Mechanisms of Neuronal Apoptosis in Vivo

描述(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经历退行性变性。这项资助以前是为了描述靶点消融引起的膝状背外侧核(DLGN)神经元退行性变的动物模型，并确定这种细胞死亡的分子介质。我们发现，这种退行性神经变性是由其结构、Bax(一个多结构域的Bcl2家族死亡效应因子)和P53以及caspase-3信号调节的细胞凋亡。这种细胞死亡是由神经元型一氧化氮合酶(NNOS)调控的，伴随着核周线粒体的聚集、DNA的氧化损伤和死亡效应分子的亚细胞移位。先前和新的实验，使用原位细胞成像，表明凋亡前的、靶标被剥夺的dLGN神经元在细胞体收缩之前积累线粒体。我们推测这些线粒体来自轴突/突触终末。在这次拨款更新中，我们将使用我们的小鼠脑细胞凋亡模型，在枕叶皮质切除后dLGN神经元经历超过7天的凋亡，以研究体内神经元凋亡的线粒体机制。在目标1中，我们将确定积累的线粒体的来源，并将测试这一假设，即线粒体通过动力蛋白马达从损伤的远程位置返回dLGN神经元细胞体，这种改变的状态由它们产生活性氧物种(ROS)的能力和仅BH3死亡蛋白(Bad、Puma和Noxa)的含量定义。新的实验还表明，凋亡前的dLGN神经元积累了细胞内的钙离子。在目标2中，我们将确定细胞内钙积聚的可能机制，以及线粒体钙摄取和钙调神经磷酸酶介导的Bad去磷酸化和线粒体易位在细胞凋亡前的作用。在目标3中，我们将检验这一假说，即靶剥夺dLGN神经元中nNOS的激活导致过氧亚硝酸盐的产生、细胞内锌离子的积累和线粒体功能障碍。这项工作可以为靶剥夺诱导的神经变性定义一种新的线粒体机制，并有助于加深对活体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