Elemental And Structural Organization Of Neurons And Gli

该项目研究神经元钙信号的细胞和生理方面，长期重点放在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，这种升高受细胞内钙储存细胞器的转运活动的强烈调控。这种转运活动在时空上塑造调控基因表达和突触可塑性等过程的信号方面发挥了重要作用。我们(和其他人)早些时候已经证明，刺激诱导的各种神经元[Ca~(2+)]i的增加会导致线粒体内钙浓度的大幅可逆升高，这反过来又对生理和病理生理过程产生重要影响。今年，我们继续探索线粒体钙摄取的后果，表明在海马锥体神经元中，这种活动至少部分地支持了缺血预适应的现象。我们还进一步鉴定了受线粒体钙调控的ERK1/2信号转导级联中的步骤。 以往的研究表明，NMDA过度刺激导致兴奋性迟发性细胞死亡(DCD)不可避免地与强烈的线粒体钙积聚有关。这种线粒体活动在空间上是异质性的，这一点很重要，因为细胞脆弱性与线粒体钙负荷的总大小和给定神经元中钙超载、受损的线粒体的数量有关。因此，线粒体受损的数量和位置被认为决定了对过量NMDA的易感性。这些观察结果使我们探索了这样一种假设，即线粒体也介导了缺血预适应(PC)的神经保护效应，这是一种鲜为人知的现象，即神经元在接受类似但更温和的非致命性挑战后，对通常致命的攻击具有抵抗力。几种公认的预适应方案的结果表明，在细胞水平上，这些处理通过诱导细胞内钙的可逆重分布和线粒体的瞬时去极化和钙负荷来概括NMDA的作用。这种影响是完全可逆的，不会导致DCD。试验方案包括：1)所谓的化学缺血(CI-PC)，即在无糖介质中暴露于2 mM氰化物中30min，24小时后致死。NMDA；和2)各种重复(2-5次)处理，通常是致死浓度的NMDA，但处理时间较短，与致死伤害前的最后24小时相隔48小时(NMDA-PC)。CI-PC后24小时暴露于致死的NMDA可使NMDA诱导的细胞死亡减少30%，而NMDA-PC的效果更好，可减少90%的细胞死亡。在这两种情况下，PC神经保护与兴奋性毒性NMDA暴露后线粒体损伤的减少是平行的。然而，NMDA-PC(多次暴露)也显著减少了Ca~(2+)内流，而CI-PC没有。这些结果支持了PC通过增加线粒体对大钙负荷的耐受性从而减轻线粒体功能障碍而发挥保护作用的工作假说。更有效的PC协议似乎招募了额外的、附加的保护机制，这些机制尚未定义。 在海马神经元中，大量的[Ca~(2+)]i升高激活了几个重要的激酶，如钙/钙调蛋白依赖的激酶(CaMKs)，这一点很重要，因为这些酶调节基因表达和神经元可塑性的中心通路。我们的实验室以前报道过，线粒体钙离子的强烈内流导致超氧阴离子自由基(O2-)的产生增加，从而上调了几种激酶的活性，包括PKA、PKC、CaMKII、核CaMKIV(从而转录因子CREB)和ERK 1/2。除PKC外，所有的上调都是通过一个共同的机制发生的，即通过O2-抑制失活的丝氨酸/苏氨酸蛋白磷酸酶来稳定活性的磷酸化形式的激酶。在所有这些情况下，线粒体是活性O2-的主要来源，但ERK1/2不同之处在于，NADPH氧化酶衍生的O2-也发挥着重要作用。今年，我们继续扩展了早期的研究，表明Ras/Raf/MEK/ERK级联反应的更复杂的调控是因为这个级联反应中的关键步骤取决于S/T蛋白磷酸酶和蛋白酪氨酸磷酸酶的活性，这两个活性都受到不同来源的O2-的不同调控。因此，NADPH氧化酶产生短暂的O2-峰值，促进RAS的初始激活，而线粒体产生持续的O2-升高，影响后来的磷酸酶依赖步骤。在ERK级联中有几条平行的通路；其中最具特点的是(RAS)/Raf-1/MEK/ERK和Rap-1/B-Raf/MEK/ERK-在MEK处汇聚，并被不同的刺激激活。由于刺激模式对于确定钙信号的靶点通常很重要，我们正在研究ERK级联的这两个分支如何对不同的高频刺激序列做出反应。免疫细胞化学定量结果显示，100 Hz/18s刺激后1min，Raf-1和B-Raf通路均被激活。相反，每隔5分钟进行三次这样的刺激，选择性地激活B-Raf，发现它依赖于PKA和PYK-2，而不依赖于Src。这表明长期诱导依赖于PKA、非依赖于Src的Rap-1/B-Raf途径，并可能抑制Raf-1途径。因此，Raf和Rap似乎是另一个检查点，在这个点上，对激酶信号的O2调制可以微调ERK级联反应的路径和时间。

This project studies cellular and physiological aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in 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 transport activity of intracellular calcium storage organelles. This transport activity plays an important role in spatio-temporally shaping the signals that regulate processes like gene expression and synaptic plasticity. We (and others) had earlier shown that stimulus-induced increases in [Ca2+]i in a variety of neurons induce large, reversible elevations in the concentration of calcium within mitochondria, which in turn has important effects on physiological and pathophysiological processes. This year we continued to explore the consequences of mitochondrial Ca2+ uptake, showing that in hippocampal pyramidal neurons this activity at least partly underlies the phenomenon of ischemic preconditioning. We have also further characterized the steps in the ERK1/2 signal transduction cascade that are regulated by mitochondrial calcium. It was previously shown that NMDA overstimulation that leads to excitotoxic delayed cell death (DCD) is inevitably associated with strong mitochondrial calcium accumulation. This mitochondrial activity is spatially heterogeneous, which is important because cell vulnerability is correlated with the aggregate size of the mitochondrial Ca load and the number of Ca-overloaded, damaged mitochondria in a given neuron. Thus, the number and location of damaged mitochondria are thought to determine vulnerability to excessive NMDA. These observations have led us to explore the hypothesis that mitochondria also mediate the neuroprotective effects of ischemic preconditioning (PC), a poorly understood phenomenon whereby neurons become resistant to a normally lethal insult after pretreatment with a similar but milder, non-lethal challenge. The results from several recognized