SGMR1/MMP9/BDNF在脑动静脉畸形发病中的分子机制

Brain arteriovenous malformations (bAVM) are characterized by vascular focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. At present, there is no specific drug for it. What’s more, the pathogenesis of bAVM remains poorly understood. SGMR1, as a molecular chaperone, locates on ER membrane and assists in protein folding and secreting. Our previous results demonstrated downregulation of SGMR1 in bAVM specimens compared to normal intracranial vessels and superficial temporal arteries. In addition, we found that active SGMR1 involves in VSMC (vescular smooth muscle cell) proliferation and BDNF secretion. BDNF is considered as an important neurotrophic factor. It has been reported that MMP-9 is one of the metalloproteinases that can promote precursor BDNF (proBDNF) conversion into mature BDNF. Nevertheless, we found that serum proBDNF level remains stable, MMP9 was significantly upregulated while BDNF was markedly downregulated in bAVM patients. Thus, we assume that the activation of SGMR1 may mediate the folding of MMP9 on ER. This project aims to, (i) investigate the molecular chaperone function of SGMR1 in bAVM on mediating MMP9 misfolding which regulates the conversion of proBDNF to BDNF; (ii) to build a HHT1 bAVM model mice, and test whether SGMR1 agonist was useful in alleviating bAVM disorder symptoms or with effective treatment in bAVM mice. The project will mainly focus on investigating the molecular mechanism, supplying potential clinical drug targets and effective pharmaceuticals for bAVM treatment.

脑动静脉畸形（bAVM）是一种局部颅内动、静脉血管异常相连的疾病，尚无有效的药物治疗bAVM。bAVM的发病机制尚不明确。激活内质网定位的SGMR1的分子伴侣功能可辅助内质网正常折叠和分泌活性蛋白。申请人前期发现SGMR1在bAVM患者畸形血管中表达显著下调，细胞中激活SGMR1影响平滑肌细胞的增殖和胞外BDNF的水平。BDNF是一种重要的神经因子。文献报道MMP9上调可促进proBDNF到BDNF的转化，但在bAVM患者血清中：MMP9表达水平显著上调，proBDNF含量不变，但BDNF表达水平却下调。因此我们推测SGMR1的激活可能介导MMP9在ER上的正常折叠。本项目将在细胞层面验证该假设，探索是否有其他蛋白酶参与该过程；完成HHT1型bAVM小鼠模型的构建，在动物水平确定SGMR1激动剂是否有潜力作为治疗bAVM的新型药物。

本项目前期研究发现，脑动静脉畸形（Brain Arteriovenous Malformations, bAVM）中SGMR1表达量降低，蛋白水解酶MMP9的表达量显著上调，但MMP9将proBDNF水解为BDNF的过程被抑制，细胞外BDNF表达量降低，促进平滑肌细胞的异常增殖。基于此结果，本项目综合应用细胞生物学、分子生物学和化学生物学等常规实验方法，发现SGMR1与MMP9共定位于内质网，且具有蛋白-蛋白相互作用；明胶酶谱实验表明过表达SGMR1后MMP9酶活性增加，促进proBDNF水解为BDNF；蛋白质组学实验发现，除MMP9外，SGMR1还可能通过调控MMP2、MMP7、TIMP1、TIMP2、丝氨酸蛋白酶糜蛋白酶和C3转化酶等参与proBDNF水解为BDNF。联合CRISPR/Cas9和立体定向注射技术成功获得3只HHT1型畸形血管模型小鼠，成功率较低，尚需改善实验操作提高模型鼠的成功率。其中1只小鼠通过灌胃方式给药SGMR1激动剂Fluvoxamine可减少脑血管数量。该研究为理解bAVM发病的分子机制提供了独特见解，也为bAVM的治疗提供了潜在的药物靶点。.MMP9可通过降解细胞外基质成分，参与多种心脑血管疾病的病理过程。在项目执行期间，我们同时收集了bAVM畸形血管、脊髓AVM畸形血管、不同年龄段健康恒河猴脑血管、以及烟雾病患者血清等脑血管疾病相关样本。通过蛋白质组学实验发现：（1）病理条件伴随着MMP9的过量表达；（2）脑血管内皮细胞的线粒体功能异常影响脑血管结构和稳态的维持，与血管破裂出血以及畸形血管形成密切相关。.SGMR1的伴侣活性降低除与bAVM有关外，SGMR1还在多种神经退行性疾病中起重要作用，如阿尔茨海默病、癫痫、帕金森、药物成瘾等。基于项目组已有的人体组织标本，本项目通过蛋白质组学技术检测了AD人脑海马、APP/PS1小鼠（AD模型小鼠）、难治性癫痫患者海马及健康捐献者脑组织中的蛋白质表达水平变化，初步探索发现：（1）与健康对照相比，AD人脑海马和小鼠海马、及癫痫患者海马组织中，SGMR1的表达水平明显降低；（2）mTOR、cytoplasmic ribosomal proteins、synaptic vesicle pathway等信号通路参与神经退行性疾病的病理过程。