Mouse Model of Myelin Basic Protein-Amyloid Beta Interactions in Brain

描述(申请人提供)：淀粉样蛋白(Ass)以实质斑块或脑血管沉积的形式在大脑中积聚，是阿尔茨海默病(AD)患者和几种相关疾病的关键病理特征。Ass多肽是由淀粉样蛋白SS-蛋白前体(ASPP)通过SS-和3-分泌酶的顺序蛋白分解而产生的。促进或阻碍Ass组装成沉积在大脑中的纤维状结构的因素在很大程度上仍不清楚。本实验室最近的体外研究表明，髓鞘碱性蛋白(MBP)是脑内髓鞘的重要组成部分，是Ass纤维组装的有效抑制物，可以保护培养的原代神经元免受Ass的毒性作用。虽然Ass在脑内的空间沉积与这一发现是一致的(即富含MBP的脑白质中几乎没有纤维状Ass沉积)，并且MBP水平降低与Ass水平升高之间存在关系，但MBP是否确实影响Ass在体内的聚集和积累仍不清楚。已经产生了几个特征良好的人类asspp转基因小鼠模型，这些模型产生了类似AD的纤维淀粉样沉积。为了研究缺乏MBP对这些已建立的ASPP转基因模型中纤维淀粉样蛋白组装和沉积的影响，人们可以将它们培育到MBP基因敲除的背景上。这种被称为颤抖鼠的模型是存在的，但也有明显的缺点，因为它们不会形成髓鞘，在出生后几个月内就会死亡。不幸的是，人类asspp转基因小鼠模型需要超过几个月的衰老才能形成显著的病理性淀粉样蛋白。我们计划采用的更复杂的方法不是敲除整个MBP蛋白的表达，而是突变MBP蛋白上的一个高度特定的区域，以使特定的功能失效。为此，在本申请的R21阶段，我们建议生成一个新的“敲入”小鼠模型，在该模型中，我们将在内源性小鼠Golli-MBP基因中的一个特定KRG基序中引入丙氨酸突变。我们最近的研究发现，这个特定的KRG基序是与天冬氨酸多肽结合并抑制其纤维组装的重要元件。由此产生的新的“敲入”模型将产生缺乏结合Ass多肽和抑制其组装的能力的MBP。这些新颖的MBP-KRG/AAA敲击小鼠将被产生，并初步鉴定其存活率、生长、行为和髓鞘形成。在成功完成本申请的R21阶段后，我们计划继续进行R33阶段，在那里我们建议用两种不同的人ASPP转基因小鼠模型进行MBP-KRG/AAA敲击，这些小鼠发展成纤维淀粉样沉积。杂交的小鼠品系将被老化，并对ASS多肽的累积、少量和沉积以及由此产生的下游病理和行为后果进行定量评估。这些研究的完成将为控制致病淀粉样蛋白组装的潜在生理机制提供新的见解，并可能为干预这一病理过程开辟新的途径。 公共卫生相关性：大脑中一种名为淀粉样蛋白(Ass)的蛋白质片段聚集和沉积是阿尔茨海默病和相关疾病的关键病理特征。调节其聚集的因素在确定ASS是否在疾病期间积累方面起着重要作用。这项建议的目的是建立一种创新和新颖的小鼠模型，以研究一种新发现的可以防止Ass在大脑中聚集和沉积的因素。

DESCRIPTION (provided by applicant): Accumulation of the amyloid ss-protein (Ass) in brain, either as parenchymal plaques or cerebrovascular deposits, is a key pathological feature of patients with Alzheimer's disease (AD) and several related disorders. The Ass peptides are derived from the amyloid ss-protein precursor (AssPP) by sequential proteolytic cleavages by ss- and 3-secretase enzymes. The factors that either promote or impede Ass assembly into fibrillar structures that deposit in brain remain largely undefined. Recent in vitro work from our laboratory has shown that myelin basic protein (MBP), a prominent component of myelin in brain, is a potent inhibitor of Ass fibrillar assembly and can protect cultured primary neurons from the toxic effects of Ass. Although the spatial deposition of Ass in brain is consistent with this finding (i.e. brain white matter rich in MBP is largely devoid of fibrillar Ass deposits) and there is a relationship between decreased MBP levels and increased Ass levels it remains unknown if MBP does indeed influence Ass assembly and accumulation in vivo. Several well-characterized human AssPP transgenic mouse models have been generated that develop AD-like fibrillar amyloid deposits. To study the consequences of the absence of MBP on fibrillar amyloid assembly and deposition in these established AssPP transgenic models one could breed them onto an MBP gene knockout background. Such a model, known as the shiverer mouse, exists but comes with the significant shortcomings in that they do not form myelin and die within several months after birth. Unfortunately, human AssPP transgenic mouse models require aging well beyond several months to develop significant pathologic amyloid formation. Instead of knocking out expression of the entire MBP protein a more sophisticated approach that we plan to employ will be to mutate a highly specific domain on the MBP protein to disable a specific function. To this end, in the R21 Phase of this application we propose to generate a novel "knock in" mouse model where we will introduce alanine mutations into a specific KRG motif in the endogenous mouse Golli-MBP gene. Our recent studies have identified this specific KRG motif as an essential element for binding to Ass peptides and inhibiting their fibrillar assembly. The resulting new "knock in" model will produce MBP that lacks the ability to bind Ass peptides and inhibit their assembly. These novel MBP- KRG/AAA knock in mice will be generated and initially characterized for viability, growth, behavior, and myelination. After successful completion of the R21 Phase of this application we plan to proceed to the R33 Phase where we propose to cross the MBP-KRG/AAA knock in mice with two different human AssPP transgenic mouse models that develop fibrillar amyloid deposition. The crossed mouse lines will be aged and quantitatively evaluated for the acumulation, asembly, and deposition of Ass peptides and the resulting downstream pathological and behavioral consequences. Completion of these studies will provide new insight into potential physiological mechanisms that govern pathogenic amyloid assembly and may lead to new avenues for intervention into this pathologic process. PUBLIC HEALTH RELEVANCE: Aggregation and deposition of a protein fragment, known as amyloid ss-protein (Ass), in the brain is a key pathological feature of Alzheimer's disease and related disorders. Factors that regulate its aggregation play an important role in determining if Ass accumulates during disease. The purpose of this proposal is to generate an innovative and novel mouse model to investigate a newly identified factor that can prevent Ass aggregation and deposition in brain.