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

 描述(由申请者提供)：突触病理学--与认知障碍最相关的疾病之一--与神经毒素Aü的寡聚体的逐渐积累有关。A？单体组装成低聚物是一个浓度依赖的过程，因此，它依赖于改变调节间质液(ISF)中A？生理水平的动态平衡机制的不利变化，例如损害正常的脑移除和/或分解代谢不足。令人惊讶的是，人们对低聚体的大脑清除和局部分解代谢知之甚少，特别是在衰老过程中。基于大量的初步/可行性数据，我们建议通过将这些过程中未被充分研究的不同方面联系起来，开始填补这一知识空白：i)大脑A？物种的高度异质性，与经典的全长二人组A？40/A？42共存的多个N-末端和C-末端截断的衍生物；ii)大脑局部驻留的酶有效地产生这些截断片段的能力；iii)这些物种在溶解性和寡聚化倾向方面的显著差异，表明参与了相反的机制，要么是淀粉样蛋白发生，要么是清除；以及iv)衰老对清除途径的解剖和功能成分造成的负面影响，例如，血管完整性受损、细胞转运体密度降低、局部蛋白分解机制的不合格表现，所有这些都可能影响血管周围引流的脑外流效率，以及通过血脑和脑脊液屏障。已经存在的淀粉样蛋白沉积是一种很少考虑的并发症，它的存在进一步模糊了清除的场景，不仅通过潜在的可溶性A？种向皮损中招募，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的发生过程超越了简单的二分法A？40/A？42，涉及局部产生的前寡聚体截断片段，以及可能由外流转运蛋白LRP-1和P-gp介导的单体和寡聚体在体内的脑清除差异，并假设这些机制受到衰老和存在预先存在的淀粉样蛋白沉积的负面调节。结合两个特定的目标，我们建议比较完整和截断的Aü物种单体和寡聚形式在体内的生理性脑清除，评估它们的局部分解代谢和Aü-外流转运体的相关性，同时评估正常衰老和已建立的淀粉样蛋白沉积在脑清除机制中的不同影响。通过使用放射性标记和同位素标记的Aü同系物、野生型小鼠的立体定向海马区注射和APPSwePS1dE9转基因技术、新的特定抗体、小鼠脑脊液中有针对性的蛋白质组/质谱学方法以及体外细胞培养范例，该项目将提供对健康和疾病中脑A？分解代谢和清除的更好了解。

 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.