接调控MAO-B表达。为了阐明Sp1在PD中的作用，本研究拟在MPTP诱导的小鼠在体PD模型及MPP+诱导的离体模拟PD细胞模型中，从mRNA、蛋白表达水平及DNA结合转录功能检测Sp1的表达和活性；继而通过重组慢病毒在PD模型中上调或下调Sp1活性后，观察多巴胺能神经元数目、纹状体中多巴胺含量、组织及细胞氧化应激水平，并结合行为学检测评估Sp1在PD模型中的保护作用；本研究进一步在PD模型中检测MAO-B的表达及活性以明确Sp1的神经保护作用机制。这些研究将阐明在PD中Sp1对MAO-B活性的调控，也为PD的治疗提供新靶点、新思路。

背景：帕金森病（PD）主要是由中脑黑质致密部多巴胺能神经元大量死亡引起，但是造成这种选择性神经元死亡的原因尚不完全清楚。在现今PD的临床治疗中，单胺氧化酶B(MAO-B)抑制剂因为可以特异性降低MAO-B活性,进而缓解PD的相关临床症状而得到了广泛应用。MAO-B的活性升高可能在PD致病中具有重要作用，但是MAO-B的活性异常增高的机制当前尚不清楚。.课题方向：本项目拟观察在PD中转录因子Sp1的表达及活性水平变化,并尝试通过调节Sp1的活性改善MAO-B的功能异常进而发挥神经保护作用。.主要内容：本课题拟通过对PD在体及离体模型中Sp1表达及活性的检测与分析，进而调节Sp1的活性后，观察Sp1在PD模型中对MAO-B表达及活性的影响，最后结合多种生物化学、分子生物学、组织学及行为学检测，探讨在PD模型中下调转录因子Sp1后通过调节MAO-B活性发挥神经保护作用的相关分子机制。.重要结果：首先，降低Sp1表达对PD离体细胞模型的神经保护效应:⑴MPP+对细胞的毒性作用主要是依赖Sp1的途径来实现的,降低Sp1活性可以逆转MPP+毒性造成的SH-SY5Y神经细胞死亡;⑵降低Sp1活性抑制了细胞核中MPP+诱导的Sp1与MAO-B启动子的DNA结合及转录活性的增强,同时逆转了MPP+导致的MAO-B水平的升高。其次，降低Sp1表达对PD在体动物模型也具有保护效应：⑴降低Sp1活性缓解了MPTP毒性诱导的C57BL/6小鼠中脑黑质致密部多巴胺能神经元的丢失及纹状体多巴胺能神经纤维密度的减少；⑵降低Sp1活性显著改善了MPTP毒性诱导的C57BL/6小鼠的行为学异常。最后，结果显示降低Sp1表达对PD产生保护效应的可能机制是：⑴降低Sp1活性逆转了C57BL/6小鼠中脑黑质致密部及纹状体中MPTP毒性诱导的异常升高的Sp1及MAO-B的表达水平；⑵降低Sp1活性逆转了C57BL/6小鼠纹状体中MPTP毒性诱导的多巴胺及其代谢产物水平的降低。.科学意义：本项目的实施将初步阐明调节Sp1活性对PD的神经保护作用及机制，为PD的发病机制研究及开发临床治疗新手段奠定理论和实验基础。