Effects of free fatty acids in neuronal survival

本实验室以前的报道发现神经生长因子分化嗜铬细胞瘤 细胞系（NGFDPC 12细胞）暴露于病理水平的外源性棕榈酸（PA）和硬脂酸（SA）脂肪酸 与缺氧/缺血期间发现的水平相当的酸导致细胞凋亡的急剧减少， 存活力和凋亡。这种细胞存活率的下降是因为这些饱和的FFA诱导了大量的 细胞凋亡（程序性细胞死亡）。相比之下，类似水平的花生四烯酸（AA）或 油酸（OL）、EPA或DMA FFA不降低这些细胞的存活力。中心假设指出， 高水平PA或SA脂肪酸诱导继发于线粒体的神经元凋亡！功能障碍这 本申请将主要集中于表征PA和SA在NGFDPC 12细胞中的凋亡作用， 将该分析扩展到初级视网膜神经节和皮质细胞神经元。具体目标1将包括 PA和SA介导的半胱天冬酶非依赖性神经元细胞死亡的表征。这一目标将决定 这些饱和FFA对培养的大鼠皮层和视网膜神经节原代细胞存活的影响。 实验设计将评估细胞活力的丧失是凋亡还是坏死的结果， 将决定这个过程是否依赖于半胱天冬酶的激活。进一步的实验将分析 哪些特定的凋亡蛋白参与了这个过程。具体目标2将确定FFA介导的 神经元细胞死亡通过线粒体途径发生。初步cDNA阵列分析 定量真实的时间PCR数据已经确定了几个候选线粒体基因， 在这个过程中。这一目标将阐明这些基因的作用，通过使用siRNA敲低， 过表达以调节细胞水平。我们预计，这些研究将有助于我们澄清 饱和FFA的病理水平如何影响神经元存活和凋亡的机制。

Previous reports from our laboratory have found that nerve growth factor differentiated pheochromacytoma cell lines (NGFDPC12 cells) exposed to pathological levels of exogenous palmitic (PA) and stearic (SA) fatty acids -- at a level comparable to that found during hypoxia/ischemia - causes a dramatic decrease in cell viability and apoptosis. This decrease in cell survival occurs because these saturated FFA induced massive apoptosis (program cell death) during 24 hours exposure. In contrast, similar levels of arachidonic (AA) or oleic (OL), EPA or DMA FFA did not decrease the viability of these cells. The central hypothesis states that high levels of PA or SA fatty acids induce neuronal apoptosis secondary to mitochondria! dysfunction. This application will primarily focus on characterizing the apoptotic effect of PA and SA in NGFDPC12 cells and extend this analysis to primary retinal ganglion and cortical cell neurons. Specific Aim 1 will include the characterization of PA and SA-mediated caspase-independent neuronal cell death. This aim will determine the effects of these saturated FFA in the survival of rat cortical and retinal ganglion primary cells in culture. The experimental design will evaluate whether the loss of cell viability is result of apoptosis or necrosis and will determine whether the process is dependent on caspase activation. Further experiments will analyze what specific apoptotic proteins are involved in the process. Specific Aim 2 will determine whether FFAmediated neuronal cell death occurs through the mitochondrial pathway. Preliminary cDNA array analysis and Quantitative real time PCR data has identified several candidate mitochondrial genes that are involved in this process. This aim will elucidate the role of these genes by using siRNA knockdown and overexpression to modulate cellular levels. We anticipate that these studies will help us to clarify the mechanisms of how pathological levels of saturated FFA affect neuronal survival and apoptosis.