CysLT1在隐球菌中枢感染引起小鼠学习记忆障碍中的作用及其机制

The patients of cryptococal meningitis often suffer from cognitive deficits such as the impairment of leaning and memory. A key factor that contributes to the difficult of prevention and treatment of the cryptococcal meningitis is our incomplete understanding of the pathogenic mechanisms of this disease. Our preliminary experiments indicated that CysLT1 play an important role in the impairment of leaning and memory caused by Cryptococcus neoformans CNS infection in mice. However, the further studies on the mechanism are needed. We have a hypothesis that C. neoformans CNS infection increase the metabolic activity of cPLA2-5-LO-CysLTs pathway in hippocampus, and that CysLTs induce the astrocyte proliferation and the activation of microglial cells via CysLT1 and MAPK pathway, which result in the dysfunction of nervous activity and the impairment of leaning and memory. To examine this hypothesis, we use the mouse model of experimental hematogenous meningoencephalitis, and the models of CysLT1 gene knock out mice, hippocampus slices, and the primary culture of nervous cells. In addition, several other techniques are also used, including Western Blot, immunohistochemistry, immunofluorescence, ELISA，chemical blocking or gene deletion, as well as the approaches of cell and molecular biology. Finally, we will determine the role and its mechanism of CysLT1 in the impairment of leaning and memory caused by the C. neoformans CNS infection, and may find a potential target, which will provide a novel strategy for the prevention and treatment of C. neoformans CNS infection.

隐球菌脑膜炎（隐脑）病人常出现学习记忆等认知功能障碍。缺乏对隐球菌脑膜炎致病机制的足够了解，是当前妨碍隐脑病防治取得进展的重要因素。我们的预实验表明，CysLT1在隐球菌中枢感染引起的小鼠学习和记忆障碍中发挥重要作用。但其作用机制还有待进一步探讨。我们提出科学假设：新生隐球菌中枢感染引起小鼠海马cPLA2 -5-LO-CysLTs代谢通路增强，CysLTs通过 CysLT1及MAPK通路介导星形胶质细胞增生和小胶质细胞活化，最终导致神经活动异常和学习记忆障碍。为了验证这一假说，我们将分别在小鼠实验性脑膜炎动物模型及CysLT1基因敲除小鼠、海马脑片及原代培养神经细胞模型上，采用Western Blot、免疫组化和免疫荧光、ELISA、药物阻断或基因敲除、以及细胞分子生物学等技术方法，确认CysLT1在隐球菌中枢感染引起学习记忆障碍中的作用及其机制，寻找有效干预靶点，为隐病的防治提供新思路。

本项研究旨在探讨隐球菌脑膜炎所致学习记忆等认知功能障碍的机制，采用小鼠实验性脑膜炎动物模型及CysLT1基因敲除小鼠、培养脑片及原代培养神经胶质细胞模型，对新生隐球菌CNS感染的病理特征及感染病灶胶质细胞相关分子标记物进行分析，确认CysLT1在隐球菌中枢感染引起小鼠学习记忆障碍中的作用，观察CysLT1基因敲除对小鼠隐球菌CNS感染胶质细胞活化增生的影响，以及CysLT1对神经胶质细胞增殖及增殖成团的影响。.研究表明：（1）CysLT1在隐球菌中枢感染引起小鼠学习记忆障碍中的发挥重要作用；（2）新生隐球菌CNS感染形成的病灶主要表现出两种主要的特征，镂空筛网状病灶以及细胞核染色较深的细胞及细胞团，细胞团的形成与感染引起的星形胶质细胞活化增殖有密切的关系；（3）CNS中至少存在GFAP+vimentin+S100β+ALDH1L1+SOX9+和GFAP- vimentin+S100β+ALDH1L1+SOX9+两类星形胶质细胞；（4）新生隐球菌CNS感染引起星形胶质细胞标志物GFAP、SOX9、vimentin、ALDH1L1、S100β等表达升高，星形胶质细胞表现出明显活化和增殖；活化的星形胶质细胞合成分泌型S100β，感染还引起CS-56免疫阳性细胞CSPGs含量的升高；（5）星形胶质细胞可表达CysLT1，新生隐球菌中枢感染病灶区CysLT1高表达。CysLT1敲除减弱星形胶质细胞的活化和增殖能力，并降低小胶质细胞的反应性，有望成为新生隐球菌CNS感染防治的靶点；（6）隐球菌中枢感染引起脑组织cPLA2 -5-LO-CysLTs代谢通路增强，CysLTs通过 CysLT1介导星形胶质细胞增生和小胶质细胞活化。因此，干预CysLT1有望减缓感染所致的病理损伤，这为新生隐球菌中枢感染的防治提供了一种可行策略，也为神经炎症相关疾病的防治提供一种新思路。