Elemental And Structural Organization Of Neurons And Gli

神经元和 Gli 的基本和结构组织

• 批准号: 6671356
• 负责人: S BRIAN Andrews
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6671356
• 关键词: Anura; biological signal transduction; cAMP response element binding protein; calcium flux; calmodulin dependent protein kinase; central nervous system; dendrites; endoplasmic reticulum; enzyme activity; fluorescent dye /probe; gene expression; glia; hippocampus; immunocytochemistry; intracellular transport; mitochondria; neurons; neurotoxins; organ culture; phosphomonoesterases; phosphorylation; protein kinase C; spectrometry; superoxides; sympathetic nervous system; synapses

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.

本计画主要研究神经元钙信号传导的生理与细胞层面，并长期着重于大型中枢神经系统神经元的突触后反应。神经元对突触刺激的反应是胞质游离Ca 2+浓度（[Ca 2 +] i）的升高，这受到细胞内Ca库活性的强烈调节。这种活性在时空塑造Ca2+信号中起着重要作用，这些信号调节重要过程，如基因表达和LTP诱导。我们先前已经表明，交感神经元中刺激诱导的[Ca2] i增加伴随着线粒体钙浓度的大的、可逆的升高。我们现在已经探索了，在海马锥体神经元，类似的线粒体反应的一系列突触刺激，并表明，在这些细胞中，线粒体Ca2+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，[Ca2 +] i的大幅增加激活几种关键激酶，而较低的[Ca2 +] i增强蛋白磷酸酶活性。这种依赖于Ca2+的磷酸化状态的再平衡的某些关键酶，例如，Ca/钙调蛋白依赖性激酶（CaMK）控制神经元可塑性的中枢途径的活性。我们以前发现，线粒体Ca积累介导的强Ca 2+进入导致超氧自由基（O2-）的产生增加，这种活动上调磷酸化的CaMKII和CaMKIV依赖的CREB通过抑制磷酸酶阵列（PP1，PP2A和/或PP2B）。相反，我们现在发现，由长时间低频刺激诱导的弱[Ca2 +] i增加优先增强磷酸酶活性，从而导致ERK 1/2激活，因为PP2A激活ERK的上游介质Raf-1。然而，在短高频（HF）刺激诱导的[Ca 2 +] i强增加后，ERK活性的开始被脑内产生的O2-抑制PP2A延迟。ERK的这种增加在刺激后10分钟达到最大，并且依赖于CaMKII活性，而CaMKII活性又通过线粒体O2介导的CaMKII去磷酸化抑制而增强。相比之下，NADPH氧化酶，质膜结合源的O2-，增强ERK激活在早期HF刺激的ERK诱导，但效果弱得多，其机制不涉及抑制PP2A。结果表明，线粒体产生的O2-和NADPH氧化酶对Ras/Raf/MEK/ERK 1/2信号通路的各个环节均有不同程度的调节作用。 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然存在争议。早期的研究表明，在海马神经元中，线粒体功能障碍取决于钙负荷的大小，因为共同应用抑制线粒体Ca2+摄取的药物，例如，FCCP大大提高了细胞活力，尽管在此类药物存在的情况下[Ca 2 +] i升高更大。因此，[Ca2 +] i单独升高，而不增加线粒体Ca，不足以诱导细胞死亡。我们通过研究损伤性刺激后线粒体的离子、结构和功能变化，继续研究线粒体参与兴奋性中毒死亡的机制。在强烈的NMDA刺激后，线粒体的Ca含量通常非常高，但更重要的是，单个线粒体的含量是高度可变的。在线粒体肿胀和膜破裂方面存在平行异质性，伴随着线粒体膜电位的丧失。令人惊讶的是，大多数线粒体耐受这些高水平的Ca而没有永久性损伤，在2小时内恢复其刺激前结构，组成和膜电位。然而，在一些细胞中，线粒体的小子集不能重新建立正常的Ca2+和体积调节。在这些线粒体中，Ca超载导致功能和形态变化，导致膜完整性丧失和可能释放促凋亡蛋白;刺激后6 - 8小时，约35%的细胞出现典型的凋亡特征。据推测，这些细胞中足够的线粒体被钙超载损伤以触发有效的促凋亡信号。这些结果支持了神经元内仅少数线粒体的兴奋性毒性损伤可导致凋亡性细胞死亡的假设。 在技术要求和对分子马达的长期兴趣驱动的其他进展中，我们使用扫描透射电子显微镜（STEM）和衍射分析来确定细菌螺原体细胞骨架带的层次和空间组织，其充当线性收缩马达。该带状物的结构单元似乎是由59 kDa蛋白质的二聚体组成的约5 nm宽的原纤维;每条带状物由7对原纤维组装而成。功能单元是一对对齐的原纤维，沿着该原纤维沿着，成对的二聚体形成四聚体环状重复。这个组织解释了细胞骨架带如何作为线性马达发挥作用：力是由四聚体亚基中的圆形到椭圆形构象变化产生的，这差异性地改变了其原纤维组分的长度。此外，我们已经开发和实施了几项技术和仪器的改进，显着提高敏感性映射细胞内钙的电子能量损失谱成像。