preconditioning protocols showed that at the cellular level these treatments recapitulate the effects of NMDA by inducing the reversible redistribution of intracellular Ca and the transient depolarization and Ca loading of mitochondria. Such effects were fully reversible and did not lead to DCD. Among the protocols tested were: 1) so-called chemical ischemia (CI-PC), that is, exposure to 2 mM cyanide in glucose-free medium for 30 min 24h before ?lethal? NMDA; and 2) various repetitive (two to five) treatments with normally lethal concentrations of NMDA but for shorter times, spaced 48 h apart with the last 24h before lethal insult (NMDA-PC). Exposure to lethal NMDA 24h after CI-PC reduced NMDA-induced cell death by >30%, whereas NMDA-PC was even more effective, reducing cell death as much as 90%. In both cases, PC neuroprotection was paralleled by reduced mitochondrial injury after excitotoxic NMDA exposure. However, NMDA-PC (multiple exposures) also substantially reduced Ca2+ entry, while CI-PC did not. These results support the working hypothesis that PC exerts its protective effect by increasing mitochondrial tolerance for large Ca loads, thereby attenuating mitochondrial dysfunction. More effective PC protocols appear to recruit additional, additive protective mechanisms that are yet to be defined. In hippocampal neurons large [Ca2+]i increases activate several important kinases, e.g., Ca/calmodulin-dependent kinases (CaMKs), which is important because these enzymes regulate pathways central to gene expression and neuronal plasticity. Our laboratory previously reported that mitochondrial calcium accumulation mediated by strong Ca2+ entry leads to an increase in the production of superoxide radicals (O2-), which up-regulates the activity of several kinases, including PKA, PKC, CaMKII, nuclear CaMKIV (and therefore the transcription factor CREB), and ERK 1/2. Up-regulation of all but PKC occurs by a common mechanism, namely, stabilization of the active, phosphorylated form of the kinase by O2- inhibition of the deactivating serine/threonine protein phosphatases. In all these cases, mitochondria are the main source of active O2-, but ERK 1/2 is different in that NADPH oxidase-derived O2- also plays a significant role. This year we have continued to expand earlier studies showing that the more complex regulation of the Ras/Raf/MEK/ERK cascade arises because key steps in this cascade depend on both S/T protein phosphatase and protein tyrosine phosphatase activities, which are differentially regulated by O2- from different sources. Thus, NADPH oxidase generates a brief spike of O2- that promotes the initial activation of Ras, whereas mitochondria produce sustained O2- elevations that affect later phosphatase-dependent steps. There are several parallel pathways in the ERK cascade; the best characterized of these -- (Ras)/Raf-1/MEK/ERK and Rap-1/B-Raf/MEK/ERK -- converge at MEK and are activated by different stimuli. Since stimulus patterns are generally important for specifying the targets of Ca2+ signals, we are examining how these two branches of the ERK cascade respond to different trains of high-frequency stimuli. Quantitative immunocytochemistry showed that at 1 min after a 100Hz/18s stimulus both Raf-1 and B-Raf pathways were activated. In contrast, three episodes of such stimulation at 5-min intervals selectively activated B-Raf, which was found to be dependent on PKA and Pyk-2, but not Src. This indicates the long-term induction of a PKA-dependent, Src-independent Rap-1/B-Raf pathway, and probably the suppression of the Raf-1 pathway. Thus, Raf vs. Rap appears to be yet another checkpoint at which O2- modulation of kinase signaling can fine-tune the route and timing of the ERK cascade.