Elemental And Structural Organization Of Neurons And Gli

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胞质Ca 2+信号中起着重要作用。我们之前已经证明，在青蛙的交感神经元？是研究细胞内Ca 2+动力学的一个很好的模型？[Ca 2 +]i的增加伴随着总线粒体钙浓度（[Ca]m）的大的、可逆的升高，其引起Ca的细胞内分布的陡峭的空间梯度。我们现在已经描述了第二个主要的Ca 2+调节细胞器，内质网（ER）的功能，其作用通常被认为是通过触发Ca 2+从其内部储存释放来放大Ca 2+信号，这一过程被称为“钙诱导的钙释放”（CICR）。然而，我们发现ER的行为更为复杂。在低水平的Ca 2+进入（因此低[Ca 2 +]i）下，ER实际上积累Ca，从而充当Ca 2+缓冲器。然而，这些神经元表现出从Ca 2+缓冲到触发的Ca 2+释放的渐进过渡，随着[Ca 2 +]i增加到约。1 μ M。此外，发现Ca 2+释放首先在外周ER池中触发，因此Ca 2+摄取和释放可以同时发生在同一细胞的不同区域。最后，ER Ca 2+运输的空间梯度是可逆的线粒体Ca 2+摄取，这表明这些细胞器之间的合作。线粒体和ER之间的功能相互依赖性的数据表明，ER的钙摄取的抑制导致线粒体的补偿性钙螯合支持。相反，在同等条件下抑制线粒体Ca积累导致ER Ca 2+摄取增加。 在包括海马锥体细胞在内的多种神经元中，[Ca 2 +]i的大幅增加激活了几种关键激酶，而较低的[Ca 2 +]i增强了磷酸酶活性。这种依赖于Ca 2+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）似乎控制神经元可塑性的中枢途径的活性，例如突触诱发的基因表达和LTP诱导。我们以前发现，线粒体钙积累，主要发生在周边位置在Ca 2+进入，导致超氧自由基（O2-）的生产增加。由于超氧化物是已知的激活蛋白激酶，并补充，以抑制磷酸酶，我们检查之间的关系[Ca 2 +]i，O2-，和磷酸化的CaMKII和蛋白激酶C（PKC），阐明这一信号通路的分子机制的目标。我们发现，线粒体Ca 2+摄取增加O2-生产依赖于刺激强度和持续时间。用荧光探针在活体海马神经元中获得的测量结果表明，弱刺激（5 Hz/18 s或更长）引起低的、持续的[Ca 2 +]i平台，但不诱导O2-产生，而较强的刺激（50或100 Hz/18 s，和90 mM K+）诱导大的Ca 2+尖峰和增加的线粒体O2-产生。使用定量免疫细胞化学，我们进一步评估了线粒体的作用，在连接胞浆Ca 2+进入细胞磷酸化的微调。一个主要途径似乎靶向CaMK的磷酸化。通过抑制一系列磷酸酶（PP 1，PP 2A和/或PP 2B），线粒体O2-增强CaMKII的自磷酸化（对LTP诱导很重要），以及CREB的CaMKIV依赖性磷酸化（对基因表达很重要）。相反，线粒体O2-似乎通过不涉及磷酸酶活性的机制来调节PKC。 线粒体Ca的过度积累被认为在兴奋性毒性中起着至关重要的作用。我们已经发现，海马神经元暴露于谷氨酸或NMDA导致这些细胞的离子含量发生显著变化，特别是游离和总胞质Ca升高，以及Na增加和K损失。处理的神经元也表现出极高水平的线粒体内Ca。这些变化在很大程度上是可逆的，因为在激动剂去除后恢复了正常的胞质离子水平，即使某些线粒体长时间维持升高的Ca。尽管FCCP增加了细胞质Ca 2+瞬变的幅度，但FCCP对线粒体Ca 2+摄取的抑制似乎具有神经保护作用。结果表明，[Ca 2 +]i单独升高，而线粒体Ca不升高，不足以诱导细胞死亡，并导致细胞命运可能取决于线粒体内Ca水平的假设。

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 cytosolic Ca2+ signals. We had earlier shown that in frog sympathetic neurons ? an excellent model for studying intracellular Ca2+ dynamics ? increases in [Ca2+]i are accompanied by large, reversible elevations in total mitochondrial calcium concentration ([Ca]m) that give rise to steep spatial gradients in the intracellular distribution of Ca. We have now characterized the function of the second major Ca2+-regulating organelle, the endoplasmic reticulum (ER), whose role is generally thought to be amplification of Ca2+ signals by triggered Ca2+ release from its internal store, a process known as "calcium-induced calcium release" (CICR). We find, however, that the behavior of the ER is more complex. At low levels of Ca2+ entry (and therefore low [Ca2+]i) the ER actually accumulates Ca, thus acting as a Ca2+ buffer. However, these neurons exhibit a progressive transition from Ca2+ buffering to triggered Ca2+ release as [Ca2+]i increases to approx. 1uM. In addition, it was found that Ca2+ release is first triggered in peripheral ER cisternae, so that both Ca2+ uptake and release can occur simultaneously different regions of the same cell. Finally, the spatial gradient of ER Ca2+ transport is reciprocal to that of mitochondrial Ca2+ uptake, suggesting cooperation between these organelles. Functional interdependence between mitochondria and ER is supported by data showing that inhibition of ER Ca2+ uptake leads to compensatory Ca sequestration