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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

基本信息

批准号：
7735253
负责人：
S BRIAN Andrews
金额：
$ 138.67万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
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：
https://reporter.nih.gov/project-details/7735253
关键词：
Accounting Alzheimer&apos s Disease Behavior Biological Brain Calcium Calcium Signaling Calcium Spikes Cell Death Cell Survival Cell physiology Cells Cessation of life Chemicals Classification Condition Conflict (Psychology)Coupling Data Detection Disease Disruption Electron Microscopy Elevation Endoplasmic Reticulum Energy-Filtering Transmission Electron Microscopy Event Functional disorder Glutamates Goals Hippocampus (Brain)Homeostasis Injury Inner mitochondrial membrane Ions Ischemic Preconditioning Lead Link Location Maps Mediating Mediator of activation protein Membrane Membrane Potentials Methodology Mitochondria Mitochondrial Swelling Modeling N-Methyl-D-Aspartate Receptors N-Methylaspartate Neuroglia Neurons Organelles Oxidative Stress Parkinson Disease Pathway interactions Permeability Phosphorus Physiological Physiology Play Predisposition Preparation Process Proteins Published Comment Rana Range Rate Rattus Receptor Inhibition Recovery Regulation Relative (related person)Residual state Role Route Rupture Signal Transduction Slice Solubility Specific qualifier value Specificity Specimen Stimulus Stroke Structure Study models Swelling Synapses Synaptic plasticity Technology Testing Thick To specify Toxic effect Traumatic Brain Injury Weight Work base cellular imaging excitotoxicity hippocampal pyramidal neuron improved inhibitor/antagonist interest mitochondrial dysfunction mitochondrial membrane mitochondrial permeability transition pore neuronal growth neuroprotection new technology novel therapeutics prevent receptor uptake

