Amyloid beta peptides and type-2 diabetes sequelae synergistically inhibit insulin signaling and trafficking at the blood brain barrier

项目摘要 2型糖尿病（T2 DM）后遗症损害脑微血管并增加阿尔茨海默氏症 通过诱导以亚生理胰岛素水平为特征的脑胰岛素抵抗和受损的 大脑中的胰岛素信号。相反，血浆中积累的可溶性淀粉样β（sAβ）肽 和大脑在阿尔茨海默病的进展加剧的影响，2型糖尿病和加重脑胰岛素 阻力迫切需要确定T2 DM后遗症和sAβ暴露如何抑制胰岛素输送， 胰岛素抵抗会加剧。长期目标是阐明脑血管和 代谢的贡献，阿尔茨海默氏病，并促进新的治疗 干预措施。本申请的总体目的是确定T2 DM后遗症的联合效应 和sAβ对胰岛素向大脑传递的影响，并确定潜在的细胞和分子机制。的 中心假设是T2 DM后遗症和sAβ肽干扰胰岛素信号传导/运输， 脑血管内皮[称为血脑屏障（BBB）]，并减少胰岛素输送到 个脑袋还假设，这些影响因以下因素之间的病理协同作用而进一步加剧： 2型糖尿病后遗症和sAβ。这项研究的基本原理是， sAβ暴露和T2 DM后遗症破坏脑胰岛素输送将使我们能够开发新的治疗方法， 解决阿尔茨海默病和2型糖尿病脑胰岛素抵抗的策略。根据初步数据， 提出了以下三个具体目标：1）确定T2 DM后遗症对胰岛素的影响 BBB的运输/信号传导; 2）确定sAβ单独和与T2 DM后遗症联合的作用 在BBB上的胰岛素运输/信号传导;和3）识别在BBB上的胰岛素运输途径， 与sAβ暴露和胰岛素信号传导受损有关。在第一和第二目标下，动态SPECT/CT成像 将用于表征表现出T2 DM的小鼠模型中BBB处的胰岛素摄取动力学， 老年痴呆症后遗症此外，BBB处胰岛素信号传导的失调将通过逆转录来捕获。 相蛋白阵列。对于第三个目标，将使用流式细胞术和TIRF显微镜来确定 sAβ ±胰岛素信号传导抑制剂对血脑屏障单层中胰岛素转胞吞作用的影响。拟议研究 是潜在的创新，因为它采用了动态成像方法与定量建模相结合 用于捕获T2 DM和阿尔茨海默病小鼠中BBB处胰岛素运输动力学变化的技术 模型这项研究具有重要意义，因为它的贡献预计将具有广泛的翻译价值。 在重新利用现有药物治疗脑胰岛素抵抗和确定候选靶点方面的重要性 来发现新药工作完成后，预计产生的新知识将具有 重要的积极影响，促进确定新的治疗策略，以打击脑 阿尔茨海默病合并2型糖尿病患者胰岛素抵抗

PROJECT SUMMARY Type-2 diabetes mellitus (T2DM) sequelae damage the cerebral microvasculature and augment Alzheimer's pathology by inducing brain insulin resistance characterized by sub-physiological insulin levels and impaired insulin-signaling in the brain. Conversely, soluble amyloid beta (sAβ) peptides that accumulate in the plasma and brain during Alzheimer's progression exacerbate the impact of T2DM and aggravate brain insulin resistance. A critical need exists to identify how T2DM sequelae and sAβ exposure inhibit insulin delivery to the brain and intensify brain insulin resistance. The long-term goal is to elucidate cerebrovascular and metabolic contributions to Alzheimer's disease and facilitate the development of novel therapeutic interventions. The overall objective in this application is to determine the combined effects of T2DM sequelae and sAβ on insulin delivery to the brain and to identify the underlying cellular and molecular mechanisms. The central hypothesis is that T2DM sequelae and sAβ peptides perturb insulin signaling/trafficking at the cerebrovascular endothelium [referred to as the blood brain barrier (BBB)] and reduce insulin delivery to the brain. It is also hypothesized that these effects are further aggravated by the pathological synergism between T2DM sequelae and sAβ. The rationale for the proposed research is that a mechanistic understanding of how sAβ exposure and T2DM sequelae disrupt brain insulin delivery will allow us to develop novel therapeutic strategies to address brain insulin resistance in Alzheimer's disease and T2DM. Guided by preliminary data, the following three specific aims are proposed: 1) Determine the effect of T2DM sequelae on insulin trafficking/signaling at the BBB; 2) Determine the effects of sAβ alone and in conjunction with T2DM sequelae on insulin trafficking/signaling at the BBB; and 3) Identify insulin trafficking pathways at the BBB, vulnerable to sAβ exposure and impaired insulin signaling. Under the first and second aims, dynamic SPECT/CT imaging will be used to characterize insulin uptake kinetics at the BBB in mouse models that exhibit T2DM and Alzheimer's sequelae. Moreover, the dysregulation in insulin signaling at the BBB will be captured by reverse phase protein arrays. For the third aim, flow cytometry and TIRF microscopy will be used to determine the effects of sAβ ± insulin signaling inhibitors on insulin transcytosis in BBB monolayers. The proposed research is potentially innovative because it employs dynamic imaging methods coupled with quantitative modeling techniques to capture changes in insulin trafficking kinetics at the BBB in T2DM and Alzheimer's mouse models. The proposed research is significant because the contribution it is expected to have broad translational importance in repurposing existing drugs to treat brain insulin resistance and in identifying candidate targets to discover novel drugs. Upon completion of the work, the new knowledge generated is expected to have an important positive impact by facilitating the identification of novel therapeutic strategies to combat brain insulin resistance in Alzheimer's patients with T2DM.