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
• 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/6432892
• 关键词: Anura; cAMP response element binding protein; calcium flux; confocal scanning microscopy; dendrites; developmental genetics; developmental neurobiology; electron microscopy; electrophysiology; endoplasmic reticulum; estradiol; gamma aminobutyrate; gene expression; glia; hippocampus; hormone regulation /control mechanism; intracellular transport; mitochondria; nerve /myelin protein; neurons; organ culture; protein biosynthesis; protein transport; synapses

This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on dendrites and dendritic spines of central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca concentration ([Ca2+]i) that is strongly modulated by the activity of intracellular Ca stores. We had earlier shown that in frog sympathetic neurons ? an excellent model for studying intracellular details of Ca dynamics ? depolarization-induced increases in [Ca2+]i are accompanied by large, reversible elevations in total mitochondrial calcium concentration ([Ca]m). This mitochondrial Ca2+ transport activity gives rise to spatial gradients in [Ca]m because it registers and retains a record of early regional differences in [Ca2+]i, and this in turn plays an important role in spatio-temporally shaping cytosolic Ca signals. .We have now characterized the function of a second major Ca2+-regulating organelle, the endoplasmic reticulum (ER), whose role is generally thought to be amplification of evoked [Ca2+]i elevations by triggered Ca2+ release from its internal store, a process known as "calcium-induced calcium release" (CICR). We find, however, that at low levels of Ca2+ entry (and therefore low [Ca2+]i) the ER actually acts as a Ca2+ buffer, albeit one whose strength is down-regulated by graded activation of a [Ca2+]i-sensitive release pathway. Theoretical simulations show that such a "reverse" mode of CICR is expected; moreover, many neurons should exhibit a progressive transition from Ca2+ buffering to triggered Ca2+ release as [Ca2+]i increases. Such a transition ? to classical CICR ? was observed when [Ca2+]i increased above approx. 1uM. In addition, such Ca2+ release is preferentially localized to peripheral ER cisternae, so that both Ca2+ uptake (centrally) and release (peripherally) can occur at the same time in 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. In hippocampal neurons, evoked [Ca2+]i transients enhance nuclear import of calmodulin (CaM), which in turn augments phosphorylation of the important transcription factor CREB. This pathway for phospho-CREB (pCREB) production is central to the synaptically-evoked gene expression that underlies long-term memory formation. It has been previously shown that superoxide (O2-) ions can enhance the stability of pCREB by inhibiting its dephosphorylation. Using quantitative immunocytochemistry and a panel of electron transport blockers, we have now evaluated the role of mitochondria in linking cytosolic Ca2+ entry to gene expression. We find that mitochondrial Ca accumulation, occurring predominantly in peripheral regions of neurons during Ca2+ entry, leads to increases in both ATP and O2- production. This, in turn, enhances nuclear import of CaM. While O2- elevation is quantitatively important for CaM transport, it appears to be essential for sustaining CREB phosphorylation.

该项目研究神经元钙信号传导的生理和细胞方面，重点关注中枢神经系统神经元的树突和树突棘。神经元对突触刺激做出反应，胞质游离 Ca 浓度 ([Ca2+]i) 升高，而胞质游离 Ca 浓度受到细胞内 Ca 储存活性的强烈调节。我们之前已经证明青蛙的交感神经元？研究细胞内 Ca 动力学细节的优秀模型？去极化诱导的 [Ca2+]i 增加伴随着线粒体总钙浓度 ([Ca]m) 的大幅可逆升高。这种线粒体 Ca2+ 转运活动会产生 [Ca2+]i 的空间梯度，因为它记录并保留了 [Ca2+]i 的早期区域差异记录，这反过来又在时空塑造胞质 Ca2+ 信号中发挥重要作用。我们现在已经确定了第二个主要 Ca2+ 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 Ca2+ 从其内部储存释放来放大诱发的 [Ca2+]i 升高，这一过程称为“钙诱导的钙释放”(CICR)。然而，我们发现，在低水平的 Ca2+ 进入（因此 [Ca2+]i 较低）下，ER 实际上充当 Ca2+ 缓冲剂，尽管其强度因 [Ca2+]i 敏感释放途径的分级激活而下调。理论模拟表明，CICR的这种“反向”模式是预期的；此外，随着 [Ca2+]i 的增加，许多神经元应该表现出从 Ca2+ 缓冲到触发 Ca2+ 释放的渐进转变。这样的转变？到经典 CICR ？当 [Ca2+]i 增加到约以上时观察到。 1微米。此外，这种Ca2+释放优先定位于外周内质网池，因此Ca2+摄取（中央）和释放（外周）可以在同一细胞的不同区域同时发生。最后，内质网 Ca2+ 转运的空间梯度与线粒体 Ca2+ 摄取的空间梯度相反，表明这些细胞器之间存在合作。在海马神经元中，诱发的 [Ca2+]i 瞬变增强钙调蛋白 (CaM) 的核输入，进而增强重要转录因子 CREB ​​的磷酸化。这种磷酸化 CREB ​​(pCREB) 产生途径对于突触诱发基因表达至关重要，而突触诱发基因表达是长期记忆形成的基础。先前的研究表明，超氧 (O2-) 离子可以通过抑制 pCREB ​​的去磷酸化来增强其稳定性。使用定量免疫细胞化学和一组电子传递阻断剂，我们现在评估了线粒体在连接细胞质 Ca2+ 进入与基因表达之间的作用。我们发现线粒体 Ca2+ 积累主要发生在 Ca2+ 进入过程中神经元的外周区域，导致 ATP 和 O2- 产量增加。这反过来又增强了 CaM 的核输入。虽然 O2- 升高对于 CaM 转运在数量上很重要，但它似乎对于维持 CREB ​​磷酸化也至关重要。