AB and ApoE in animal models of Alzheimer's disease

阿尔茨海默病 (AD) 是一种影响全球超过 2000 万人的痴呆症。 在美国，AD 是老年人的第四大死因。 遗传、化学和形态学研究强烈表明，β 淀粉样蛋白 (Abeta) 肽在 AD 发病机制中具有重要作用。 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-β1 (TGF-β1) 转基因 (tg) 小鼠的成功创建为研究 Abeta 淀粉样蛋白生成的病理生理学提供了机会。 Tg 小鼠在 AD 治疗剂（例如抗 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 治疗方法中广泛采用 tg 小鼠模型，但尚未尝试对遗传和表型多样化的 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）由于大脑的寿命、系统发育距离、功能组织和应激状况的差异，人类和小鼠之间的Abeta结构和代谢会存在显着的量和质的差异； 2) tg小鼠中Abeta从大脑到血液的清除失败导致可溶性和不溶性Abe​​ta在大脑中逐渐积累； 3) ApoE的一些氨基酸结构域的部分丢失，在富含蛋白水解酶的环境中，例如AD脑，导致结构变化，这会影响ApoE对Abeta的亲和力以及复合物的清除。 该研究计划将表征 tg 小鼠中 Abeta 肽的化学结构并对其进行定量：APP23、2576、PDAPP、PS1、TGF-β1 以及双 tg PS1/betaAPP 和 TGF-beta1/betaAPP。 与 Abeta 相关的 ApoE 将在 tg 小鼠大脑中进行量化和表征。 此外，将根据可溶性和不溶性Abe​​ta40和Abeta42负载来评估双PS1/betaAPP tg小鼠中Abeta疫苗接种的效果。 为了实现这些目标，将通过快速高效的液相柱色谱法从脑匀浆或血浆中从 tg 小鼠中分离出水溶性和水不溶性 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。肽的定量将通过蛋白质印迹或敏感铕免疫测定进行。 这些重要的表征和比较将为进化、生理和病理学术语提供重要的见解，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.