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

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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.