Elemental & Structural Organization Of Neurons And Glia

元素

• 批准号: 6842425
• 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/6842425
• 关键词: Anura; biological signal transduction; cAMP response element binding protein; calcium flux; calmodulin dependent protein kinase; dendrites; electron microscopy; endoplasmic reticulum; enzyme activity; free radical oxygen; gene expression; glia; hippocampus; immunocytochemistry; intracellular transport; mitochondria; neuroanatomy; neurons; neurotoxins; organ culture; phosphomonoesterases; phosphorylation; spectrometry; 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. The latter 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 a variety of neurons induce large, reversible elevations in the concentration of calcium within mitochondria, which in turn has several important physiological and pathophysiological effects. We have now further explored the consequences of mitochondrial Ca2+ uptake, showing that in hippocampal pyramidal neurons this activity profoundly affects regulatory kinase cascades and programmed cell death. In hippocampal neurons large increases in [Ca2+]i activate several important 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 serine/threonine protein phosphatases (PP1, PP2A and/or PP2B). Similarly, both mitochondrial and NADPH oxidase-derived O2- enhance Ca2+-dependent activation of the prototypical signaling kinase ERK, but only mitochondrial O2- operates by an analogous mechanism, i.e., inhibition of S/T protein phosphatases. Since O2- can also block protein tyrosine phosphatases (PTPs), recent investigations have focused on Raf-1, an up-stream mediator of ERK. A 100Hz/18s stimulus activated Raf-1, resulting in an increase in ERK. Inhibition of the Ca2+-dependent, non-receptor tyrosine kinases Pyk-2 and Src suppressed Raf-1 and ERK, indicating that in hippocampal neurons Raf-1 is up-regulated by Pyk-2 and Src. Inhibition of either mitochondrial or NADPH oxidase-derived O2- attenuated stimulus-induced phosphorylation of both Pyk-2 and Src, implying a mechanism involving O2- inhibition of PTPs. Up-regulation of Pyk-2 and Src phosphorylation by the PTP blockers vanadate and dephostatin, which are normally without effect because PTPs are already constitutively blocked by O2-, was observed when O2- from either source was inhibited. These results indicate that both mitochondrial and NADPH oxidase-derived O2- promote the Raf-1 pathway for ERK activation by inhibiting PTPs, and provide yet another example of physiological regulation by reactive oxygen species. We have continued to investigate mechanisms of mitochondrial involvement in excitotoxic death of cultured hippocampal neurons. We previously showed that delayed apoptotic death of neurons following NMDA overstimulation is mediated by mitochondrial calcium overload, which is variable from one mitochondria to another and correlates with mitochondrial injury. There is parallel variability among mitochondria with regard to swelling, membrane rupture, and loss of membrane potential, leading to the hypothesis that a proapoptotic signal is released only by a subpopulation of damaged mitochondria. Evidence that mitochondrial swelling was caused by Ca overload include: 1) a correlation between swelling and the level of elevated mitochondrial Ca; 2) the absence of mitochondrial swelling in zero Ca medium; and 3) the absence of mitochondrial swelling after substitution of extracellular Ca by Ba. Using EM immunocytochemistry we found that the known apoptogen cytochrome c, normally localized exclusively to mitochondria, was released into the cytoplasm following overstimulation but, importantly, also retained in many mitochondria. Even at 6h post-stimulation, clearly apoptotic neurons still retained cytochrome c in their mitochondria (which were, however, fewer in number). FCCP pretreatment, which decreased mitochondrial Ca accumulation and improved survival, prevented cytochrome c release but not mitochondrial swelling, indicating that both elevated mitochondrial Ca and swelling are essential for cytochrome c release. Our results indicate that neuronal apoptosis induced by excitotoxic stimulation involves release of apoptogens from only a subpopulation of mitochondria that have been injured by Ca overload. Consequently, undamaged mitochondria can continue to function normally and produce enough ATP to support downstream apoptotic reactions.

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。后者在调节基因表达和LTP诱导等重要过程的钙信号的时空形成中起着重要的作用。我们早些时候已经证明，刺激诱导的多种神经元内[Ca~(2+)]i的升高导致线粒体内钙浓度的大幅可逆升高，这反过来又具有几个重要的生理和病理生理效应。我们现在已经进一步探索了线粒体钙摄取的后果，表明在海马锥体神经元中，这种活动深刻地影响调节性激酶级联和程序性细胞死亡。 在海马神经元中，[Ca~(2+)]i的大量增加激活了几个重要的激酶，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，线粒体钙离子的大量聚集导致超氧阴离子自由基(O2-)的产生增加，并且这种活性通过抑制一系列丝氨酸/苏氨酸蛋白磷酸酶(PP1、PP2A和/或PP2B)来上调CaMKII和CaMKIV依赖的CREB的磷酸化。类似地，线粒体和NADPH氧化酶衍生的O2-都促进了典型的信号激酶ERK的钙依赖激活，但只有线粒体O2-通过类似的机制起作用，即抑制S/T蛋白磷酸酶。由于O2-还可以阻断蛋白酪氨酸磷酸酶(PTPs)，最近的研究集中在ERK的上游介质Raf-1上。100 Hz/18s刺激激活了Raf-1，导致ERK增加。抑制钙依赖的非受体酪氨酸激酶PYK-2和Src抑制Raf-1和ERK，表明在海马神经元中，PYK-2和Src上调了Raf-1的表达。抑制线粒体或NADPH氧化酶衍生的O2-减弱刺激诱导的PYK-2和Src的磷酸化，暗示了一种涉及O2-抑制PTPs的机制。当抑制两种来源的O2-时，PTP阻断剂钒酸和去磷他汀上调PTP受体PYK-2和Src的磷酸化，这通常是无效的，因为PTPs已经被O2-结构性地阻断了。这些结果表明，线粒体和NADPH氧化酶衍生的O2-都通过抑制PTPs来促进Raf-1途径的ERK激活，并为活性氧的生理调节提供了又一个例子。 我们继续研究线粒体参与培养的海马神经元兴奋性毒性死亡的机制。我们以前的研究表明，NMDA过度刺激后神经元的迟发性凋亡是由线粒体钙超载所介导的，线粒体钙超载在不同的线粒体之间是不同的，并与线粒体损伤相关。线粒体在肿胀、膜破裂和膜电位丧失方面存在平行的变异性，这导致了一种假设，即促凋亡信号只由一小部分受损的线粒体释放。钙超载引起线粒体肿胀的证据包括：1)肿胀与线粒体钙升高有关；2)在无钙介质中线粒体不肿胀；3)用细胞外钙替代细胞外钙后线粒体不肿胀。利用EM免疫细胞化学，我们发现已知的细胞色素c，通常仅定位于线粒体，在过度刺激后释放到细胞质中，但重要的是，也保留在许多线粒体中。即使在刺激后6h，明显的凋亡神经元仍在其线粒体中保留细胞色素c(然而，其数量较少)。FCCP预处理减少了线粒体钙的积累，提高了存活率，但抑制了细胞色素c的释放，但不能阻止线粒体肿胀，表明线粒体钙的升高和线粒体的肿胀都是细胞色素c释放所必需的。我们的结果表明，兴奋性毒性刺激诱导的神经细胞凋亡只涉及钙超载损伤的线粒体亚群中的凋亡素的释放。因此，未受损的线粒体可以继续正常运作，并产生足够的ATP来支持下游的凋亡反应。