项目摘要

NMDA receptors play important and diverse roles in CNS function, ranging from the regulation of synaptic plasticity and neuronal growth and survival to the initiation of cell death. It is generally agreed that mitochondrial calcium (Ca2+) overload and subsequent dysfunction, due to excessive Ca2+ entry through glutamate-overactivated NMDA receptors (NMDARs), are crucial early events in excitotoxic injury. However, there are diverse and seemingly conflicting viewpoints concerning the mechanisms underlying NMDAR susceptibility to overactivation. The overall goals of this project are: 1) to establish the cellular mechanisms by which mitochondria mediate glutamate-induced, calcium-dependent toxicity; and 2) to determine the mechanism of mitochondrial injury per se and devise ways to prevent it. This project is generally informed by the unifying hypothesis that calcium-induced mitochondrial dysfunction is the indispensable, central event in excitotoxicity, so that the effects of other known factors can be explained by their common, convergent impact on calcium load-dependent mitochondrial injury. Aim #1: To determine whether mitochondrial calcium overload can account for the role of NMDAR location and subunit composition in excitotoxic injury. Excitotoxicity depends on the activation of specific routes of Ca2+ entry. For example, NMDAR location appears to play an important role in specifying the induction of survival vs. death pathways, with extrasynaptic NMDARs being linked to excitotoxic death, while synaptic NMDARs promote cell survival. Alternatively, it has been suggested that excitotoxicity is triggered by the selective activation of those NMDARs containing the NR2B subunit. This projects working hypothesis proposes, and preliminary data support, the idea that the effects of factors such as location and composition can be accounted for by a common, convergent impact on calcium-induced mitochondrial injury. Thus, exposure of cultured rat hippocampal neurons to 100 uM NMDA normally leads to massive calcium accumulation and cell death. In cells expressing comparable levels of NR2A- and NR2B-containing NMDARs, inhibition of both receptor subtypes was necessary to reduce NMDA-evoked calcium elevations, preserve mitochondrial structure and function, and achieve satisfactory neuroprotection. This indicates that that toxic calcium loading can be mediated by receptors of either subtype, and therefore that subunit composition alone is not sufficient to specify signal coupling. Selective activation of extrasynaptic NMDARs resulted in calcium loads and toxicity that were comparable to global activation, while synaptic stimulation produced only low-amplitude calcium spikes that were pro-survival. These results imply that extrasynaptic NMDARs represent the major mediator of excitotoxic stimuli, precisely because they are the dominant route of calcium entry. The results support the hypothesis that excessive calcium loading and mitochondrial dysfunction are common, obligatory steps along the pathway to excitotoxic injury. Aim #2: To test the hypothesis that calcium overloadinduced mitochondrial damage is greater in CA1 neurons than in CA3 neurons, thereby explaining why CA1 neurons are more vulnerable to ischemic injury. To determine whether ischemic preconditioning works by reducing mitochondrial Ca overload in CA1 neurons. Slice cultures of hippocampus are a good model for studying neuronal tolerance, since pyramidal neurons of the CA1 region are quite sensitive to excitotoxic stimuli, while neurons in the CA3 region show a high level of endogenous neuroprotection. Present data indicate that NMDA exposure selectively induces large calcium elevations in CA1 neurons, but not in CA3 neurons. Consistent with the general principle that mitochondrial damage is a key event in excitotoxic vulnerability, mitochondrial calcium overload and damage were also much more severe in CA1. The NMDA antagonist MK-801 prevented CA1 calcium elevation and was neuroprotective. Considering results described in Aim #1, it is of interest to examine the role of route specificity in CA1 relative to CA3 to determine if mitochondrial calcium overload is generally responsible for the excitotoxic vulnerability of CA1 neurons. Aim #3: To determine if mitochondrial swelling during excitotoxic overstimulation is caused by calcium-induced opening of the mitochondrial permeability transition (MPT) pore. Calcium loading and oxidative stress are observed clinically during ischemic episodes. Although this suggests a role for MPT in excitotoxic injury, this idea remains controversial. In isolated mitochondria MPT is associated with the loss of the mitochondrial membrane potential (MMP), permeabilization of the inner mitochondrial membrane, swelling of the matrix, and outer membrane rupture, followed by release of apoptogenic proteins. These are all hallmarks of mitochondrial injury leading to excitotoxic death. Like mitochondria in intact neurons, isolated brain mitochondria are capable of accumulating large amounts of calcium when exposed to submicromolar concentrations of this ion. Accumulated calcium is stored in the matrix as phosphorus-rich precipitates, the chemical composition of which is largely unknown. Also unknown is the fate of these precipitates when the inner mitochondrial membrane is breached. This study aims to determine how the amount and rate of mitochondrial calcium uptake relate to MPT, precipitate composition and precipitate retention. Various inhibitors of MPT altered the Ca/P ratio of formed precipitates, indicating that precipitate chemical form and solubility vary with the conditions of loading. Following MPT the release of accumulated Ca2+ is rapid but incomplete; significant residual calcium is retained in damaged mitochondria for prolonged periods. Since mitochondrial Ca2+ transport significantly influences cell signaling, it seems desirable to examine whether prolonging Ca2+ release will have a significant impact during post-stimulus recovery periods. Aim #4: To develop new technology, based on energy filtering transmission electron microscopy (EFTEM), for the quantitative mapping of intracellular calcium distributions at the single organelle level. To apply this technology to elucidating spatio-temporal calcium dynamics in a model neuron. Because calcium plays such a major role in physiological and pathophysiological processes, quantitative mapping of Ca distributions in the analytical electron microscopy at the level of cellular organelles would be very helpful for elucidating mechanisms of calcium regulation and calcium-dependent dysfunction. However, since physiological concentrations of calcium are very low this task is challenging. In this project we propose mapping calcium using EFTEM. With EFTEM one can obtain megapixel images from cells with relatively short (<1 min) exposures, which greatly improves throughput and efficiency relative to alternative approaches. The main hurdle to producing reliable results from biological specimens is that thickness-dependent systematic errors in subtracting the spectral background from the weak, calcium-specific core edge signal must be eliminated. Present results show that by modeling the behavior of the spectrum background as a function of specimen thickness and inelastic mean free path, we can correct for plural scattering and detect calcium in neuronal preparations at concentrations below 10 mmol/kg dry weight, which is in the high physiological range. Results also suggest that is will be quite feasible to further reduce these detection limits. The first proposed biological application of this methodology will be directed toward understanding calcium dynamics during recovery from stimulated calcium entry in frog sympathetic neurons, a model that we have studied in in the past.

