急性高眼压介导视网膜Müller细胞AQP4内化／降解及其信号转导

Aquaporin-4(AQP4) is the predominant aquaporins and abundantly expressed in retina with a polarized pattern targeted to specific membrane domains of Müller cells，which plays important roles in the water and ion balance and the regulation of visual function. It is reported that the anchoring proteins of AQP4, such as laminin and agrin, are responsible for the polarized expression of AQP4 in the Müller cells. The preliminary experiments in our study initially provided the evidences that AQP4 was internalized into early and late endosome in the retinae induced by acute ocular hypertension, and followed by its sorting to lysosome for degradation, accompanying by the downregulation of water transport, which suggested that internalization of retinal glial AQP4 can be induced by hydrostatic pressure and/or hypoxia-ischemia；In vivo study showed exogenous laminin, one of extracellular matrix molecules, can affect the occurrence of AQP4 internalization induced by hydrostatic pressure, which suggested AQP4 internalization may be related to its anchoring mechanism. Some studies showed the activation of several protein kinases also can result in AQP4 internalization. However, the details of AQP4 internalization followed by its lysosomal degradation induced by hydrostatic pressure and/or hypoxia-Ischemia and the potential signal transduction mechanism remain unknown. Therefore, the research works on rat models of acute ocular hypertension and Müller cells treated by oxygen-glucose deprivation will be carried out, in which the relationship between the AQP4 internalization and its anchoring mechanism and the effect induced by the changes of several protein kinases on AQP4 internalization and retinal edema will be investigated by means of biochemistry and molecular biology techniques and methods. The results from this study will clarify the molecular mediators of retinal edema induced by acute ocular hypertension in depth and supply an experimental basis for the development of relevant therapeutic drugs for the treatment of retinal edema.

水通道蛋白4（AQP4）是视网膜内含量最多、分布最广的水通道蛋白，呈极性分布于Müller细胞特殊膜域，与视网膜水电解质运输平衡及视觉活动调节有联系。AQP4的极性分布与laminin等锚定蛋白有关。课题组发现急性高眼压可导致AQP4内化（internalization，即AQP4进入内涵体）及分选至溶酶体，并伴随其水转运功能的降低，压力/缺血缺氧可能参与该过程；外源性laminin可影响加压时AQP4的内化，提示AQP4内化可能与锚定蛋白有关。文献证实，某些蛋白激酶的激活也可导致AQP4内化。然而，压力/缺血缺氧介导AQP4内化/降解的规律及其信号通路尚不明确。为此，本研究将应用分子生物学等方法，从在体模型和加压/氧-糖剥夺培养Müller细胞中研究AQP4锚定蛋白与其内化的关系及蛋白激酶活性改变对AQP4内化及水肿的影响，以阐明高眼压介导视网膜损伤的分子机制，并为新药开发提供实验基础。

水通道蛋白-4（aquaporin-4，AQP4）是中枢神经系统（central nervous system，CNS；包括脑和眼球）中含量最多、分布最广的一种水通道蛋白，呈极性分布于胶质细胞（包括星形胶质细胞及视网膜Müller细胞）的特殊膜域，使水沿着渗透压和静水压梯度从高向低流动。在CNS水肿相关的疾病中，AQP4既可在细胞毒性水肿阶段参与水肿的产生，也可以在血管源性水肿阶段参与水肿的清除。项目申请人已发现，急性高眼压可导致AQP4内化（internalization）进入内涵体及分选至溶酶体降解，并伴随其水转运功能的降低。AQP4内化/降解与视网膜水肿的发生密切相关，但该现象是否存在于CNS其它疾病及其发生机制均不清楚。因此，课题组以急性高眼压、大脑中动脉栓塞、细菌性脑膜炎、胶原酶/自体血所致脑出血等多种CNS疾病大鼠模型作为研究对象，针对AQP4内化/降解的发生及其相关机制展开研究。结果表明：⑴锚定蛋白α-syntrophin及热休克蛋白70在急性高眼压所致的APQ4内化/降解中发挥作用；⑵在大脑中动脉栓塞所致脑缺血模型中，存在AQP4内化和溶酶体分选现象；蛋白激酶C的激动剂佛波酯可下调AQP4的表达，同时减轻缺血早期细胞毒性脑水肿的程度，但此作用并非通过促进AQP4内化和溶酶体分选实现的；⑶在链球菌诱导的脑膜炎模型中，AQP4及其锚定蛋白α-DG 和β-DG表达增高，且存在AQP4内化和溶酶体分选现象；⑷在胶原酶诱导的脑出血模型中，伴随着脑水肿的发生及AQP4 mRNA和蛋白水平的上调，发现AQP4内化至晚期内涵体，部分内化的AQP4可以分选至溶酶体被降解。离体实验表明，锚定蛋白α-syntrophin的表达减少在AQP4内化／降解中发挥作用；⑸自体血注入所致脑出血模型中，伴随着AQP4蛋白和mRNA的表达上调，出血脑组织AQP4极性表达缺失，该种变化与β-DG表达下调密切相关；以上改变可能促进了出血性脑水肿的发展。综合以上各动物模型的实验结果，可总结出本项目的科学意义：⑴AQP4内化／降解的发生是CNS各种与水肿相关疾病的一个普遍规律，其在多种疾病所致水肿的发生、发展过程中发挥重要作用；⑵锚定蛋白的表达变化在AQP4内化／降解过程中发挥作用，在不同疾病中，锚定蛋白的变化规律并不完全一致；⑶对AQP4内化／降解的调控可作为CNS水肿治疗的潜在靶点。