by mitochondria. Reciprocally, suppression of mitochondrial Ca accumulation under equivalent conditions results in increased ER Ca2+ uptake. In a variety of neurons, including hippocampal pyramidal cells, large increases in [Ca2+]i activate several key kinases, whereas lower [Ca2+]i enhances phosphatase activity. This Ca2+-dependent rebalancing of the phosphorylation status of certain key enzymes, e.g., Ca/calmodulin-dependent kinases (CaMKs), appears to control the activity of pathways central to neuronal plasticity, examples being synaptically-evoked gene expression and LTP induction. We previously found that mitochondrial Ca accumulation, occurring mainly in peripheral locations during Ca2+ entry, leads to an increase in the production of superoxide radicals (O2-). As superoxide is known to activate protein kinases and, complementarily, to inhibit phosphatases, we examined the relationship between [Ca2+]i, O2-, and phosphorylation of CaMKII and protein kinase C (PKC), with the goal of elucidating the molecular mechanisms underlying this signaling pathway. We find that mitochondrial Ca2+ uptake that augments O2- production is dependent on stimulus strength and duration. Measurements obtained with fluorescent probes in living hippocampal neurons demonstrate that weak stimuli (5Hz/18s or longer) elicit low, sustained [Ca2+]i plateaus, but do not induce O2- generation, while stronger stimuli (50 or 100Hz/18s, and 90 mM K+) induce large Ca2+ spikes and increased mitochondrial O2- production. Using quantitative immunocytochemistry, we have further evaluated the role of mitochondria in linking cytosolic Ca2+ entry to the fine-tuning of cellular phosphorylation. One major pathway appears to target the phosphorylation of CaMKs. By inhibiting an array of phosphatases (PP1, PP2A and/or PP2B), mitochondrial O2- enhances autophosphorylation of CaMKII (important for LTP induction), as well as CaMKIV-dependent phosphorylation of CREB (important for gene expression). In contrast, mitochondrial O2- appears to modulate PKC by a mechanism that does not involve phosphatase activity. Excessive mitochondrial Ca accumulation is thought to play a crucial role in excitotoxicity. We have found that exposure of hippocampal neurons to glutamate or NMDA results in dramatic changes in the ionic content of these cells, specifically, an elevation of free and total cytosolic Ca, as well as a gain in Na and a loss of K. Treated neurons also exhibited extremely high levels of intramitochondrial Ca. These changes were largely reversible, in that normal cytosolic ion levels were restored after agonist removal, even though certain mitochondria maintained elevated Ca for prolonged periods. Inhibition of mitochondrial Ca2+ uptake by FCCP appeared to be neuroprotective, despite the fact that FCCP increased the amplitude of cytosolic Ca2+ transients. The results suggest that elevation of [Ca2+]i alone, without elevation of mitochondrial Ca, is not sufficient to induce cell death, and leads to the hypothesis that cell fate may depend on the level of Ca within mitochondria.