NMDA 受体在中枢神经系统功能中发挥着重要而多样的作用，从突触可塑性的调节、神经元生长和存活到细胞死亡的启动。人们普遍认为，由于过量的 Ca2+ 通过谷氨酸过度激活的 NMDA 受体 (NMDAR) 进入，导致线粒体钙 (Ca2+) 过载和随后的功能障碍，是兴奋性毒性损伤的关键早期事件。然而，关于 NMDAR 对过度激活的敏感性的机制存在多种且看似相互矛盾的观点。该项目的总体目标是：1）建立线粒体介导谷氨酸诱导的钙依赖性毒性的细胞机制； 2）确定线粒体损伤本身的机制并设计预防方法。该项目通常基于以下统一假设：钙诱导的线粒体功能障碍是兴奋性毒性中不可或缺的核心事件，因此其他已知因素的影响可以通过它们对钙负荷依赖性线粒体损伤的共同、聚合影响来解释。目标#1：确定线粒体钙超载是否可以解释 NMDAR 位置和亚基组成在兴奋性毒性损伤中的作用。兴奋性毒性取决于特定 Ca2+ 进入途径的激活。例如，NMDAR 位置似乎在指定诱导存活与死亡途径中发挥重要作用，突触外 NMDAR 与兴奋性毒性死亡有关，而突触 NMDAR 则促进细胞存活。或者，有人认为兴奋性毒性是由含有 NR2B 亚基的 NMDAR 的选择性激活引发的。该项目的工作假设提出并得到了初步数据支持，即位置和组成等因素的影响可以通过对钙诱导的线粒体损伤的共同、聚合影响来解释。因此，将培养的大鼠海马神经元暴露于 100 uM NMDA 通常会导致大量钙积累和细胞死亡。在表达相当水平的含有 NR2A 和 NR2B 的 NMDAR 的细胞中，抑制两种受体亚型对于减少 NMDA 引起的钙升高、保护线粒体结构和功能以及实现令人满意的神经保护是必要的。这表明有毒的钙负载可以由任一亚型的受体介导，因此仅亚基组成不足以指定信号耦合。突触外 NMDAR 的选择性激活导致钙负荷和毒性与整体激活相当，而突触刺激仅产生有利于生存的低幅度钙峰值。这些结果意味着突触外 NMDAR 代表了兴奋性毒性刺激的主要介质，正是因为它们是钙进入的主要途径。结果支持这样的假设：过量的钙负荷和线粒体功能障碍是兴奋性毒性损伤途径中常见的、必然的步骤。目标#2：检验钙超载引起的线粒体损伤在 CA1 神经元中比在 CA3 神经元中更大的假设，从而解释为什么 CA1 神经元更容易受到缺血性损伤。确定缺血预处理是否通过减少 CA1 神经元线粒体 Ca 超载起作用。海马切片培养物是研究神经元耐受性的良好模型，因为 CA1 区的锥体神经元对兴奋性毒性刺激非常敏感，而 CA3 区的神经元则表现出高水平的内源性神经保护作用。目前的数据表明，NMDA 暴露选择性地诱导 CA1 神经元中的钙离子大幅升高，但不会引起 CA3 神经元中的钙离子大幅升高。与线粒体损伤是兴奋性毒性脆弱性的关键事件这一一般原则相一致，CA1 中线粒体钙超载和损伤也更为严重。 NMDA 拮抗剂 MK-801 可防止 CA1 钙升高并具有神经保护作用。考虑到目标 #1 中描述的结果，有必要检查 CA1 相对于 CA3 中路径特异性的作用，以确定线粒体钙超载是否通常导致 CA1 神经元的兴奋毒性脆弱性。目标#3：确定兴奋毒性过度刺激期间线粒体肿胀是否是由钙诱导的线粒体通透性转变 (MPT) 孔打开引起的。临床上观察缺血发作期间的钙负荷和氧化应激。尽管这表明 MPT 在兴奋性毒性损伤中发挥作用，但这一想法仍然存在争议。在分离的线粒体中，MPT 与线粒体膜电位 (MMP) 丧失、线粒体内膜透化、基质肿胀和外膜破裂以及随后凋亡蛋白的释放有关。这些都是线粒体损伤导致兴奋性中毒死亡的标志。与完整神经元中的线粒体一样，分离的脑线粒体在暴露于亚微摩尔浓度的钙离子时能够积累大量的钙。累积的钙以富磷沉淀物的形式储存在基质中，其化学成分很大程度上未知。同样未知的是，当线粒体内膜被破坏时，这些沉淀物的命运。本研究旨在确定线粒体钙摄取量和速率与 MPT、沉淀物成分和沉淀物保留的关系。 MPT 的各种抑制剂改变了形成的沉淀物的 Ca/P 比，表明沉淀物的化学形态和溶解度随负载条件的变化而变化。 MPT 后，积累的 Ca2+ 释放迅速但不完全；大量残留的钙会长时间保留在受损的线粒体中。由于线粒体 Ca2+ 转运显着影响细胞信号传导，因此似乎需要检查延长 Ca2+ 释放是否会在刺激后恢复期间产生显着影响。目标#4：开发基于能量过滤透射电子显微镜 (EFTEM) 的新技术，用于在单个细胞器水平上定量绘制细胞内钙分布。应用该技术来阐明模型神经元中的时空钙动力学。由于钙在生理和病理生理过程中起着如此重要的作用，因此在分析电子显微镜中在细胞器水平上定量绘制 Ca 分布对于阐明钙调节和钙依赖性功能障碍的机制非常有帮助。然而，由于钙的生理浓度非常低，这项任务具有挑战性。在这个项目中，我们建议使用 EFTEM 绘制钙图。使用 EFTEM，人们可以通过相对较短（<1 分钟）的曝光时间从细胞中获得百万像素图像，相对于其他方法，这大大提高了通量和效率。从生物样本中产生可靠结果的主要障碍是，必须消除从微弱的钙特异性核心边缘信号中减去光谱背景时与厚度相关的系统误差。目前的结果表明，通过将光谱背景的行为建模为样本厚度和非弹性平均自由程的函数，我们可以校正多重散射并检测神经元制剂中浓度低于 10 mmol/kg 干重（处于高生理范围）的钙。结果还表明，进一步降低这些检测限是非常可行的。该方法的第一个提议的生物学应用将旨在了解青蛙交感神经元受刺激钙进入恢复过程中的钙动态，这是我们过去研究过的一个模型。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.

