Impact of aging, sex, vitamin D deficiency and high cholesterol on beta-amyloid peptide accumulation in brains of guinea pigs

衰老的一个主要难题是大脑功能的丧失。40和42个氨基酸长度的ß-淀粉样肽（Aß40和Aß42）是淀粉样前体蛋白（APP）随着年龄增长而越来越多地形成的病理裂解产物。其他调节因素可能是性别、维生素d缺乏和高胆固醇。APP通过ß-和？-分泌酶。亚ß单体发展为低聚体，然后聚集形成斑块。通过p -糖蛋白（P-gp，多药耐药蛋白1的基因产物，MDR1）和低密度脂蛋白受体相关蛋白1 （LRP1）在血脑屏障的外排来清除asas；ß通过晚期糖基化终产物受体（RAGE）返回大脑。其他外排转运蛋白——乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白（MRP1/4）——也能促进ß从大脑外排。as2主要被neprilysin降解，然后是胰岛素降解酶，并被小胶质细胞吞噬。随着正常和病理性衰老，ß-分泌酶蛋白（BACE-1）向淀粉样变途径升高，neprilysin、P-gp、LRP1和维生素D水平降低，RAGE升高。高胆固醇水平催化Aß42的形成，降低大脑中CYP46A1的表达，有利于大脑BACE-1的活性。虽然外周胆固醇和脑胆固醇是独立的池，但外周胆固醇水平与脑β积累密切相关，而不是脑胆固醇水平。选择豚鼠（GP）进行研究。它的大小非常适合连续收集血浆和脑脊液。GP α肽序列100%对应，Mdr1与人类具有高度同源性。GP以低密度脂蛋白的形式携带大部分胆固醇，并具有胆固醇酯转移蛋白和脂蛋白脂肪酶活性，导致胆固醇逆向转运和类似于男性的脱脂级联反应。该计划的重点是定义GP酶和转运体的细胞变化，这些转运体随着衰老和维生素D/胆固醇状态的改变而形成和去除Aßs，这项科学研究属于NSERC的范围。我们的目标1：开发一种改进的固相萃取法（SPE-ELISA）检测Aß40和Aß42的方法；目标2：检测不同年龄、性别和维生素D/胆固醇状况的全科医生中与asin稳态相关的基因的变化；目标3：检测不同年龄、性别和维生素D/胆固醇状况的全科医生脑和肝脏中asin的药代动力学；建立一个基于生理的药代动力学模型，用于预测衰老和维生素D/胆固醇状态改变时asas的变化。这些目标共同验证了年龄、维生素D缺乏和高胆固醇是asas在大脑中积累的危险因素的假设。科学数据将导致补救措施，以改善结果，以改善大脑健康。

A major puzzle with aging is the loss of brain function. ß-Amyloid peptides of 40 and 42 amino acid lengths (Aß40 and Aß42) are pathologic cleavage products that are being formed increasingly from the amyloid precursor protein (APP) with aging. Other modulating factors may be sex, vitamin D-deficiency and high cholesterol. The synthesis of Aß from APP occurs via sequential cleavage by the ß- and ?- secretases. The Aß monomers progress to oligomers then aggregate to plaques. Removal of Aß occurs via efflux by P-glycoprotein (P-gp, gene product of the multidrug resistance protein 1, MDR1) and the low-density lipoprotein receptor-related protein 1 (LRP1) at the blood brain barrier; Aß returns to the brain via the receptor for advanced glycation end products (RAGE). Other efflux transporters - the breast cancer resistance protein (BCRP) and multidrug resistance associated proteins (MRP1/4) - also facilitate Aß efflux from brain. Aß is degraded mostly by neprilysin, then the insulin degrading enzyme, and is phagocytosed by the microglia. With normal and pathological aging, ß-secretase protein (BACE-1) is elevated towards the amyloidogenic pathway and neprilysin, P-gp, LRP1 and vitamin D levels are decreased, and RAGE, elevated. High cholesterol levels catalyze Aß42 formation and render lower CYP46A1 expression in brain that benefits brain BACE-1 activity. Although the peripheral and brain cholesterol are independent pools, peripheral but not brain cholesterol levels correlate strongly with brain Aß accumulation. The guinea pig (GP) is chosen for study. Its size is well-suited for serial plasma and CSF collection. There is 100% sequence correspondence of the GP Aß peptide and a high homology of the Mdr1 with that in humans. The GP carries most of its cholesterol in low density lipoprotein and possesses cholesterol ester transfer protein and lipoprotein lipase activities that result in reverse cholesterol transport and delipidation cascades similar to those in men. This proposed program focuses on defining the cellular changes in GP enzymes and transporters that form and remove Aßs with aging and altered vitamin D/cholesterol status, and the scientific investigation falls under the purview of NSERC. We will Aim 1: develop an improved method to assay for Aß40 and Aß42 by solid phase extraction, SPE-ELISA Aim 2: examine the changes in genes involved in Aß homeostasis among GPs of different ages, sexes, and vitamin D/cholesterol status Aim 3: examine the pharmacokinetics of Aß in brain and liver among GPs of different ages, sexes, and vitamin D/cholesterol status Aim 4: develop a physiologically-based pharmacokinetic model for the prediction of Aß changes in aging and altered vitamin D/cholesterol status  The aims collectively test the hypothesis that age, vitamin D deficiency and high cholesterol are risk factors for Aß accumulation in the brain. The scientific data will lead to remedial measures to ameliorate the outcomes to improve brain health.