ATP13A2通过调节锌离子稳态参与线粒体内Aβ产生和聚集的机制研究

Alzheimer’s disease (AD) pathogenesis is believed to be triggered by the amyloid-β (Aβ) accumulation. Overproduction of Aβ and the failure of Aβ clearance have been observed in mitochondria of AD brains. Mitochondrial Aβ presence causes oxidative damage and mitochondrial respiratory dysfunction, activating a cascade of events leading to neurodegeneration. Zinc binding with Aβ induces aggregation and extracellular plaque formation. More importantly, a role for cytosolic zinc with Aβ accumulation has been shown to correlate with reactive oxygen species (ROS) production rates, and alters mitochondrial membrane potential, which results in mitochondrial dysfunction and fragmentation. Recently, ATP13A2 is found to be associated with zinc homeostasis and remediates mitochondrial dysfunction. An increased accumulation of failing mitochondria in ATP13A2−/− cells in conditions of zinc exposure may contribute to the increased sensitivity of zinc. ATP13A2, a transmembrane protein of 1180 amino acids, belongs to the P5 type pump ATPases and is highly expressed in the brain. The promoter region of the human ATP13A2 gene contains hypoxia response elements, which can bind to the transcription factor hypoxia inducible factor1α (HIF-1α). Hypoxic conditions up-regulate transcription of the ATP13A2 gene. Several physical interactors of ATP13A2 are involved inregulation of mitochondrial bioenergetics, and evidence from genetic interaction studies in yeast also provides a link between YPK9 and mitochondrial clearance. Therefore, in the present project, we would like to generate new animal models, ATP13A2 conditional-knockout and human APP/PS1 transgenic (ATP13A2flox/flox:CAMKII/APP/PS1) mice, to analyze whether ATP13A2 could maintain the balance of zinc in cytoplasm to protect the mitochondrial function and inhibit Aβ accumulation. In addition, we will use genome editing technology to knockdown ATP13A2 in familial Alzheimer’s disease (fAD) iPSC-derived neurons and explore the mechanism of the ATP13A2 involved in zinc transport and Aβ accumulation/clearance in mitochondria. This study will be helpful to understand whether ATP13A2 is involved in brain zinc metabolism and to provide a clue for ATP13A2 to AD prevention and therapy.

β-淀粉样蛋白(Aβ)在线粒体内沉积是导致阿尔茨海默病(AD)脑内神经元死亡及其功能障碍的重要原因之一。研究表明，细胞内锌离子稳态失衡是导致线粒体电子传递链损伤，诱发氧化应激，并促进Aβ的聚集的关键因素。因此，调控细胞内锌离子稳态成为抑制Aβ在线粒体内沉积的有效途径。新近研究表明，ATP13A2具有维持细胞内锌离子的稳态、保护线粒体的重要功能。我们前期研究发现，ATP13A2在AD脑内及体外培养的AD细胞模型中均显著升高，但其是否参与AD病理过程及其具体机制尚未见报道。本项目拟采用ATP13A2基因敲除的APP/PS1转基因小鼠，以及体外构建的ATP13A2基因缺失的神经元为研究对象，旨在阐明“ATP13A2调控细胞内锌离子稳态→减少线粒体内Aβ的聚集→稳定线粒体功能→改善AD病理进程”的具体机制，为确立ATP13A2作为AD防治的新靶点提供充分的理论和实验依据。

阿尔茨海默病的重要病理变化是由β-淀粉样蛋白前体剪切产生的Aβ在细胞沉积形成外老年斑。研究表明，Aβ是一个拥有多金属键合位点的金属蛋白。细胞内锌离子稳态失衡是导致线粒体电子传递链损伤，诱发氧化应激，并促进Aβ的聚集的关键因素。新近研究表明，ATP13A2具有维持细胞内锌离子的稳态、保护线粒体的重要功能。然而，ATP13A2是否参与Aβ的产生和沉积的具体机制尚不清楚。本研究证实了1）ATP13A2可以调节APP及其剪接片段的表达；ATP13A2的缺失可能是影响溶酶体降解APP及剪切产物和引起细胞自噬功能受损。2）ATP13A2通过调控锌转运蛋白，促使锌离子转运进入溶酶体或其他细胞器中，维持细胞内锌离子稳态；ATP13A2缺失引起细胞内锌离子浓度升高导致的线粒体功能损伤。3）ATP13A2与APP相互作用，通过影响自噬溶酶体功能调控APP的代谢从而影响Aβ老年斑的形成和沉积。综上所述，通过三部分的独立又互有联系的研究，我们得出ATP13A2在阿尔茨海默病的病理进程中起到主要的神经保护作用，其机制可能通过线粒体或溶酶体自噬通路调控APP的代谢影响Aβ老年斑的形成，并通过调节锌离子转运体从而维持细胞内锌离子的稳态，保护线粒体功能，从而改善AD小鼠的学习记忆能力。本项目为ATP13A2做为AD的靶向治疗的重要因子提供重要的理论和实验基础。