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Cytosolic and mitochondrial Calcium-independent phospholipase A2 (VIA and VIB iPLA2): Non-canonical iPLA2 functions in calcium regulation und mitochondrial functionality as mediator for neuroprotection

胞质和线粒体钙非依赖性磷脂酶 A2（VIA 和 VIB iPLA2）：非经典 iPLA2 在钙调节和线粒体功能中发挥神经保护介质的作用

基本信息

批准号：
236346012
负责人：
Professor Dr. Georg Reiser
金额：
--
依托单位：
Institut für Inflammation und Neurodegeneration
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2016-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/236346012?language=en
关键词：
Cytosolic mitochondrial Calcium independent phospholipase

项目摘要

In numerous brain pathologies, like stroke, trauma, and inflammation, the neuronal death is caused by oxidative stress, disturbance of Ca2+ homeostasis, and mitochondrial dysfunction. These pathological conditions lead also to enhanced activity of iPLA2 (group VI Ca2+-independent PLA2). The iPLA2, designated group VI, has two major isoforms, VIA and VIB. We propose that non-canonical functions of iPLA2 are of unique importance for neurodegenerative processes. Non-canonical functions of iPLA2 are defined as those, which extend beyond the classic housekeeping of phospholipid homeostasis. Our previous data and analysis of the literature lead to the hypothesis that the non-canonical functions of VIA and VIB iPLA2 are a key point for the pathological states in CNS, which are linked to oxidative stress. We explore this hypothesis in the proposed study, which has two major parts. Part 1 investigates the fundamental biochemical mechanisms, the non-canonical iPLA2 functions, i.e. the role of the VIA and VIB iPLA2 isoforms in regulation of Ca2+ homeostasis and mitochondrial functions in brain cells. We use specific pharmacological inhibitors and corresponding molecular biology methods (RNA silencing and in-situ analysis) for identifying the isoforms involved. Part 2 analyzes how control of iPLA2 can stabilize Ca2+ and the functions of mitochondria in brain cells in different models of oxidative stress, focusing on the stroke model (oxygen-glucose deprivation) and glutamate excitotoxicity paradigm. This will help to envisage neuroprotective mechanisms based on control of specific iPLA2 isoforms in hippocampal neurons / astrocytes, and organotypic hippocampal slice cultures. These conditions are associated with increased iPLA2 activity. Therefore, we aim to establish iPLA2 (VIA and VIB) as novel molecular targets to improve brain cell survival in oxidative stress and mitochondrial dysfunction. On the other side, the hereditary neurodegenerative human infantile neuroaxonal dystrophy (INAD) is clearly linked to the genetically caused deficits in VIA iPLA2. Therefore, in addition we will investigate the molecular consequences of chronic deficit in iPLA2 activity using two animal models of the human INAD. We will use the brain cells from two gene-modified mouse strains, one is VIA iPLA2 hypomorph, and the other has the inactive VIA iPLA2 G373R mutant. They serve to investigate the molecular basis for INAD pathology due to VIA iPLA2 insufficiency. We will study INAD-driven changes in Ca2+-regulation, mitochondrial functions and changes in oxidative stress to derive possible neuroprotective mechanisms. Moreover, the 2 mutant mice will allow to distinguish whether the non-canonical functions of iPLA2 possibly have direct connections to its phospholipase activity. Overall, the project yields fundamental knowledge concerning the role of iPLA2 isoforms in regulation of mitochondrial functions, ROS generation, and cellular Ca2+ homeostasis.

在许多脑病中，如中风、创伤和炎症，神经元死亡是由氧化应激、Ca2+稳态紊乱和线粒体功能障碍引起的。这些病理条件也导致iPLA2（组VI Ca2+独立PLA2）的活性增强。iPLA2，被称为第六族，有两个主要的同工异构体，VIA和VIB。我们认为iPLA2的非规范功能在神经退行性过程中具有独特的重要性。iPLA2的非规范功能被定义为那些超出了磷脂稳态的经典管理的功能。我们之前的数据和对文献的分析得出假设，VIA和VIB iPLA2的非规范功能是中枢神经系统病理状态的关键，这些病理状态与氧化应激有关。我们在提出的研究中探讨了这一假设，该研究分为两个主要部分。第一部分研究了基本的生化机制，非规范iPLA2功能，即VIA和VIB iPLA2亚型在脑细胞中调节Ca2+稳态和线粒体功能中的作用。我们使用特定的药理学抑制剂和相应的分子生物学方法（RNA沉默和原位分析）来鉴定所涉及的亚型。第2部分分析了iPLA2的控制如何在不同的氧化应激模型中稳定Ca2+和线粒体在脑细胞中的功能，重点是中风模型（氧-葡萄糖剥夺）和谷氨酸兴奋毒性范式。这将有助于设想基于控制海马神经元/星形胶质细胞和器官型海马切片培养中特定iPLA2亚型的神经保护机制。这些情况与iPLA2活性增加有关。因此，我们的目标是建立iPLA2 （VIA和VIB）作为新的分子靶点来改善氧化应激和线粒体功能障碍下的脑细胞存活。另一方面，遗传性神经退行性人类婴儿神经轴突营养不良（INAD）显然与遗传引起的VIA iPLA2缺陷有关。因此，此外，我们将使用两种人类INAD动物模型来研究iPLA2活性慢性缺陷的分子后果。我们将使用两种基因修饰小鼠的脑细胞，一种是VIA iPLA2亚型，另一种是失活的VIA iPLA2 G373R突变体。它们用于研究VIA iPLA2不足引起的INAD病理的分子基础。我们将研究inad驱动的Ca2+调节、线粒体功能和氧化应激变化，以得出可能的神经保护机制。此外，2个突变小鼠将允许区分iPLA2的非规范功能是否可能与其磷脂酶活性有直接联系。总的来说，该项目获得了关于iPLA2亚型在线粒体功能、ROS生成和细胞Ca2+稳态调节中的作用的基本知识。