中脑microRNA-133b对帕金森病模型小鼠铜蓝蛋白表达的调控作用及其机制研究

Parkinson’s disease (PD) is an age related neurodegenerative disease characterized by the loss of dopaminergic neurons and iron deposition in the substantia nigra. Ceruloplasmin (CP) is a multi-copper enzyme with ferroxidase activity which is very important for brain iron metabolism. Many results had shown that both the CP level and oxidative activity in serum and cerebral spinal fluid (CSF) were significantly lower in PD patients than that in the age- and sex-matched healthy controls. In addition, lower serum ceruloplasmin levels correlated with younger age of onset in PD. Furthermore, our and other previous studies showed that CP dysmetabolism specifically exacerbates iron deposition in the substantia nigra of midbrain in PD patients. However, little is known about the cause of low CP level in PD patients. Our recent study examined the CP gene variations in PD patients and found no single nucleotide polymorphism of CP gene was corelated with CP levels, and no copy number variants of CP gene was found in PD patients. MicroRNAs (miRNAs) have recently emerged as an important class of small RNAs (22 nucleotides long) that act as post-transcriptional regulators of gene expression. MiRNAs are increasingly being recognized not only as regulators of developmental processes but contributors to pathological states. Downregulation of miR-133b in midbrain of PD patients as well as in mouse models of PD had been reported in several studies. Moreover, our recent study had shown that serum miR-133b was significantly correlated with the CP levels in PD patients. According to the analysis of the miRNAs database, including miRbase, TargetScan and PicTar, the ribosomal protein L13a (RPL13A)-which is the upstream molecular of CP, is the target gene of miR-133b. Several studies had reported that the RPL13A could inhibit CP expression by targeting the 3′UTR GAIT(IFN-gamma activated inhibitor of translation) element of CP mRNA. The goal of this study is to clarify the regulatory role of miR-133b on the expression of CP in PD brain and explore the mechanism by which miR-133b regulate CP expression. MPTP（1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine）induced sub-chronic PD mouse model was used to detect the expression of miR-133b and CP. Moreover, we will give the PD mouse midbrain miR-133b contained virus injection in order to elevate the expression of miR-133b, and observe whether miR-133b could increase the CP levels, improve the performance of behavior, decrease the iron deposition, and alleviate the pathology in PD mice brain. In addition, we will verify the target gene of miR133b by Luciferase report system. And primary cultured astrocyte was used to detect the role of miR-133b on the expression of CP under the condition of normal RPL13A plasmid or the miR-133b-resistant mutant RPL13A plasmid. This study will help us to understanding the mechanism of the low CP levels in PD brain, and also indicate us a new way for the therapeutic strategy of PD.

铜蓝蛋白（Ceruloplasmin，CP）是存在于大脑星形胶质细胞表面的一种铁氧化酶。帕金森病（Parkinson’s disease， PD）患者血清和脑内CP水平显著下降，但原因未明。我们既往的研究发现PD患者血清miR-133b的表达水平与CP水平显著相关。为了明确PD模型脑内miR-133b调控CP表达的功能及机制，我们拟研究PD模型小鼠中脑miR-133b、CP的表达；并给予模型小鼠中脑立体定位注射包被miR-133b的病毒载体，观察miR-133b对CP表达、脑铁沉积及PD病理改变的影响。根据miRNAs数据库的分析，CP的上游调控分子RPL13A（Ribosomal protein L13a）是miR-133b的靶基因。我们将利用Luciferase报告系统进行miR-133b靶基因的验证。本研究将阐明PD患者CP水平降低的原因及可能机制，并为探索PD的治疗策略提供帮助。