AB and ApoE in animal models of Alzheimer's disease

阿尔茨海默病（AD）是一种痴呆症，影响全球超过2000万人。 在美国，AD是老年人死亡的第四大原因。 遗传学、化学和形态学研究强烈表明淀粉样蛋白β（Abeta）肽在AD发病机制中的重要作用。 β-淀粉样前体蛋白（betaAPP）、早老素1（PS1）、载脂蛋白E（ApoE）和转化生长因子-β 1（TGF-β 1）转基因（tg）小鼠的成功建立为研究A β淀粉样蛋白形成的病理生理学提供了机会。 Tg小鼠在AD治疗剂如抗Abeta聚集化合物、分泌酶抑制剂和抗Abeta疫苗接种的测试中至关重要。尽管在寻找有效的AD治疗中广泛采用tg小鼠模型，但尚未尝试对遗传和表型多样性tg小鼠品系产生的Abeta进行严格的化学表征和定量研究。假设：1）由于寿命、系统发育距离、脑的功能组织和痛苦状况的差异，在人和小鼠之间的Abeta结构和代谢中将发现显著的定量和定性差异; 2）在tg小鼠中，Abeta从脑到血液的清除失败导致可溶性和不溶性Abeta在脑中的进行性积累; 3）在富含蛋白水解酶的环境如AD脑中，ApoE的一些氨基酸结构域的部分丢失导致结构变化，这将影响ApoE对Abeta的亲和力和复合物的清除。 该研究计划将表征化学结构并量化tg小鼠中的Abeta肽：APP 23，2576，PDAPP，PS1，TGF-β 1和双tg PS1/betaAPP和TGF-β 1/betaAPP。 将在tg小鼠脑中定量和表征与Abeta相关的ApoE。 此外，将根据可溶性和不溶性Abeta 40和Abeta 42负荷评价Abeta疫苗接种在双PS1/betaAPP tg小鼠中的作用。 为了实现这些目标，将通过快速和高效液相柱色谱法从脑匀浆或血浆中分离水溶性和水不溶性Abeta和ApoE肽。 所有纯化的分子和衍生的酶促肽将通过质谱法、氨基酸分析和氨基酸测序进行化学表征。肽的定量将通过蛋白质印迹法或灵敏的铕免疫测定法进行。 这些重要的表征和比较将提供重要的见解，在进化，生理和病理方面，这将是有用的理解AD和减轻治疗的解释在tg小鼠模型。

Alzheimer disease (AD) is a dementia affecting over 20 million people worldwide. In the USA, AD represents the fourth greatest cause of death in the elderly. Genetic, chemical and morphological studies strongly suggest a seminal role for amyloid-beta (Abeta) peptides in the pathogenesis of AD. The successful creation of beta-amyloid precursor protein (betaAPP), presenilin 1 (PS1), apolipoprotein E (ApoE) and transforming growth factor-beta1 (TGF-beta1) transgenic (tg) mice has provided an opportunity to investigate the pathophysiology of Abeta amyloidogenesis. Tg mice are of paramount importance in the testing of AD therapeutic agents such as anti-Abeta aggregating compounds, secretase inhibitors and anti-Abeta vaccination. Despite the wide adoption of tg mice models in the search for effective AD treatments, a rigorous chemical characterization and quantification study of the Abeta produced by the genetically and phenotypically diverse tg mice strains has not been attempted. It is hypothesized that: 1) due to differences in lifespan, phylogenetic distance, functional organization and distress conditions of the brain significant quantitative and qualitative differences will be found in Abeta structure and metabolism between humans and mice; 2) failure in the clearance of Abeta from the brain to the blood in tg mice causes the progressive accumulation of both soluble and insoluble Abeta in the brain; 3) the partial loss of some of amino acid domains of ApoE, in an environment rich in proteolytic enzymes such as the AD brain, results in structural changes which would affect the affinity of ApoE for Abeta and the clearance of the complexes. The research plan will characterize the chemical structure and quantify Abeta peptides in the tg mice: APP23, 2576, PDAPP, PS1, TGF-beta1 and the double tg PS1/betaAPP and TGF-beta1/betaAPP. The ApoE associated to Abeta will be quantified and characterized in the tg mice brains. In addition, the effects of Abeta vaccination in the doubly PS1/betaAPP tg mice will be evaluated in terms of the soluble and insoluble Abeta40 and Abeta42 load. To achieve these goals the water-soluble and water-insoluble Abeta and ApoE peptides will be isolated from the tg mice by fast- and high- performance liquid column chromatographies from either brain homogenates or plasma. All purified molecules and derived enzymatic peptides will be chemically characterized by mass spectrometry, amino acid analysis and amino acid sequencing. Quantification of peptides will be performed by Western blot or by sensitive europium immunoassay. These significant characterizations and comparisons will provide important insights in evolutionary, physiological and pathological terms that will be useful for understanding AD and for the interpretation of mitigating treatments in tg mouse models.