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，大脑神经元(哪些细胞器)中主要产生淀粉样多肽(Aüx)的位置&是否由于AD中处理APP的淀粉样前体蛋白(APP)和/或处理APP的？分泌酶的错误提取/错误路由而导致A？x的过度产生。在阿尔茨海默病中，存在脑酸中毒(例如，pH 6.6)&细胞内pH降低。1由于？分泌酶具有最适的酸性pH，因此确定细胞内的酸中毒是否也导致处理APP的细胞器的pH降低是很重要的。这项拟议工作的目的是(1)从计算机模拟中获得分子水平的理解，了解粗细胞器模型(脂质体)管腔pH的变化如何影响A？42自聚集和A？42-脂类相互作用，(2)建立胚胎和成年小鼠脑神经元中早期和晚期内小体(LE)、溶酶体(LY)和高尔基体(A？x可能在其中产生的细胞器)的基线pH分布，这是(A)每种细胞器类型与细胞体的距离的函数，(3)表征这些分布在家族性阿尔茨海默病(FAD)转基因小鼠模型(5XFAD)中作为(B)小鼠年龄和(C)大脑区(海马体和等皮质)的函数的变化，以及(4)表征APP，A？x，和 目标(1)将通过粗粒分子动力学模拟被包裹的脂质体来实现。目标(2)-(4)将使用荧光显微镜技术来实现。所提出的工作是由以下假设驱动的：(1)降低的pH将促进A？x的自聚集以及A？x与脂质体壁上的相互作用，导致脂质体的完整性被破坏。(2)非阿尔茨海默病患者神经细胞器的pH分布呈空间梯度，胞体近端的细胞器比远端的细胞器更具酸性。(3)随着5XFAD小鼠年龄的增长，(1)高尔基体和早期内小体(EES)的酸中毒；(2)与AD模型的LES和Lys相比，高尔基体和EES中A？x和？分泌酶的共存显著增加。变化(I)和(II)都会出现在海马区的神经元中，然后出现在等皮质的神经元中。这项拟议的工作旨在使NIH的NIA更接近其目标，即“了解衰老的本质和衰老过程，以及与变老相关的疾病和状况”，因为它与AD有关，AD是一种无情的、目前无法阻止的世界性流行病。

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.