Fractalkine-mediated neuronal protection in the diabetic retina

项目摘要 在糖尿病视网膜中，Fractalkine（FKN）信号传导的中断诱导纤维蛋白（原）沉积，增加了纤维蛋白（原）沉积。 小胶质细胞产生IL-1β，血管和神经元损伤。但是，FKN及其受体CX 3CR 1， 调节小神经胶质细胞活化和视网膜病理学是未知的。在人类中，两个单核苷酸 CX 3CR 1基因座（hCX 3CR 1-I249/M280）的多态性显示与FKN的结合缺陷， 糖尿病期间的炎症。在表达人CX 3CR 1 - 1的小鼠模型中诱导糖尿病 I249/M280显示视网膜中神经元损失加速，全身炎症引起小胶质细胞减少。 促炎细胞因子的聚集和上调。因此，FKN/CX 3CR 1信号传导通路发挥了重要作用。 在糖尿病视网膜病变（DR）中的作用未得到充分重视，可能是通过炎症过程。但有一个 关于FKN/CX 3CR 1信号传导、小胶质细胞活化和 糖尿病宿主的细胞损伤（神经元和血管），其中高血糖和反复发作的 发生全身性炎症。中心假设是FKN通过阻断小胶质细胞对DR具有神经保护作用 活化和随后的血管通透性和炎症变化是DR的特征。 以下具体目的将测试纤维蛋白（原）持续激活小胶质细胞的机制。 增强神经元和血管内皮细胞损伤。利用糖尿病实验小鼠模型 结合全身性内毒素血症，提出了以下具体目标。 具体目标1。确定小胶质细胞在糖尿病的发生和发展中的作用 视网膜病变这一目标将检验FKN与CX 3CR 1结合使小胶质细胞极化为抗CX 3CR 1的假设。 炎症通路早期疾病。因此，a）急性和慢性糖尿病小鼠中的小胶质细胞耗竭 疾病的不同阶段，和B）外周血纤维蛋白（原）消耗，以证明1）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， 神经元损伤和2）小胶质细胞活化的调节可用于预防视力丧失。 具体目标2。确定可溶性FKN减轻小胶质细胞活化的神经保护作用 挽救神经和血管损伤。FKN作为跨膜蛋白在神经元上表达， 作为膜结合粘附分子（mFKN）或作为可溶性蛋白（sFKN）， 细胞表面腺相关病毒载体将用于在神经元中表达sFKN或mFKN，以测试神经元中sFKN或mFKN的表达。 假设通过其可溶性结构域增强FKN信号传导的途径是神经保护性的， 防止视力丧失。还将实施方法以确定sFKN和抗-IFN-γ的协同作用。 纤维蛋白方法预防缺乏CX 3CR 1或表达人CX 3CR 1 I249/M280的小鼠的视力丧失 变量。

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.