去极化诱导的交感神经元线粒体 Ca 积累：空间和时间特征。

DOI：
10.1523/jneurosci.19-15-06372.1999
发表时间：
1999
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
0
作者：
Pivovarova,NB;Hongpaisan,J;Andrews,SB;Friel,DD
通讯作者：
Friel,DD

传入突触刺激后 CA3 海马树突线粒体和内质网的相关钙吸收和释放。

DOI：
10.1523/jneurosci.22-24-10653.2002
发表时间：
2002
期刊：
The Journal of neuroscience : the official journal of the Society for Neuroscience
影响因子：
0
作者：
Pivovarova,NataliaB;Pozzo-Miller,LucasD;Hongpaisan,Jarin;Andrews,SBrian
通讯作者：
Andrews,SBrian

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines.

DOI：
10.1054/ceca.1999.0043
发表时间：
1999-06
期刊：
Cell calcium
影响因子：
4
作者：
S. Yagodin;N. Pivovarova;S. Andrews;D. B. Sattelle
通讯作者：
S. Yagodin;N. Pivovarova;S. Andrews;D. B. Sattelle

Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium?

神经垂体神经末梢的刺激-分泌耦合：膀胱内钠的作用？

DOI：
10.1073/pnas.96.6.3206
发表时间：
1999
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Thirion,S;Troadec,JD;Pivovarova,NB;Pagnotta,S;Andrews,SB;Leapman,RD;Nicaise,G
通讯作者：
Nicaise,G

Mass distribution and spatial organization of the linear bacterial motor of Spiroplasma citri R8A2.

柑橘螺原体 R8A2 线性细菌运动的质量分布和空间组织。