Fractalkine-mediated neuronal protection in the diabetic retina

项目摘要 在糖尿病视网膜中，FKN信号诱导纤维蛋白(原)沉积，增加 小胶质细胞、血管和神经元损伤产生IL-1β。但是，FKN及其受体CX3CR1是如何， 调节小胶质细胞的激活和视网膜的病理尚不清楚。在人类中，两个单核苷酸 CX3CR1基因座(hCX3CR1-I249/M280)与FKN结合缺陷的多态起着关键作用 在糖尿病期间的炎症中。表达人CX3CR1-的小鼠糖尿病模型的建立 I249/M280显示视网膜神经元加速丢失，全身炎症导致小胶质细胞 促炎症细胞因子的聚集和上调。因此，FKN/CX3CR1信号通路在 在糖尿病视网膜病变(DR)中的作用被低估，可能是通过炎症过程。然而，有一个 关于FKN/CX3CR1信号、小胶质细胞激活和FKN/CX3CR1信号之间确切关系的认识差距 高血糖和反复发作的糖尿病宿主的细胞损伤(神经元和血管) 全身性炎症会发生。中心假设是FKN通过阻断小胶质细胞对糖尿病视网膜病变具有神经保护作用。 活化和随后的血管通透性和炎性变化是DR的特征。 以下特定目标将测试纤维蛋白(原)持续激活小胶质细胞的机制 加强神经元和血管内皮细胞的损伤。利用实验性糖尿病小鼠模型 结合全身性内毒素血症，提出以下具体目标。 具体目的1.确定小胶质细胞在糖尿病发病和发展中的作用 视网膜病变。这一目标将检验FKN与CX3CR1极化小胶质细胞结合的假说。 炎症途径在疾病早期。因此，a)急性和慢性糖尿病小鼠的小胶质细胞耗竭 疾病分期，以及b)外周纤维蛋白(原)耗竭，以证明1)调节失调 小胶质细胞对纤维蛋白(原)的反应诱导促炎作用和氧化损伤，从而促进 神经损伤和2)小胶质细胞激活的调节可以被利用来防止视力丧失。 特定目的2.测定可溶性FKN减轻小胶质细胞活化的神经保护作用 并抢救神经元和血管损伤。FKN在神经元上以跨膜蛋白的形式表达， 作为膜结合的黏附分子(MFKN)或作为可溶性蛋白质(SFKN)在裂解 细胞表面。将使用腺相关病毒载体在神经元中表达sFKN或mFKN，以测试 假设通过其可溶性结构域增强FKN信号的通路具有神经保护作用，并将 防止视力丧失。还将采用方法来确定sFKN和抗-FKN的协同效应 纤维蛋白预防CX3CR1缺失或表达人CX3CR1I249/M280小鼠的视力丧失 变种。

Project Summary In the diabetic retina disruption of fractalkine (FKN) signaling induces fibrin(ogen) deposition, increased production of IL-1β by microglia, vascular and neuronal damage. But, how FKN and its receptor, CX3CR1, regulate microglia activation and retinal pathology is unknown. In humans, two single nucleotide polymorphisms in the CX3CR1 locus (hCX3CR1-I249/M280) that show defective binding to FKN, play a key role in inflammation during diabetes. Induction of diabetes in a mouse model expressing the human CX3CR1- I249/M280 revealed accelerated neuronal loss in the retina, and systemic inflammation caused microglial clustering and upregulation of pro-inflammatory cytokines. Thus, the FKN/CX3CR1 signaling pathway plays an under-appreciated role in diabetic retinopathy (DR), perhaps via inflammatory processes. However, there is a gap in knowledge regarding the exact relationship among FKN/CX3CR1 signaling, microglial activation and cell damage (neuronal and vascular) in a diabetic host in which hyperglycemia and repeated episodes of systemic inflammation occur. The central hypothesis is that FKN is neuroprotective in DR by blocking microglia activation and the subsequent vascular permeability and inflammatory changes that are characteristics of DR. The following specific aims will test the mechanism by which sustained microglial activation by fibrin(ogen) potentiates neuronal and vascular endothelial cell damage. Utilizing experimental mouse models of diabetes combined with systemic endotoxemia the following specific aims are proposed. Specific Aim 1. Determine the role of microglia in the initiation and progression of diabetic retinopathy. This aim will test the hypothesis that FKN binds to CX3CR1 polarizing microglia towards an anti- inflammatory pathway early in disease. Therefore, a) microglia depletion in diabetic mice at acute and chronic stages of disease, and b) peripheral fibrin(ogen) depletion will be used, to demonstrate that 1) dysregulated microglia in response to fibrin(ogen) induce pro-inflammatory actions and oxidative damage that contribute to neuronal damage and 2) that regulation of microglia activation can be harnessed to prevent vision loss. Specific Aim 2. Determine the neuroprotective effects of soluble FKN to mitigate microglia activation and rescue neuronal and vascular damage. FKN is expressed on neurons as a transmembrane protein, acting as a membrane-bound adhesion molecule (mFKN) or as a soluble protein (sFKN) upon cleavage from cell surfaces. Adeno-associated viral vectors will be used to express sFKN or mFKN in neurons to test the hypothesis that pathways that enhance FKN signaling via its soluble domain are neuroprotective and will prevent vision loss. Approaches will be implemented to also determine the synergistic effect of sFKN and anti- fibrin approaches to prevent vision loss in mice lacking CX3CR1 or expressing the human CX3CR1I249/M280 variant.