Organellar pH shifts in Alzheimer's disease from simulation & experiment

描述（由申请人提供）：尚不清楚在阿尔茨海默病（AD）中在脑神经元（哪些细胞器）内淀粉样蛋白-β肽（A β x）主要产生于何处&发生的A β x的过度产生是否是由于淀粉样蛋白前体蛋白（APP）和/或在AD中加工APP的β-分泌酶的误作用/错误路由。在AD中，存在脑酸中毒（例如，由于β-分泌酶具有酸性pH最适值，因此确定细胞内酸中毒是否也导致APP在其中加工的细胞器的pH降低是重要的。本论文的主要目的是：（1）通过计算机模拟，从分子水平上了解细胞器模型内部pH值的变化（脂质体）影响A β 42自聚集和A β 42-脂质相互作用，以（2）建立早期和晚期内体（LE），溶酶体（LY），高尔基体（Aßx可能在其中产生的细胞器）在胚胎和成年小鼠脑神经元内的变化，作为（a）每种细胞器类型与细胞体的接近度，（3）表征这些分布在家族性阿尔茨海默病（FAD）的转基因小鼠模型（5XFAD）中如何作为（B）小鼠年龄和（c）总脑区的函数而变化（海马和同皮质）相比，（4）表征APP，Aßx， β-分泌酶在这些细胞器内的变化与a、B和c有关。目的（1）将使用具有包封的A β x的脂质体的粗粒度分子动力学模拟来实现。目的（2）-（4）将使用荧光显微镜技术实现。所提出的工作是由以下假设驱动的：（1）降低的pH将促进Aßx的自聚集以及Aßx与脂质体壁的相互作用，导致脂质体完整性的破坏。(2)在所有非AD病例中神经元细胞器的pH分布将表现出空间梯度，其中细胞体近端的细胞器将比远端的细胞器酸性更强。(3)随着5XFAD小鼠年龄的增长，将存在（i）高尔基体和早期核内体（EE）的酸中毒;（ii）与AD模型中的LE和LY相比，高尔基体和EE中的A β x和β-分泌酶的共定位在统计学上显著增加。变化（i）和（ii）都将出现在海马的神经元中，然后出现在同皮质的神经元中。拟议的工作旨在使NIH的NIA更接近其目标，即“了解衰老的本质和衰老过程，以及与衰老相关的疾病和状况”，因为它涉及到AD，一种无情的，目前不可阻挡的全球流行病。

DESCRIPTION (provided by applicant): It is unclear where within brain neurons (which organelles) the amyloid-ß peptide (Aßx) is predominantly produced in Alzheimer's disease (AD) & whether or not the overproduction of Aßx that occurs is due to a mistrafficking/misrouting of the amyloid precursor protein (APP) &/or the ß-secretase enzyme that processes APP in AD. In AD, there is cerebral acidosis (e.g., pH 6.6) & a decreased intracellular pH.1 Since ß-secretase has an acidic pH optimum, it is important to determine if the intracellular acidosis also leads to decrease in the pH of organelles within which APP is processed. The Aims of the proposed work are to (1) gain a molecular level understanding from computer simulations of how changes in the lumenal pH of crude organelle models (liposomes) impact the Aß42 self-aggregation & Aß42-lipid interactions, to (2) establish the baseline pH distribution of the early & late endosome (LE), lysosome (LY), & Golgi apparatus (the organelles within which Aßx is potentially produced) within embryonic & adult mouse brain neurons as a function of (a) each organelle- type's proximity to the cell body, to (3) characterize how these distributions change in a transgenic mouse model (5XFAD) of familial Alzheimer's disease (FAD) as a function of (b) the age of the mice & (c) gross brain region (hippocampus & isocortex) when compared to controls, & to (4) characterize how the colocalization of APP, Aßx, & ß-secretase changes within these organelles with respect to a, b, & c. Aim (1) will be achieved using coarse-grained molecular dynamics simulations of liposomes with encapsulated Aßx. Aims (2)-(4) will be achieved using fluorescence microscopy techniques. The proposed work is driven by the following Hypotheses: (1) a decreased pH will promote self-aggregation of Aßx & interactions of Aßx with the liposome walls, leading to disruption of the integrity of the liposome. (2) The pH distribution of neuronal organelles in all non-AD cases will exhibit a spatial gradient in which the organelles proximal to the cell body will be more acidic than distal organelles. (3) As the 5XFAD mice age, there will be (i) acidosis of the Golgi & early endosomes (EEs) & (ii) a statistically significant increase in th colocalization of Aßx & the ß-secretase enzyme in the Golgi & EEs as compared to the LEs & LYs in the AD model. Changes (i) & (ii) will both appear in neurons of the hippocampus before appearing in neurons of the isocortex. The proposed work seeks to bring the NIH's NIA one step closer to its goal of "understand[ing] the nature of aging & the aging process, & diseases & conditions associated with growing older" as it relates to AD, a ruthless, currently unstoppable, worldwide epidemic.