Influence of myelin basic protein on neuronal A Beta assembly and toxicity

描述（由申请人提供）：淀粉样蛋白是阿尔茨海默病（AD）和相关疾病发病机制中的关键致病分子。A？具有自组装成可溶性低聚物并最终形成存款于中枢神经系统中的不溶性纤维状结构的强烈倾向。虽然可溶性A寡聚体组装和A？沉积物主要存在于CNS的细胞外室，但越来越多的证据表明，A？的初始组装阶段及其毒性活性的潜在位点发生在神经元内，这表明这可能是靶向破坏该过程的早期位点。然而，我们目前的理解是什么调节这些过程中的中枢神经系统，特别是在神经元，仍然是不完整的。最近，我们发现髓鞘碱性蛋白（MBP）作为一种新的因素，从大脑中分离，可以强烈结合到A肽，并有效地抑制其组装成纤维和其神经毒性。此外，MBP的这种特殊功能定位于N-末端残基1-64（MBP 1 -64），这是一个也包含在大多数相关Golli蛋白中的区域。除了其在髓鞘鞘形成中的突出作用之外，Golli-MBP蛋白存在于许多细胞（包括神经元）中，并且已经被提出作为细胞内多功能支架，其可以结合许多影响不同细胞过程的细胞内蛋白质和小分子配体。 根据这些观点，该探索性提议的总体假设是Golli-MBP蛋白与细胞内A肽相互作用以影响它们在神经元内和脑细胞外的组装、积累和毒性。在本提案中，我们计划实施多方面的方法来研究生物活性MBP片段如何在体外和体内与A肽相互作用，以调节其细胞内和细胞外组装，沉积和病理后果。首先，我们将利用从Tg-5xFAD小鼠制备的培养的皮层神经元，其在体外产生高水平的细胞内和细胞外A肽。我们将在这些神经元中表达MBP 1 -64片段，以研究它如何影响细胞内A肽的命运。其次，我们将使用充分表征的Tg-5xFAD小鼠，其表现出神经元内A <$并产生丰富的年龄依赖性细胞外A <$斑块沉积，伴随神经炎症和行为缺陷。Tg-5xFAD小鼠将与新产生的在神经元中表达生物活性MBP 1 -64片段的转基因小鼠杂交，以研究其与A肽的相互作用以及这可能如何改变病理结果。这些研究的完成将为细胞内Golli-MBP与A？相互作用的生物学提供新的见解，这可能有助于A？水平的空间和定量调节、组装和脑中的沉积以及伴随的下游病理后果。 公共卫生相关性：蛋白质片段（称为淀粉样蛋白（A））在大脑中的组装和沉积是阿尔茨海默病和相关疾病的关键病理特征。调节其组装的因素在确定疾病期间A？是否以及在何处积聚方面起着重要作用。本提案的目的是研究一种新鉴定的因子，髓鞘碱性蛋白（MBP），在培养的神经元细胞和脑中使用神经元内和细胞外A <$积累的小鼠模型的A <$组装和沉积的活性。

DESCRIPTION (provided by applicant): The amyloid ¿-protein is implicated as a key pathogenic molecule in the pathogenesis of Alzheimer's disease (AD) and related disorders. A¿ possesses the strong propensity to self-assemble into soluble, oligomeric species and ultimately into insoluble, fibrillar structures that deposit in the central nervous system. Although soluble A oligomeric assemblies and A¿ deposits exist largely in the extracellular compartment of the CNS growing evidence suggests that initial assembly stages of A¿ and potential sites for its toxic activities occurs within neurons suggesting that this may be an early site for targeting the disruption of this process. However, our present understanding of what regulates these processes in the CNS, in particular within neurons, remains incomplete. Recently, we identified myelin basic protein (MBP) as a novel factor isolated from brain that can strongly bind to A¿ peptides and potently inhibit their assembly into fibrils and its neurotoxicity. Moreover, this particular function of MBP was mapped to N-terminal residues 1-64 (MBP1-64), a region that is contained in most related Golli proteins as well. In addition to its prominent role in myelin sheat formation, Golli-MBP proteins are present in numerous cells, including neurons, and have been proposed as intracellular multifunctional scaffolds that can bind a number of intracellular proteins and small molecule ligands affecting diverse cellular processes. In light of these points the overall hypothesis of this exploratory proposal is that Golli-MBP proteins interact with intracellular A¿ peptides to influence their assembly, accumulation, and toxicity within neurons and extracellularly in the brain. In the present proposal we plan to implement a multi-faceted approach to investigate how a biologically active MBP fragment interacts with A¿ peptides both in vitro and in vivo to regulate their intracellular and extracellular assembly, deposition, and pathological consequences. First, we will utilize cultured cortical neurons prepared from Tg-5xFAD mice that produce high levels of intracellular and extracellular A¿ peptides in vitro. We will express the MBP1-64 fragment in these neurons to investigate how it influences the fate of intracellular A¿ peptide. Second, we will use the well-characterized Tg-5xFAD mice that exhibit intraneuronal A¿ and develop abundant, age-dependent extracellular A¿ plaque deposits with accompanying neuroinflammation and behavioral deficits. The Tg-5xFAD mice will be crossed with newly generated transgenic mice that express the biologically active MBP1-64 fragment in neurons to investigate its interaction with A¿ peptides and how this might alter pathological outcomes. Completion of these studies will provide new insight into the biology of intracellular Golli-MBP interactions with A¿ that may contribute to the spatial and quantitative regulation of A¿ levels, assembly, and deposition in brain as well as the accompanying downstream pathological consequences. PUBLIC HEALTH RELEVANCE: Assembly and deposition of a protein fragment, known as amyloid ¿ -protein (A¿), in the brain is a key pathological feature of Alzheimer's disease and related disorders. Factors that regulate its assembly play an important role in determining if and where A¿ accumulates during disease. The purpose of this proposal is to investigate the activity of a newly identified factor, myelin basic protein (MBP), on A¿ assembly and deposition in cultured neuronal cells and in brain using a mouse model of intraneuronal and extracellular A¿ accumulation.