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（蛋白MFG-E8的片段），是否可能有助于淀粉样蛋白β的聚集。在阿尔茨海默病的两个小鼠模型中，我们现在发现i）MFG-E8水平随着脑淀粉样蛋白-β沉积而增加，并且MFG-E8染色定位于淀粉样蛋白纤维，并且ii）MFG-E8的遗传缺陷减少脑淀粉样蛋白变性并可能改变淀粉样蛋白构象。由于我们不能纯化重组medin用于体内给药，我们寻找内源性来源，发现iii）medin的细胞外聚集发生在老年野生型（但不是Mfge 8敲除）小鼠的主动脉中，强烈地使人联想到人类的情况，和iv）脑内注射老年野生型主动脉匀浆诱导预沉积APP转基因动物中的淀粉样蛋白-β聚集。进一步的数据表明，与年龄匹配的对照相比，v）全长MFG-E8和~6 kDa片段（其可对应于medin）在人类阿尔茨海默病患者中增加。由于已经报道MFG-E8可以增强淀粉样蛋白-β的吞噬作用，我们还分析了小胶质细胞淀粉样蛋白摄取，但发现MFG-E8缺乏没有改变。 因此，我们的数据强烈表明MFG-E8/medin和淀粉样蛋白-β之间的直接相互作用增强淀粉样蛋白-β聚集。在当前资助期内的实验将旨在确认我们的初步数据，例如APP x Mfge 8敲除小鼠中脑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，它们将包括鼠/人脑和脑脊液中medin水平的定量（该分析迄今为止由于可用的抗medin抗体与淀粉样蛋白-β在蛋白质印迹上的交叉反应以及肽的相似分子量（即4/5 kDa）而被阻止;然而，我们刚刚获得了一种新的单克隆medin特异性抗体，这将使我们能够进行这些实验）。 此外，我想申请一年的项目延期，以牢固地建立淀粉样蛋白β和medin的体内相互作用。为此，我建议通过脑内注射i）年轻/年老野生型主动脉和显示低/高水平medin的人主动脉或ii）富集medin或medin耗尽的主动脉匀浆来分析淀粉样蛋白β聚集的诱导，并且iii）进行共免疫沉淀实验以确定medin/MFG-E8是否直接与脑中的淀粉样蛋白β相互作用。由于几乎在60岁以上的任何人的血管中都发现了medin聚集体，因此很容易想象medin聚集体也在衰老的大脑中形成，特别是因为大脑MFG-E8水平随着衰老而增加。我们的研究结果将证明medin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.