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.

在许多脑部病理（如中风，创伤和炎症）中，神经元死亡是由氧化应激，CA2+稳态障碍以及线粒体功能障碍引起的。这些病理条件也导致IPLA2的活性增强（VI CA2+独立的PLA2）。 IPLA2（指定为VI组）具有两个主要的同工型Via和Vib。我们建议IPLA2的非规范功能对于神经退行性过程至关重要。 IPLA2的非典型功能被定义为那些，其范围超出了磷脂稳态的经典家政服务。我们先前对文献的数据和分析导致了以下假设：VIB和VIB IPLA2的非规范函数是CNS中病理状态的关键点，CNS与氧化应激有关。我们在拟议的研究中探讨了这一假设，该假设有两个主要部分。第1部分研究了基本的生化机制，非规范的IPLA2功能，即VIB和VIB IPLA2同工型在调节Ca2+稳态和脑细胞中线粒体功能中的作用。我们使用特定的药理抑制剂和相应的分子生物学方法（RNA沉默和原位分析）来识别所涉及的同工型。第2部分分析了IPLA2的控制如何稳定Ca2+以及在不同氧化应激模型中脑细胞中线粒体的功能，重点是中风模型（氧气 - 葡萄糖剥夺）和谷氨酸兴奋性毒性范式。这将有助于基于对海马神经元 /星形胶质细胞中特定IPLA2同工型的控制和器官型海马片培养物的控制神经保护机制。这些条件与IPLA2活性增加有关。因此，我们旨在将IPLA2（通过和VIB）建立为新的分子靶标，以改善氧化应激和线粒体功能障碍中的脑细胞存活。另一方面，遗传性神经退行性人类婴儿神经性营养不良（INAD）显然与通过IPLA2造成的遗传缺陷有关。因此，此外，我们将使用人INAD的两个动物模型研究IPLA2活性中慢性缺陷的分子后果。我们将使用来自两个基因修饰的小鼠菌株的脑细胞，一种是通过IPLA2 Hypomorph，另一个是通过IPLA2 G373R突变体具有不活性的。由于通过IPLA2不足，它们用于研究INAD病理学的分子基础。我们将研究Ca2+调节，线粒体功能以及氧化应激的变化的INAD驱动变化，以得出可能的神经保护机制。此外，这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直接连接。总体而言，该项目产生有关IPLA2同工型在调节线粒体功能，ROS产生和细胞Ca2+稳态中的作用的基本知识。