脑微出血通过凋亡相关微粒蛋白介导脑淀粉样血管病病理生理的实验研究

Cerebral microbleeds are common in cerebral amyloid angiopathy (CAA) and associated with cognitive impairments. The pathophysiology of CAA-associated microbleeds remain largely unknown. CAA is caused by deposition of amyloid β protein in the vessel walls. Paravascular glymphatic system is a major clearance pathway of amyloid β. Apoptosis-associated speck-like protein (ASC) is an adopter of inflammasome and owns prion-like features. ASC is recently reported to be able to bind with amyloid β and increase the formation of amyloid-β aggregates. We found that microbleeds induced chronically sustained inflammation. ASC was significantly increased in the area outside of the lesion core and some ASCs were released to form a speck extracellularly. In addition, synthesized ASC could be detected within Paravascular space when it was injected into the cisterna magna. We hypothesize that extracellular ASC may spread through glymphatic system and propagate inflammation via its prion-like activity, impair glymphatic clearance of A beta, thereby deteriorating CAA. The activation and extracellular release of ASC is tightly regulated. NF-κB can upregulate inflammasome which in turn enhance intracellular aggregation of ASC whereas autophagy eliminates ASC.  We found that inhibition of immunoproteasome significantly reduces ischemia-induced activation of NF-κB and attenuate post-stroke inflammation. Therefore, we propose to study the role of ASC in CAA and explore whether inhibiting immouoproteasome or enhancing autophagy can reduce ASC and diminish microbleeds-mediated brain damage in CAA. We believe that our proposed study is likely to provide insight into the pathogenesis of cerebral microbleeds in CAA and may identify new therapeutic targets.

脑微出血在脑淀粉样血管病(CAA)常见。但脑微出血是否影响CAA尚无定论。CAA是β淀粉样蛋白（Aβ）沉积在血管的脑血管病。脑类淋巴系统是Aβ的重要清除通路。凋亡相关微粒蛋白（ASC）是炎症小体接头蛋白，具有朊蛋白样特性传播炎症。最新发现ASC可直接与Aβ结合促进其斑块形成。我们预实验发现脑微出血小胶质细胞持续炎症，ASC胞外释放。注射ASC重组蛋白可沉积在血管旁间隙。推测胞外ASC可通过朊蛋白样特性介导炎症，并进入脑类淋巴系统促进Aβ斑块形成阻碍Aβ清除，加重CAA。ASC斑点形成及胞外释放受机体调控。其中NF-κB上调炎症小体促进ASC生成而自噬则参与ASC降解。我们前期发现免疫蛋白酶体可抑制脑梗死所致的NF-κB。我们将利用细胞及动物模型结合双光子激光共聚焦显微镜，研究ASC参与CAA微出血的机制，探讨自噬与免疫蛋白酶体的调控以及治疗靶点，为阐明CAA微出血提供新思路及治疗策略。

脑淀粉样血管病严重危害人类健康。β淀粉样蛋白（Aβ）沉积在脑血管是CAA的主要病理特点。脑类淋巴系统是Aβ的重要清除通路。凋亡相关微粒蛋白（ASC）是炎症小体(NLRP3)中重要成分。我们前期发现脑微出血小胶质细胞持续炎症，ASC胞外释放。注射ASC重组蛋白可沉积在血管旁间隙。我们在前期的基础上进一步研究脑微出血在CAA发病中的作用机制，探索ASC/NLRP3在其中的作用，并利用临床队列验证实验研究中脑类淋巴系统的发现。根据计划引进了CAA模型鼠(Tg-SwDI小鼠)以及NLRP3-/-小鼠。也建立了化学诱导的CAA模型，对微出血通过ASC/NLRP3参与CAA的类淋巴系统病理生理机能及干预靶点进行研究。通过动物实验及细胞模型，我们发现NLRP3是ASC介导CAA的脑类淋巴系统障碍的关键分子。在肠脑轴机制研究中，发现肠道慢性炎症刺激会经脑肠轴导致脑内持续炎症，小胶质细胞和星形胶质细胞活化增加，ASC 聚集增加，AQP4极性损害，脑内IFN+阳性的T细胞增多，类淋巴系统清除功能障碍，脑内Aβ沉增加，认知损害发生。在ASC介导炎症传播的具体机制中研究发现，小鼠脑内ASC通过NLRP3 进一步引起caspase-1及1-IL1β通路的活化，导致小胶质细胞炎症播散。利用小胶质细胞敲除NLRP3的转基因小鼠，发现小胶质细胞的nlrp3敲除可以抑制ASC/NLRP3 通路，减轻小胶质细胞炎症，改善类淋巴系统，减少Aβ沉积、神经元凋亡和认知损害。我们发现间充质干细胞来源的外泌体能显著改善脑类淋巴系统的功能，减少Aβ沉积。充质干细胞外泌体中含有能特异性抑制ASC/NLRP3的miRNA（miR-22-3p及时miR-223-p)，进一步证实ASC/NLRP3是CAA的潜在治疗靶点。利用广州健康老年人与AD队列，我们探索类淋巴功能障碍导致Aβ沉积的临床转化潜力，初步结果发现AD 患者中存在类淋巴功能障碍，严重程度和Aβ沉积及脑胶质炎症呈正相关，类淋巴功能障碍可以通过测定磁共振的ALPS来判断，ALPS结合外周血标志物可以预测脑内Aβ沉积。提示本研究结果具有一定的临床应用潜力。本研究为发现CAA的致病机理及新治疗靶点提供基础。在研期间，我们共发表论文SCI论文6篇，其中4篇影响因子大于5分， 完成目标。

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