Insights into the Brain Clearance Mechanisms of Oligomeric Beta-Amyloid Species

 描述（由申请人提供）：突触病理学-与认知障碍的最强相关性之一-与神经毒性A4的寡聚体形式的进行性积累有关。A β单体组装成寡聚体是一个浓度依赖性过程，因此，它依赖于改变调节间质液（ISF）中A β生理水平的稳态机制的不利变化，例如受损的正常脑清除和/或缺乏catalysts。令人惊讶的是，很少有人知道的脑清除和局部catalysts的寡聚体，特别是在老化过程中。基于大量的初步/可行性数据，我们建议通过将这些过程的不同的未充分研究的方面连接在一起来开始填补知识上的这一空白：i）脑Aß 40物种的认识不足的高度异质性，其中多个N-和C-末端截短的衍生物与经典的全长二聚体A β 40/A β 42共存; iii）这些种类在溶解性和寡聚化倾向方面的显著不同，表明参与淀粉样蛋白生成或清除的相反机制;和iv）老化对清除途径的解剖学和功能组分的负面影响，例如受损的血管完整性、细胞AAPs转运蛋白的较低密度、局部蛋白水解机制的不合格性能，所有这些都可能影响血管周围引流以及通过血脑和脑CSF屏障的脑流出效率。已经建立的淀粉样蛋白沉积物的存在（很少考虑的并发症）进一步模糊了清除情况，这不仅是通过可溶性淀粉样蛋白物质潜在募集到病变，而且还通过额外限制血管功能，促进淀粉样蛋白生成环的自我延续。我们假设淀粉样蛋白形成的过程超出了简单的二分法A β 40/A β 42，涉及局部产生的前寡聚体截短片段和可能由外排转运蛋白LRP-1和P-gp介导的单体和寡聚体A β种类的体内脑清除差异，并假设这些机制受到老化和预先存在的淀粉样蛋白沉积物的负面调节。组装在两个特定的目标，我们建议比较生理在体内脑清除的单体和寡聚体形式的完整和截短的AAPs物种，评估其局部catalysts和相关的AAPs-外排转运蛋白，同时评估的差异效应，正常老化和已经建立的淀粉样蛋白沉积物的存在下发挥在脑清除机制。通过使用放射性标记和同位素标记的ARAPINE同源物，在野生型小鼠和APPswePS 1dE 9转基因小鼠中进行立体定位海马内注射，新型特异性抗体，小鼠CSF中的靶向蛋白质组学/质谱法，以及体外细胞培养范例，该项目将更好地了解健康和疾病中的脑ARAPINE catalysts和清除。

 DESCRIPTION (provided by applicant): Synaptic pathology- one of the strongest correlates to cognitive impairment- is related to the progressive accumulation of oligomeric forms of neurotoxic Aß. The assembly of Aß monomers into oligomers is a concentration-dependent process and as such, it is dependent on adverse changes that alter homeostatic mechanisms regulating Aß physiologic levels in the interstitial fluid (ISF), such us impaired normal brain removal and/or deficient catabolism. Surprisingly, very little is known about the brain clearance and local catabolism of Aß oligomers, particularly during the aging process. Based on a wealth of preliminary/feasibility data we propose to start filling this gap in knowledge by bridging together different understudied aspects of these processes: i) the poorly recognized high heterogeneity of the brain Aß species, with multiple N- and C- terminally truncated derivatives co-existing with the classic full-length duo Aß40/Aß42; ii) the ability of local brain resident enzymes to efficiently generate these truncated fragments; iii) the remarkable dissimilarities of these species in solubility and oligomerization propensity, suggesting engagement in opposite mechanisms either amyloidogenesis or clearance; and iv) the negative impact that aging imposes to anatomical and functional components of the clearance pathway e.g. compromised vascular integrity, lower density of cellular Aß transporters, substandard performance of the local proteolytic machinery all likely affecting the brain efflux efficiency of the perivascular drainage, as well as through the blood-brain and brain-CSF barriers. The presence of already established amyloid deposits a seldom considered complication further obscures the clearance scenario, not only through the potential recruitment of soluble Aß species to the lesions but by additionally restricting vessel functionality, contributing to the self-perpetuation of the amyloidogenic loop. We hypothesize that the process of amyloidogenesis goes beyond the simplistic dichotomy Aß40/Aß42, involving locally generated pro-oligomeric truncated fragments and differential in vivo brain clearance for monomeric and oligomeric Aß species likely mediated by the efflux transporters LRP-1 and P-gp, and postulate that these mechanisms are negatively modulated by aging and by the presence of pre- existing amyloid deposits. Assembled in two specific aims, we propose to compare the physiologic in vivo brain clearance of monomeric and oligomeric forms of intact and truncated Aß species, evaluate their local catabolism and relevance of Aß-efflux transporters while assessing the differential effect that normal aging and the presence of already established amyloid deposits exert in the brain removal mechanisms. Through the use of radiolabeled and isotopically-labeled Aß homologues, stereotaxic intra-hippocampal injections in wild-type mice and APPswePS1dE9 transgenics, novel specific antibodies, targeted proteomic/mass spectrometry approaches in mouse CSF, and in vitro cell culture paradigms, the project will provide a better understanding of brain Aß catabolism and clearance in health and disease.