铜蓝蛋白在选择性损伤中脑多巴胺能神经元中的作用和机制研究

Dysregulation of iron metabolism and oxidative stress due to redox-active iron in the brain is considered to be involved in pathogenesis of Parkinson's disease (PD).In our previous study, we found that decreased levels of serum ceruloplasmin may specifically exacerbate nigral iron deposition in patients with PD, but the mechanism remain unknown. Ceruloplasmin (Cp) is found in the brain in both a secreted form and a glycosylphosphatidylinositol (GPI)-linked isoform on the membrane of astrocytes and regulates iron efflux. Astrocytes play an important role in the maintain the function of neurovascular unit (NVU).The aim of the present study was to verify whether the Cp metabolic abnormalities can induce selective damage of dopaminergic neurons and accumulation of brain iron through glial cells activation and damage the function of neurovascular unit (NVU) which can contribute to selective damage of dopaminergic neurons..In our project, we intend to build glial cells - neurons - brain vascular endothelial cell co-culture model in vitro and Cp transgenic mouse model of Parkinson's disease in vivo .We use gene over-expression and gene silencing technology to investigate dysregulation of Cp can induce microglia activation，aggravate brain iron deposition ,then the activated glia cells and iron can induce oxidative stress, inflammation, and mitochondrial damage which play inportant roles in PD pathogenesis. Besides, we will study the difference of iron metabolism in the substantia nigra and other brain regions with or without Cp,and find out its role in brain. These data provide the phathophisiological basis for understanding the role of Cp deficiency in PD pathogenesisa and suggest a new therapeutic strategy in PD.

脑铁代谢紊乱与帕金森病（PD）病理损害机制关系密切。我们的前期研究发现铜蓝蛋白（CP）代谢异常选择性加重PD患者黑质铁沉积,但具体的机制尚不明确。脑内CP通过磷脂酰肌醇锚定在血脑屏障周围的星形胶质细胞表面，与铁代谢密切相关，而星性胶质细胞又在神经血管单元（NVU）中发挥重要作用。为确定CP代谢异常是否通过诱发胶质细胞和神经血管单元功能异常致中脑黑质多巴胺能神经元选择性损伤和铁异常沉积，本课题拟构建离体胶质细胞-神经元-脑血管内皮细胞共培养模型和CP转基因帕金森病小鼠模型，运用基因过表达和基因沉默等技术阐明CP代谢异常通过促进胶质细胞激活并由此诱发氧化应激、炎症及线粒体损害促使帕金森病发生的病理损害机制以及中脑黑质与其他脑区铁代谢的差异。本课题将为PD的神经保护治疗提供新的靶点和实验依据。

帕金森病（Parkinson’s disease, PD）是一种常见的神经退行性病变，但迄今为止病因未明。近年来随着对氧化应激损伤认识的深入，铁在PD发病机制中的作用越来越被重视。我们前期研究工作提示铜蓝蛋白（Ceruloplasmin, CP）代谢异常可能是PD危险因素并与黑质铁沉积相关，但铜蓝蛋白代谢异常选择性加重黑质铁沉积和相关区域的神经元损害的机制尚不清楚，且导致帕金森病患者CP水平异常的原因亦不清楚。本研究通过铜蓝蛋白（CP）干扰、细胞共培养等体外细胞学方法阐明了CP代谢异常对中枢铁代谢的调节及铁诱导DA能神经元损伤的影响，同时收集临床PD样本对CP基因、CNV和miRNA进行研究了解PD患者低CP的潜在调控机制。结果发现：1、铁诱导神经元炎症反应及ROS生成，而CP对神经元具有保护作用，其机制与CP抑制炎症反应及减少ROS生成有关；2、铁抑制神经元Beclin1途径的自噬，并在α-Synuclein及pTau病理改变中发挥作用；3、CP可通过调控神经血管单元星形胶质细胞Cx43磷酸化水平及内皮细胞通透性，从而参与脑铁运输清除机制；4、PD患者低血清CP可能与CP基因启动子区SNP、miR-152、miR-133b及miRNA-520d-5p水平异常有关，进一步研究显示miRNA-520d-5p可能通过负向调控CP表达，间接影响脑铁代谢途径。我们的结果提示：CP对铁诱导的中枢神经毒性具有保护作用；miRNA-520d-5p通过调控CP基因启动子，抑制CP表达，继而影响脑铁代谢途径，中枢铁排出障碍，引起脑内异常铁沉积，从而加剧PD病理损伤。本课题的研究成果进一步阐明脑铁代谢紊乱在帕金森病发病机制的作用，也可能为帕金森病诊断标志物的发现及新的治疗靶点提供实验依据。