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

描述（由申请人提供）：淀粉样蛋白ss （Ass）在大脑中以实质斑块或脑血管沉积物的形式积累，是阿尔茨海默病（AD）和一些相关疾病患者的关键病理特征。Ass肽是由淀粉样蛋白ss-蛋白前体（AssPP）通过ss-和3-分泌酶的连续蛋白水解裂解而产生的。促进或阻碍Ass组装成纤维状结构并沉积在大脑中的因素在很大程度上仍未明确。我们实验室最近的体外研究表明，髓鞘碱性蛋白（MBP）是大脑中髓鞘的主要成分，是一种有效的Ass纤维组装抑制剂，可以保护培养的初级神经元免受Ass的毒性作用。尽管Ass在大脑中的空间沉积与这一发现一致（即富含MBP的脑白质在很大程度上缺乏纤维状Ass沉积物），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在体内的组装和积累仍然未知。已经产生了几种具有良好特征的人类AssPP转基因小鼠模型，这些模型具有ad样纤维淀粉样沉积。在这些已建立的AssPP转基因模型中，为了研究缺乏MBP对纤维淀粉样蛋白组装和沉积的影响，可以在MBP基因敲除的背景下培育它们。这种被称为“颤抖小鼠”的模型已经存在，但存在明显的缺陷，即它们不能形成髓磷脂，并且在出生后几个月内死亡。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产生显著的病理性淀粉样蛋白形成。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，而是使MBP蛋白上的高度特异性区域发生突变，从而使特定功能失效。为此，在本申请的R21阶段，我们建议生成一种新的“敲入”小鼠模型，我们将在内源性小鼠Golli-MBP基因的特定KRG基序中引入丙氨酸突变。我们最近的研究已经确定了这个特定的KRG基序是与Ass肽结合并抑制其纤维组装的基本元件。由此产生的新“敲入”模型将产生缺乏结合Ass肽并抑制其组装能力的MBP。这些新的MBP- KRG/AAA敲在小鼠中产生，并最初以生存能力、生长、行为和髓鞘形成为特征。在成功完成该申请的R21阶段后，我们计划进入R33阶段，我们建议在小鼠中交叉MBP-KRG/AAA敲入两种不同的人类AssPP转基因小鼠模型，这些小鼠产生纤维状淀粉样蛋白沉积。将对杂交小鼠进行老化并定量评估Ass肽的积累、组装和沉积以及由此产生的下游病理和行为后果。这些研究的完成将为控制致病性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提供新的途径。

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.