MYORG基因突变介导星形胶质细胞功能异常及血脑屏障破坏导致原发性家族性脑钙化症的致病机制

Primary familial brain calcification (PFBC) is a neurogenetic disorder characterized by symmetrical and bilateral calcium deposits in multiple brain regions. PFBC have high clinical and genetic heterogeneity, and there are no effective drugs for PFBC to date. In 2018, we identified that biallelic mutations in the gene MYORG was a novel cause for PFBC, however, the gene function of MYORG and the underlying mechanism of MYORG mutations on PFBC is still unknown. In the preliminary study, we found that MYORG was specially expressed in brain astrocytes, which played an important role in the control of blood brain barrier (BBB) function. Therefore, we speculated that MYORG mutations may induce the dysfunction of astrocytes, subsequently destroy the BBB integrity and eventually lead to calcium deposit in brain. In this study, we first aim to establish eukaryotic expression plasmid and detect the effect of identified MYORG mutations on protein expression and localization; Then, we will use shRNA knockdown model, in vitro BBB model and Myorg-/- mice to explore the effect of MYORG on astrocyte, vascular endothelial cell and BBB function. This study will clarify the pathogenesis mechanism of MYORG mutations on PFBC.

原发性家族性脑钙化症（PFBC）是一种以多个脑区出现双侧对称性钙化灶为特点的神经遗传病，其具有高度的临床异质性和遗传异质性，目前尚缺乏有效的治疗药物。MYORG基因为2018年最新发现的PFBC的致病基因，目前关于MYORG基因的功能及其突变后的具体致病机制仍不明确。我们在前期研究中发现MYORG蛋白特异性的表达在脑内的星形胶质细胞中，而星形胶质细胞与正常血脑屏障功能的维持有重要联系。由此，我们推测MYORG基因突变可能引起星形胶质细胞功能障碍，进而破坏血脑屏障的完整性，最终导致脑钙化的形成。本项目拟首先通过构建MYORG真核表达质粒，明确前期已发现的不同类型突变影响蛋白表达的方式；另外，通过shRNA基因敲减模型，体外血脑屏障模型并结合Myorg-/-小鼠，全面分析MYORG表达异常对星形胶质细胞、血管内皮细胞及血脑屏障正常功能的影响，阐明MYORG基因突变的致病机制。

MYORG基因已被发现为原发性家族性脑钙化症(PFBC)最常见的隐性致病基因，本项目旨在通过研究MYORG基因突变对星形胶质细胞功能及血脑屏障的影响，揭示脑钙化形成的机制。本项目研究发现MYORG基因截短突变能够显著降低mRNA表达水平，错义突变则主要导致蛋白半衰期明显缩短。通过成功筛选MYORG shRNA, 发现敲减MYORG蛋白表达后，星形胶质细胞炎性相关因子IL-6及TNF-α表达升高，终板蛋白AQP4表达下降，同时糖基化水平及各种血脑屏障相关因子表达明显下降；上述结果提示MYORG蛋白对于星形胶质细胞正常功能有重要作用。本项目通过体外构建血脑屏障模型，发现MYORG表达降低后，内皮细胞跨膜电容值变化不大，但电阻值明显降低，提示内皮细胞连接破坏；同时内皮细胞连接蛋白OCLN表达水平下降；通过Myorg敲除小鼠，初步发现基因敲除小鼠中血脑屏障完整性破坏。本项目研究结果证实MYORG基因突变能够破坏星形胶质细胞正常功能，为血脑屏障破坏在脑钙化形成中的作用提供了新的证据。