The role of medin, the most common human amyloid, in the pathology of Alzheimer disease

在过去的1.5年里，我们一直在研究最常见的人类淀粉样蛋白Medin(MFG-E8蛋白的一段)是否可能有助于淀粉样β蛋白的聚集。在两种阿尔茨海默病小鼠模型中，我们现在发现：i)MFG-E8水平随着大脑淀粉样β蛋白的沉积而增加，并且MFG-E8染色仅局限于淀粉样纤维，并且ii)MFG-E8的遗传缺陷会减少大脑β-淀粉样变性，并可能改变淀粉样蛋白的构象。由于我们无法纯化重组Medin用于体内给药，我们寻找了内源性来源，发现iii)老年野生型(但不是Mfge8基因敲除)小鼠的主动脉中存在Medin的细胞外聚集，这与人类的情况非常相似；iv)脑内注射老年野生型主动脉匀浆可诱导预沉积APP转基因动物的淀粉样β聚集。进一步的数据表明，与年龄匹配的对照组相比，人类阿尔茨海默病患者的全长MFG-E8和~6 kDa片段(可能对应于Medin)增加。由于有报道称MFG-E8可以增强淀粉样β蛋白的吞噬功能，我们也分析了小胶质细胞对淀粉样蛋白的摄取，但发现MFG-E8缺乏对小胶质细胞的淀粉样蛋白摄取没有影响。因此，我们的数据强烈表明，MFG-E8/Medin和淀粉样β蛋白之间存在直接相互作用，从而增强了淀粉样β蛋白的聚集。目前授权期内的实验将旨在确认我们的初步数据，例如APP x Mfge8基因敲除小鼠脑部和血管淀粉样蛋白沉积的减少以及淀粉样蛋白构象的变化。此外，它们还将包括量化小鼠/人脑和脑脊液中的Medin水平(到目前为止，这种分析是由于现有的抗Medin抗体与淀粉样β蛋白在Western blotts上的交叉反应以及这些肽的类似分子量，即4/5 kDa；然而，我们刚刚获得了一种新的针对Medin的单抗，这将使我们能够进行这些实验)。此外，我想申请延长一年的项目，以牢固地建立淀粉样β蛋白和麦德林在体内的相互作用。为此，我建议通过脑内注射i)年轻/老年野生型主动脉和显示低/高水平Medin的人主动脉或ii)富含Medin或缺乏Medin的主动脉匀浆来分析诱导淀粉样β聚集，并iii)进行免疫共沉淀实验以确定Medin/MFG-E8是否直接与大脑中的淀粉样β作用。由于在几乎所有60岁以上的人的血管中都发现了Medin聚集体，很容易想象Medin聚集体也会在老化的大脑中形成，特别是因为大脑MFG-E8的水平随着年龄的增长而增加。我们的研究结果将证明梅丁是否可以促进淀粉样蛋白-β聚集。因此，该项目产生的见解可以为阿尔茨海默病的新治疗方法提供基础。

Over the last 1.5 years, we have been investigating whether the most common human amyloid, medin (a fragment of the protein MFG-E8), may contribute to the aggregation of amyloid-beta. In two mouse models of Alzheimer disease, we now found that i) MFG-E8 levels increase with cerebral amyloid-beta deposition and MFG-E8 staining is localized to amyloid fibrils, and that ii) genetic deficiency in MFG-E8 decreases cerebral beta-amyloidosis and may alter amyloid conformation. As we could not purify recombinant medin for in vivo administration, we looked for an endogenous source and found that iii) extracellular aggregation of medin occurs in the aorta of aged wildtype (but not Mfge8 knockout) mice, strongly reminiscent of the human situation, and iv) intracerebral injection of aged wildtype aorta homogenate induces amyloid-beta aggregation in pre-depositing APP transgenic animals. Further data indicate that v) full-length MFG-E8 and a ~6 kDa fragment (which may correspond to medin) are increased in human Alzheimer patients compared to age-matched controls. As it has been reported that MFG-E8 may enhance phagocytosis of amyloid-beta, we also analysed microglial amyloid uptake but found it unchanged by MFG-E8 deficiency. Thus, our data strongly suggest a direct interaction between MFG-E8/medin and amyloid-beta that enhances amyloid-beta aggregation. Experiments in the current grant period will aim to confirm our preliminary data, e.g. the reduction of cerebral and vascular amyloid deposition and a change in amyloid conformation in APP x Mfge8 knockout mice. Further, they will include the quantification of medin levels in murine/human brain and cerebrospinal fluid (this analysis has so far been prevented by cross-reaction of available anti-medin antibodies with amyloid-beta on Western Blots and the similar molecular weight of the peptides, i.e. 4/5 kDa; however, we have just obtained a new monoclonal medin-specific antibody, which will allow us to perform these experiments). In addition, I would like to apply for a one-year project extension to firmly establish the in vivo interaction of amyloid-beta and medin. To this end, I propose to analyse the induction of amyloid-beta aggregation through intracerebral injection of i) young/old wildtype aorta and human aorta showing low/high levels of medin or of ii) enriched medin or medin-depleted aorta homogenate, and to iii) perform co-immunoprecipitation experiments to determine if medin/MFG-E8 interact directly with amyloid-beta in the brain. As medin aggregates are found in blood vessels of virtually anyone above 60 years of age, it is easily conceivable that medin aggregates also form in the ageing brain, particularly since brain MFG-E8 levels increase with ageing. The results of our studies will demonstrate if medin can promote amyloid-beta aggregation. Thus, the insights generated from this project could provide the basis for new therapeutic approaches for Alzheimer disease.