Mechanisms of Neuronal Apoptosis in Vivo

描述（由申请人提供）：神经系统中的神经元在神经退行性疾病和急性神经损伤后发生退行性变性。该基金先前被授予表征由靶向消融诱导的背外侧膝状体核（dLGN）神经元退行性变性的动物模型，并确定这种细胞死亡的分子介质。我们发现，这种逆行性神经变性是细胞凋亡，明确定义其结构，介导的Bax（多域Bcl-2家族死亡效应）和p53，和caspase-3信号。这种细胞死亡伴随着核周线粒体的积累、对DNA的氧化损伤和死亡效应物的亚细胞易位而出现，并且由神经元型一氧化氮合酶（nNOS）调节。以前和新的实验，使用原位细胞成像，表明凋亡前，目标剥夺dLGN神经元积累线粒体细胞体收缩之前。我们假设这些线粒体来源于轴突/突触末梢。在这次资助更新中，我们将使用我们的小鼠脑细胞凋亡模型，其中dLGN神经元在枕叶皮层消融后7天内发生凋亡，以研究体内神经元凋亡的线粒体机制。在目标1中，我们将确定线粒体积累的来源，并将测试线粒体通过动力蛋白马达从损伤的远端部位以改变的状态返回dLGN神经元细胞体的假设，所述改变的状态由其产生活性氧（ROS）的能力和仅BH 3死亡蛋白（Bad、Puma和Noxa）的含量定义。新的实验还表明，凋亡前dLGN神经元积累细胞内Ca 2+。在目标2中，我们将确定细胞内Ca 2+积累的可能机制和线粒体Ca摄取和钙调神经磷酸酶介导的Bad去磷酸化和线粒体易位的凋亡前作用。在目标3中，我们将研究的假设，nNOS激活的目标剥夺dLGN神经元导致过氧亚硝酸盐的生产，细胞内锌2+的积累，和线粒体功能障碍。这项工作可以为靶点剥夺诱导的神经变性定义一个新的线粒体机制，并可以提高对体内神经元凋亡的细胞和分子机制的理解

DESCRIPTION (provided by applicant): Neurons in the nervous system undergo retrograde degeneration in neurodegenerative diseases and after acute neurological insults. This grant was awarded previously to characterize an animal model of retrograde degeneration of neurons in the dorsal lateral geniculate nucleus (dLGN) induced by target ablation, and to identify molecular mediators of this cell death. We found that this retrograde neurodegeneration is apoptosis, unequivocally defined by its structure, mediation by Bax (a multidomain Bcl-2 family death effector) and p53, and caspase-3 signaling. This cell death emerges with accumulation of perikaryal mitochondria, oxidative damage to DNA, and subcellular translocations of death effectors, and is modulated by neuronal nitric oxide synthase (nNOS). Previous and new experiments, using in situ cell imaging, show that preapoptotic, target-deprived dLGN neurons accumulate mitochondria prior to cell body shrinkage. We hypothesize that these mitochondria are derived from the axon/synaptic terminals. In this grant renewal we will use our model of apoptosis in mouse brain, in which dLGN neurons undergo apoptosis over 7 days after occipital cortex ablation, to study mitochondrial mechanisms of apoptosis in neurons in vivo. In Aim 1 we will identify sources of the accumulating mitochondria and will test the hypothesis that mitochondria return via dynein motors to the dLGN neuron cell body from the remote site of injury in an altered state defined by their capacity for generating reactive oxygen species (ROS) and content of BH3-only death proteins (Bad, Puma, and Noxa). New experiments also suggest that preapoptotic dLGN neurons accumulate intracellular Ca2+. In Aim 2 we will identify possible mechanisms of intracellular Ca2+ accumulation and the preapoptotic roles of mitochondrial Ca uptake and calcineurin-mediated Bad dephosphorylation and mitochondrial translocation. In Aim 3 we will examine the hypothesis that nNOS activation in target-deprived dLGN neurons leads to peroxynitrite production, intracellular Zn2+ accumulation, and mitochondrial dysfunction. This work can define a new mitochondrial mechanism for target deprivation-induced neurodegeneration and can improve the understanding of the cellular and molecular mechanisms of neuronal apoptosis in vivo