AB and ApoE in animal models of Alzheimer's disease

阿尔茨海默病（AD）是一种影响全球2000多万人的痴呆症。在美国，老年痴呆症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 -淀粉样蛋白前体蛋白（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β -淀粉样蛋白形成的病理生理机制提供了机会。Tg小鼠在抗abeta聚集化合物、分泌酶抑制剂和抗abeta疫苗等AD治疗药物的测试中具有至关重要的意义。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不同的tg小鼠菌株产生的β进行严格的化学表征和定量研究。假设：1)由于人与小鼠在寿命、系统发育距离、脑功能组织和应激条件等方面的差异，在Abeta结构和代谢方面存在显著的数量和质量差异；2)在tg小鼠中，β从大脑到血液的清除失败导致大脑中可溶性和不可溶性β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，从而影响ApoE对Abeta的亲和力和复合物的清除。本研究计划将对tg小鼠：APP23、2576、PDAPP、PS1、TGF-beta1以及双tg PS1/betaAPP和TGF-beta1/betaAPP中的β肽进行化学结构表征和定量。与β相关的ApoE将在tg小鼠大脑中被量化和表征。此外，将根据可溶性和不可溶性Abeta40和Abeta42的负荷来评估Abeta疫苗接种在双PS1/betaAPP tg小鼠中的效果。为了实现这些目标，将采用快速高效液相色谱法从tg小鼠脑匀浆或血浆中分离出水溶性和不水溶性的Abeta和ApoE肽。所有纯化的分子和衍生的酶肽将通过质谱、氨基酸分析和氨基酸测序进行化学表征。多肽的定量将通过Western blot或灵敏的铕免疫分析法进行。这些重要的特征和比较将在进化、生理和病理方面提供重要的见解，这将有助于理解AD和解释tg小鼠模型中的缓解治疗。

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.