弓形虫酪氨酸羟化酶对小鼠脑多巴胺代谢的调控作用及机制研究

Chronic infection of Toxoplasma gondii could result in the change of behavior and cognitive ability of its hosts, and the pathological mechanism was not known, but the metabolic abnormalities of dopamine in the brain were associated with it. Tyrosine hydroxylase is the key synthase of the metabolic pathway of dopamine in the brain. In ourselves previous research, we found that the dopamine concentration was increased in the brain of chronic Toxoplasma-infected mice, and the variation was correlated with behavior change of the mice. Toxoplasma gondii could encode and express tyrosine hydroxylase 2 (TgAaaH2) , and the expression level of TgAaaH2 was improved in the chronic infected mice. But it was unknown that whether TgAaaH2 was involved in the dopamine metabolism in the brain. Therefore, we put forward the hypothesis that “ Toxoplasma gondii could express and secrete TgAaaH2 into the neural cells in the mice to rugulate the metabolic pathway of dopamine, and then the increased dopamine concentration could give rise to the behavior change of the infected mice”. The following research would been done to verify the hypothesis. Firstly, transgenic Toxoplasma with fluorescence labelled TgAaaH2 protein would be constructed, through fluorescence tracer method and metabolite detection, the research would analyze the expression,the position and enzymatic reaction of TgAaaH2 in cysts and in host nerve cells in vivo and in vitro.Secondly, TgAaaH2-overexpression strains, and TgAaaH2-defect strains of transgenic Toxoplasma would be constructed respectively, combined with behavioral experiment of the mice, it would be analyzed that the influence of TgAaaH2 on host dopamine metabolism and behavior change in vivo and in vitro. The research objectives was to illuminate the regulation mechanism of TgAaaH2 on dopamine in the brain of the infected mice, and to explore the molecular pathological basis of chronic infection of Toxoplasma gondii changing host behavior.

弓形虫慢性感染可以导致宿主的行为和认知能力发生改变，其病理机制尚不清楚，但宿主脑内多巴胺代谢异常与之相关。酪氨酸羟化酶是脑多巴胺代谢通路的关键合成酶，申请者前期研究发现弓形虫慢性感染小鼠脑内多巴胺浓度升高且与小鼠行为改变相关，而弓形虫自身编码的酪氨酸羟化酶2(TgAaaH2)的表达量也升高，其是否参与调控脑多巴胺代谢尚需研究。由此，本课题提出弓形虫表达分泌的TgAaaH2进入小鼠神经细胞调控代谢通路导致多巴胺浓度升高，从而引起小鼠行为改变的假说。并拟通过以下研究进行验证：①荧光标记TgAaaH2蛋白，体内外试验明确其在包囊及小鼠神经细胞内的表达、定位及酶促活性；②分别构建TgAaaH2蛋白过表达和基因功能缺陷转基因弓形虫株，结合小鼠行为学试验，分析其对小鼠脑多巴胺代谢和行为改变的影响。研究目标是阐明TgAaaH2对小鼠脑多巴胺代谢的调控机制，探求弓形虫慢性感染改变宿主行为的分子病理基础。

弓形虫慢性感染可以导致宿主的行为和认知能力发生改变，其病理机制尚不清楚，但宿主脑内多巴胺代谢异常与之相关。酪氨酸羟化酶是脑多巴胺代谢通路的关键合成酶，弓形虫自身编码2种酪氨酸羟化酶AAH1和AAH2，其是否参与调控脑多巴胺代谢尚需研究。课题组利用qPCR技术分析得出AAH1和AAH2基因在弓形虫中均为单拷贝，为转基因敲除研究提供依据。采用TALEN技术，构建弓形虫AAH2荧光标记株PRU/AAH2-eGFP，通过分析PRU/AAH2-eGFP在体外和体内的缓殖子转化状况，用活体成像系统观察AAH2在小鼠慢性感染模型中的荧光定位，结果显示eGFP标记的AAH2转基因弓形虫可在体内进行成像研究。验证了TALEN技术在弓形虫基因编辑中的可行性，同时为弓形虫缓殖子阶段蛋白的体内研究提供了一种活体成像方法。通过简便高效的CRISPR／Cas9技术分别构建了AAH1和AAH2基因缺陷虫株：RH△AAH1、PRU△AAH1，PRU△AAH2，转基因虫株的体外细胞培养和小鼠体内试验结果显示，AAH1基因的敲除不影响弓形虫RH株和PRU株对宿主细胞的入侵、在宿主细胞内的增殖和生长能力，以及对弓形虫感染宿主的毒力作用。同时AAH1基因的敲除不影响PRU株慢性感染小鼠在旷场实验中的探索行为和在Morris水迷宫中的学习相关行为，但会影响弓形虫PRU株慢性感染小鼠脑内整体包囊负荷和局部包囊形成，以及在Morris水迷宫中的记忆相关行为。弓形虫PRU△AAH2虫株感染小鼠的动物学实验正在进行和完善中，初步结果显示对小鼠的部分行为改变有影响。研究中课题组建立了弓形虫包囊激光显微切割技术，不仅可以快速分离纯化包囊，而且可以得到单个包囊，最大限度除去宿主组织污染，利用单细胞技术立即提取单包囊cDNA，更能反映包囊内缓殖子在体内的真实表达状况。通过电镜观察包囊，发现包囊内缓殖子可分泌出大小不等的囊泡，结合单包囊缓殖子差异表达基因的初步数据，提出慢性感染小鼠不同脑组织中包囊可分泌不同蛋白从而影响宿主功能的假说。研究结果为深入探索弓形虫慢性感染影响宿主行为的分子病理机制奠定基础。