Elemental And Structural Organization Of Neurons And Gli
神经元和 Gli 的基本和结构组织
基本信息
- 批准号:7143827
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:NAD(P)H dehydrogenasebiological signal transductioncAMP response element binding proteincalcium fluxcalcium ioncalmodulin dependent protein kinasedendriteselectron microscopyexcitatory aminoacidfree radical oxygengene expressiongliaguanine nucleotide binding proteinhippocampusimmunocytochemistryintracellular transportischemic preconditioningmitochondriamitogen activated protein kinaseneuroanatomyneuroprotectantsneurotoxinsphosphoprotein phosphatasephosphorylationprotein signal sequence
项目摘要
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.
本计画主要研究神经元钙信号的细胞与生理方面,并长期着重于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,其强烈地受到细胞内钙储存细胞器的转运活性的调节。这种转运活动在时空塑造调节基因表达和突触可塑性等过程的信号中起着重要作用。我们(和其他人)早先已经表明,刺激诱导的[Ca 2 +]i在各种神经元中的增加诱导线粒体内钙浓度的大的、可逆的升高,这反过来又对生理和病理生理过程具有重要影响。今年,我们继续探索线粒体Ca 2+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成为缺血预适应现象的基础。我们还进一步表征了由线粒体钙调节的ERK 1/2信号转导级联中的步骤。
以前的研究表明,NMDA过度刺激导致兴奋性毒性延迟性细胞死亡(DCD)不可避免地与线粒体钙积累有关。这种线粒体活性在空间上是不均匀的,这是重要的,因为细胞的脆弱性与线粒体Ca负荷的总大小和给定神经元中Ca超载、受损线粒体的数量相关。因此,受损线粒体的数量和位置被认为决定了对过量NMDA的脆弱性。这些观察结果使我们探索线粒体也介导缺血预处理(PC)的神经保护作用的假设,缺血预处理是一种知之甚少的现象,即神经元在用类似但更温和的非致死性挑战预处理后对正常致死性损伤具有抵抗力。几种公认的预处理方案的结果表明,在细胞水平上,这些处理通过诱导细胞内Ca的可逆再分布和线粒体的瞬时去极化和Ca负载来概括NMDA的作用。这些影响是完全可逆的,不会导致DCD。其中测试的协议是:1)所谓的化学缺血(CI-PC),即暴露于2 mM的氰化物在无葡萄糖培养基中30分钟,24小时前?致命的?NMDA;(2)用正常致死浓度的NMDA进行各种重复(2 - 5次)处理,但时间较短,间隔48 h,致死损伤前的最后24 h(NMDA-PC)。在CI-PC后24小时暴露于致死性NMDA使NMDA诱导的细胞死亡减少> 30%,而NMDA-PC甚至更有效,使细胞死亡减少多达90%。在这两种情况下,PC的神经保护作用,减少兴奋毒性NMDA暴露后线粒体损伤。然而,NMDA-PC(多次暴露)也大大减少了Ca 2+的进入,而CI-PC没有。这些结果支持PC通过增加线粒体对大钙负荷的耐受性,从而减弱线粒体功能障碍来发挥其保护作用的工作假设。更有效的PC协议似乎招募额外的,添加剂的保护机制,尚未被定义。
在海马神经元中,大的[Ca 2 +]i增加激活几种重要的激酶,例如,钙/钙调蛋白依赖性激酶(CaMK),这是很重要的,因为这些酶调节途径的核心基因表达和神经元可塑性。我们的实验室先前报道,由强Ca 2+进入介导的线粒体钙积累导致超氧化物自由基(O2-)产生增加,其上调几种激酶的活性,包括PKA、PKC、CaMKII、核CaMKIV(因此转录因子CREB)和ERK 1/2。除了PKC以外,所有蛋白的上调都是通过一种共同的机制发生的,即通过O2抑制失活的丝氨酸/苏氨酸蛋白磷酸酶来稳定激酶的活性磷酸化形式。在所有这些情况下,线粒体是活性O2-的主要来源,但ERK 1/2是不同的,NADPH氧化酶衍生的O2-也发挥了重要作用。今年,我们继续扩展早期的研究,表明Ras/Raf/MEK/ERK级联反应的调节更加复杂,因为该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,这些活性受到来自不同来源的O2-的差异调节。因此,NADPH氧化酶产生一个短暂的尖峰O2-,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的步骤。在ERK级联中有几个平行的通路;其中最具特征的-(Ras)/Raf-1/MEK/ERK和Rap-1/B-Raf/MEK/ERK -会聚在MEK处,并被不同的刺激激活。由于刺激模式通常对于指定Ca 2+信号的目标很重要,因此我们正在研究ERK级联的这两个分支如何响应不同的高频刺激。定量免疫细胞化学显示,在100 Hz/18 s刺激后1 min,Raf-1和B-Raf途径均被激活。相反,以5分钟的间隔进行三次这样的刺激选择性地激活B-Raf,发现B-Raf依赖于PKA和Pyk-2,但不依赖于Src。这表明长期诱导PKA依赖性、Src非依赖性Rap-1/B-Raf途径,并可能抑制Raf-1途径。因此,Raf vs. Rap似乎是另一个检查点,在该检查点,激酶信号的O2调节可以微调ERK级联的途径和时间。
项目成果
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S BRIAN Andrews其他文献
S BRIAN Andrews的其他文献
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{{ truncateString('S BRIAN Andrews', 18)}}的其他基金
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 - 财政年份:
- 资助金额:
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ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA
神经元和神经胶质细胞的基本和结构组织
- 批准号:
6163016 - 财政年份:
- 资助金额:
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ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA
神经元和神经胶质细胞的基本和结构组织
- 批准号:
6111844 - 财政年份:
- 资助金额:
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Elemental And Structural Organization Of Neurons And Glia
神经元和神经胶质细胞的元素和结构组织
- 批准号:
8746767 - 财政年份:
- 资助金额:
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Elemental And Structural Organization Of Neurons And Glia
神经元和神经胶质细胞的元素和结构组织
- 批准号:
7735253 - 财政年份:
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STRUCTURAL AND ELEMENTAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES
大分子组装体的结构和元素分析
- 批准号:
2579609 - 财政年份:
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ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA
神经元和神经胶质细胞的基本和结构组织
- 批准号:
6432892 - 财政年份:
- 资助金额:
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Elemental And Structural Organization Of Neurons And Glia
神经元和神经胶质细胞的元素和结构组织
- 批准号:
8342197 - 财政年份:
- 资助金额:
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Elemental And Structural Organization Of Neurons And Glia
神经元和神经胶质细胞的元素和结构组织
- 批准号:
8557000 - 财政年份:
- 资助金额:
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