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

在许多脑病理中，如中风、创伤和炎症，神经元死亡是由氧化应激、钙稳态失调和线粒体功能障碍引起的。这些病理状态也导致iPLA2活性增强(第VI组非钙非依赖性PLA2)。IPLA2被命名为VI族，有两个主要的亚型，VIA和VIB。我们认为iPLA2的非正则功能在神经退行性变过程中具有独特的重要性。IPLA2的非典型功能被定义为那些超出经典的磷脂动态平衡的内务管理的功能。我们以前的数据和对文献的分析导致了这样的假设，即VIA和VIB iPLA2的非规范功能是中枢神经系统病理状态的关键，而这些病理状态与氧化应激有关。我们在这项拟议的研究中探讨了这一假设，该研究包括两个主要部分。第一部分探讨了iPLA2的基本生化机制、非规范的iPLA2功能，即VIA和VIB iPLA2亚型在调节脑细胞内钙稳态和线粒体功能中的作用。我们使用特定的药物抑制剂和相应的分子生物学方法(RNA沉默和原位分析)来鉴定所涉及的异构体。第二部分分析了在不同的氧化应激模型中，iPLA2的控制如何稳定脑细胞中的钙离子和线粒体的功能，重点是中风模型(缺氧-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/星形胶质细胞中特定iPLA2亚型的神经保护机制，以及器官型海马片培养。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，以改善氧化应激和线粒体功能障碍中脑细胞的存活。另一方面，遗传性神经退行性婴儿神经轴索营养不良(INAD)显然与遗传导致的VIA iPLA2缺陷有关。因此，此外，我们还将使用两种人类INAD的动物模型来研究iPLA2活性慢性缺陷的分子后果。我们将使用两个转基因小鼠品系的脑细胞，一个是通过iPLA2低晶型，另一个是通过iPLA2 G373R突变体具有非活性。它们用于研究由于iPLA2缺乏引起的INAD病理的分子基础。我们将研究内源性钙离子调节、线粒体功能和氧化应激的变化，以得出可能的神经保护机制。此外，这两个突变的小鼠将允许区分iPLA2的非规范功能是否可能与其磷脂酶活性有直接联系。总体而言，该项目获得了关于iPLA2亚型在调节线粒体功能、ROS生成和细胞内钙稳态中所起作用的基础知识。

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.