Elemental And Structural Organization Of Neurons And Gli

该项目研究神经元钙信号的生理和细胞方面，长期重点放在大型中枢神经系统神经元的突触后反应上。神经元对突触刺激的反应是胞内游离钙浓度([Ca~(2+)]i)的升高，这种升高受到细胞内钙储存库活动的强烈调控。这种活动在调节基因表达和LTP诱导等重要过程的时空钙信号的形成中发挥着重要作用。我们早些时候已经证明，刺激诱导的交感神经元[Ca~(2+)]i的增加伴随着线粒体钙浓度的大幅可逆升高。我们现在已经探索了在海马锥体神经元中，线粒体对一系列突触刺激的类似反应，并表明在这些细胞中，线粒体钙转运活动具有重要的生理和病理生理效应。 在海马锥体神经元中，[Ca~(2+)]i的大量增加激活了几个关键的激酶，而较低的[Ca~(2+)]i则增强了蛋白磷酸酶的活性。这种依赖于钙离子的某些关键酶，如钙/钙调蛋白依赖的激酶(CaMKs)的磷酸化状态的再平衡，控制着神经元可塑性中心通路的活性。我们先前发现，由强烈的钙离子内流介导的线粒体钙积累导致超氧阴离子自由基(O2-)的产生增加，这种活性通过抑制一系列磷酸酶(PP1、PP2A和/或PP2B)上调CaMKII和CaMKIV依赖的CREB的磷酸化。相反，我们现在发现，由于PP2A激活了ERK的上游介质Raf-1，所以持续低频刺激引起的弱[Ca+]i升高优先增强了磷酸酶活性，从而导致了ERK1/2的激活。然而，在短波高频刺激引起细胞内[Ca~(2+)]i强烈升高后，线粒体产生的O2-对PP2A的抑制延迟了ERK活性的启动。这种ERK的增加在刺激后10min达到最大，并且依赖于CaMKII的活性，而CaMKII的活性又被线粒体O2介导的抑制CaMKII去磷酸化所增强。相反，在HF刺激的ERK诱导早期，质膜结合的O2-来源NADPH氧化酶能增强ERK的激活，但其作用要弱得多，其机制不涉及抑制PP2A。结果表明，在Ras/Raf/MEK/ERK1/2途径中，线粒体产生的O2-和NADPH氧化酶调节特定而不同的步骤。 线粒体功能障碍在谷氨酸诱导的兴奋性毒性中起着核心作用，但导致细胞死亡的机制仍存在争议。早期的工作表明，在海马神经元中，线粒体功能障碍取决于钙负荷的大小，因为联合应用抑制线粒体钙摄取的药物，如FCCP，可以极大地提高细胞存活率，即使在存在此类药物的情况下，[Ca2+]i升高更大。因此，单靠[Ca~(2+)]i升高而不增加线粒体Ca~(2+)不足以诱导细胞死亡。我们继续通过研究损伤刺激后线粒体的离子、结构和功能变化来研究线粒体参与兴奋性死亡的机制。在强烈的NMDA刺激下，线粒体的钙含量普遍很高，但更重要的是，单个线粒体的含量变化很大。线粒体在肿胀和膜破裂方面存在平行的异质性，伴随着线粒体膜电位的丧失。令人惊讶的是，大多数线粒体都能耐受这些高水平的钙而不会造成永久性的损伤，并在2小时内恢复其刺激前的结构、组成和膜电位。然而，在一些细胞中，线粒体的小亚群并不能重建正常的钙离子和容量调节。在这些线粒体中，钙超载导致功能和形态的改变，导致膜完整性的丧失和可能的凋亡蛋白的释放；刺激后6-8h，约35%的细胞出现典型的凋亡特征。据推测，这些细胞中有足够多的线粒体被钙超载损伤，从而触发了有效的促凋亡信号。这些结果支持这样一种假设，即神经元内只有少数线粒体受到兴奋性毒性损伤就会导致细胞凋亡。 在技术要求和对分子马达的长期兴趣推动的其他进展中，我们使用扫描透射电子显微镜(STEM)和衍射分析来确定作为线性收缩马达的螺旋体的细胞骨架带的等级和空间组织。这条带的结构单元似乎是一个纤维，~5 nm宽，由59 kDa蛋白质的二聚体组成；每条带由7对纤维对组成。功能单元是一对排列的纤维，沿其对的二聚体形成四聚体环状重复序列。这个组织解释了细胞骨架带如何作为线性马达发挥作用：力是通过四聚体亚基从圆形到椭圆形的构象变化产生的，这种变化不同地改变了其原纤维成分的长度。此外，我们开发和实施了几项技术和仪器改进，显著提高了通过电子能量损失谱成像绘制细胞内钙的灵敏度。

This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in large central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration ([Ca2+]i) that is strongly modulated by the activity of intracellular Ca stores. This activity plays an important role in spatio-temporally shaping Ca2+ signals that regulate important processes such as gene expression and LTP induction. We had earlier shown that stimulus-induced increases in [Ca2]i in sympathetic neurons are accompanied by large, reversible elevations in mitochondrial calcium concentration. We have now explored, in hippocampal pyramidal neurons, analogous mitochondrial responses to a range of synaptic stimuli, and show that in these cells, mitochondrial Ca2+ transport activity has important physiological and pathophysiological effects. In hippocampal pyramidal neurons, large increases in [Ca2+]i activate several key kinases, whereas lower [Ca2+]i enhances protein phosphatase activity. This Ca2+-dependent rebalancing of the phosphorylation status of certain key enzymes, e.g., Ca/calmodulin-dependent kinases (CaMKs), controls the activity of pathways central to neuronal plasticity. We previously found that mitochondrial Ca accumulation mediated by strong Ca2+ entry leads to an increase in the production of superoxide radicals (O2-), and that this activity up-regulates the phosphorylation of CaMKII and CaMKIV-dependent CREB by inhibiting an array of phosphatases (PP1, PP2A and/or PP2B). Conversely, we now find that weak [Ca2+]i increases induced by prolonged low-frequency stimulation preferential enhance phosphatase activity, thereby leading to ERK1/2 activation because PP2A activates Raf-1, an up-stream mediator of ERK. Following strong [Ca2+]i increases induced by short high-frequency (HF) stimulation, however, the onset of ERK activity is delayed by