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

描述(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。具有强烈的自我组装成可溶的低聚物种的倾向，最终形成沉积在中枢神经系统中的不可溶的纤维状结构。尽管可溶的A寡聚体和A？沉积主要存在于中枢神经系统的胞外隔室，但越来越多的证据表明，A？的初始组装阶段和其毒性活动的潜在部位发生在神经元内，这表明这可能是破坏这一过程的早期靶点。然而，我们目前对是什么在中枢神经系统，特别是在神经元中调节这些过程的了解仍然不完整。最近，我们发现髓鞘碱性蛋白(MBP)是从大脑中分离出来的一种新的因子，它能与A肽强烈结合，并能有效地抑制它们组装成纤维及其神经毒性。此外，MBP的这一特殊功能被定位到N-末端残基1-(MBP1-)，这一区域也包含在大多数相关的高尔基蛋白中。除了在髓鞘形成中的重要作用外，Golli-MBP蛋白还存在于包括神经元在内的众多细胞中，并被认为是细胞内多功能支架，可以结合许多细胞内蛋白质和影响不同细胞过程的小分子配体。根据这些观点，这一探索性建议的总体假设是，Golli-MBP蛋白与细胞内A？肽相互作用，影响它们在神经元内和细胞外的组装、蓄积和毒性。在目前的提案中，我们计划采用多方面的方法来研究具有生物活性的MBP片段如何在体外和体内与A？肽相互作用，以调节它们在细胞内和细胞外的组装、沉积和病理后果。首先，我们将利用从TG-5xFAD小鼠制备的体外培养的皮质神经元，这些神经元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MBP1-片段，以研究它如何影响细胞内A肽的命运。其次，我们将使用特征良好的TG-5xFAD小鼠，这些小鼠表现出神经元内的A？，并发展出丰富的、与年龄相关的细胞外A？斑块沉积，并伴随着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具有生物活性的MBP1-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？水平的空间和数量调节、在脑中的组装和沉积以及伴随的下游病理后果。

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.