mitochondrially-produced O2- inhibition of PP2A. This increase in ERK is maximal at 10-min post-stimulation, and depends on CaMKII activity, which is, in turn, enhanced by mitochondrial O2-mediated inhibition of CaMKII dephosphorylation. In contrast, NADPH oxidase, a plasma membrane-bound source of O2-, enhances ERK activation during early HF-stimulated ERK induction, but the effect is much weaker and the mechanism does not involve suppression of PP2A. The results indicate that O2- produced by mitochondria and NADPH oxidase modulate specific and distinct steps in Ras/Raf/MEK/ERK1/2 pathway. Mitochondrial dysfunction plays a central role in glutamate-induced excitotoxicity, but mechanisms leading to cell death remain controversial. Earlier work had shown that in hippocampal neurons mitochondrial dysfunction depends on the size of the calcium load, since co-application of agents that inhibit mitochondrial Ca2+ uptake, e.g., FCCP, greatly improve cell viability even though [Ca2+]i elevations are larger in the presence of such drugs. Thus, [Ca2+]i elevation alone, without an increase in mitochondrial Ca, is not sufficient to induce cell death. We have continued to investigate mechanisms of mitochondrial involvement in excitotoxic death by studying ionic, structural and functional changes in mitochondria following injurious stimuli. Following strong NMDA stimulation, the Ca content of mitochondria is in general very high, but more importantly, the content of individual mitochondria is highly variable. There is parallel heterogeneity among mitochondria with regard to swelling and membrane rupture, accompanied by the loss of mitochondrial membrane potential. Surprisingly, the majority of mitochondria tolerate these high levels of Ca without permanent damage, recovering their prestimulus structure, composition and membrane potential within 2h. In some cells, however, small subsets of mitochondria do not re-establish normal Ca2+ and volume regulation. In these mitochondria Ca overload leads to functional and morphological changes resulting in loss of membrane integrity and the probable release of apoptogenic proteins; by 6-8h post-stimulation, typical apoptotic features had developed in ~35% of cells. Presumably, enough mitochondria in those cells were injured by Ca overload to trigger an effective proapoptotic signal. These results support the hypothesis that excitotoxic injury to only a few mitochondria within a neuron can lead to apoptotic cell death. In other progress driven by technical requirements and a longstanding interest in molecular motors, we have used scanning transmission electron microscopy (STEM) and diffraction analysis to determine the hierarchical and spatial organization of cytoskeletal ribbon of the bacterium Spiroplasma, which acts as a linear, contractile motor. The structural unit of this ribbon appears to be a fibril, ~5 nm wide, composed of dimers of a 59 kDa protein; each ribbon is assembled from seven fibril pairs. The functional unit is a pair of aligned fibrils along which pairs of dimers form tetrameric ring-like repeats. This organization explains how the cytoskeletal ribbon functions as a linear motor: Force is generated by a circular-to-elliptical conformational change in the tetrameric subunits, which differentially changes the length of its fibril components. In addition, we have developed and implemented several technical and instrumental refinements that significantly improve sensitivity for mapping intracellular Ca by electron energy loss spectrum imaging.