Functional implications of the heterogeneity of the lipid second messenger-metabolizing enzyme, diacylglycerol kinase, in neurons

神经元中脂质第二信使代谢酶二酰甘油激酶异质性的功能意义

• 批准号: 14380351
• 负责人: GOTO Kaoru
• 金额: $ 8万
• 依托单位: Yamagata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380351/
• 关键词: phospholipid; diacylglycerol; diacylglycerol kinase; neuron; nucleus; signal transduction; second messenger; シナプス; 細胞内局在; 核細胞質移行

We have so far isolated and characterized several isozymes of diacylglycerol kinase(DGK) from rat brain cDNA library. Our series of studies revealed the heterogeneity of the enzyme in terms of molecular structure, tissue distribution, enzymatic activity, and the localization of gene expression, and provided functional implications for DGK family. Interestingly, most of the isozymes show abundant expression in distinct patterns in the brain, suggesting the importance of this enzyme in neuronal function. Among these, mRNA for DGKbeta is detected uniquely in restricted regions of the brain, such as the caudate-putamen, accumbens nucleus, olfactory tubercle, olfactory bulb, and hippocampal pyramidal cell layer. In our previous experiments using transfected COS-7 cells, DGKbeta is shown to be distributed throughout the cytoplasm in a filamentous pattern. However, detailed subcellular localization of this isozyme remains unclear in neurons. To address this point, we performed in the present … More study immunohistochemical examination on rat brain with a specific antibody against DGKbeta, together with cDNA-transfection into primary cultured neurons using GFP expression vector. Under the con focal laser scanning microscopy, the immunoreactivity for DGKbeta was detected in neurons of the corresponding brain regions that express the mRNA abundantly and appeared as ring-like structures. To examine the presice localization of DGKbeta, we employed immunoelectron microscopy to localize the immunolabelled structures. Dark immunoreaction products in pre-embedding immunoperoxidase electron microscopy were distributed diffuselly within dendrites and spines in neurons. Furthermore, most of the particulate metal marker in pre-embedding silver-intensified immunogold electron microscopy were deposited in association with the cytoplasmic face of the cell membrane in dendrites and spines in neurons. In the transfected hippocampal neurons in culture, GFP-DGKbeta was predominantly detected as button-like structures in the presumed dendritic processes. These results suggest that DGKbeta may be involved in synaptic transmission at postsynaptic site and signal transduction in neuronal cells.In addition, it should be also noted that two of the isozymes, DGKzeta and DGKiota, contain a nuclear localization signal, suggesting possible roles of these isozymes in nuclear processes. We have revealed selective translocation of DGKzeta in hippocampal neurons under transient forebrain ischemia. We found that DGKzeta is translocated from the nucleus to the perikaryal cytoplasm in hippocampal pyramidal cells immediately after 20 min of ischemic insult. Previous studies have shown that neuronal death in the hippocampus occurs after 48-72 h reperfusion, but not immediately, under transient ischemia, a phenomenon called "delayed neuronal death." Therefore we speculate that the nucleo-cytoplasmic translocation of DGKzeta in the hippocampal neurons at an early phase of ischemic insult may be one of the triggering events of the delayed neuronal death, but not a result of dying process. Less

我们从大鼠脑cDNA文库中分离并鉴定了二酰甘油激酶（DGK）的几种同工酶。我们的一系列研究揭示了该酶在分子结构、组织分布、酶活性和基因表达定位等方面的异质性，并为DGK家族的功能研究提供了理论依据。有趣的是，大多数同工酶在大脑中以不同的模式显示出丰富的表达，表明这种酶在神经元功能中的重要性。其中，DGKbeta的mRNA仅在大脑的限制区域中检测到，例如尾壳核、丘脑核、嗅结节、嗅球和海马锥体细胞层。在我们先前使用转染的COS-7细胞的实验中，显示DGKbeta以丝状模式分布在整个细胞质中。然而，这种同工酶的详细亚细胞定位在神经元中仍不清楚。为了解决这一点，我们在现在进行了 ...更多信息 用DGK β特异性抗体对大鼠脑组织进行免疫组化检测，并用GFP表达载体将cDNA转染原代培养的神经元。在激光共聚焦显微镜下，DGK β在相应脑区的神经元中表达丰富，呈环状结构。为了检查DGKbeta的前定位，我们采用免疫电子显微镜定位免疫标记的结构。包埋前免疫过氧化物酶电镜下免疫反应产物呈弥漫性分布于神经元的树突和棘内。此外，大多数的颗粒金属标记物在预包埋银增强免疫金电子显微镜沉积在与细胞膜的细胞质面在树突和神经元的棘。在培养的转染的海马神经元中，GFP-DGK β主要在假定的树突状突起中检测到纽扣样结构。这些结果提示，DGK β可能参与突触后部位的突触传递和神经细胞内的信号转导，另外，DGKzeta和DGKiota两种同工酶含有核定位信号，提示它们可能参与核过程。我们已经揭示了短暂前脑缺血海马神经元DGKzeta的选择性易位。我们发现，DGKzeta易位从细胞核到核周细胞质海马锥体细胞缺血损伤后20分钟立即。先前的研究表明，海马神经元死亡发生在再灌注48-72小时后，但不是立即发生，在短暂缺血下，这种现象称为“迟发性神经元死亡”。“因此，我们推测，缺血损伤早期海马神经元中DGKzeta的核质易位可能是延迟性神经元死亡的触发事件之一，但不是死亡过程的结果。少

项目成果

Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung

二酰甘油激酶同工酶在大鼠肺中的表达和定位及酶学特征

• 期刊: Am. J. Physiol. Lung Cell Mol. Physiol. (in press)
• 作者: Katagiri;Y.;Ito;T.;Saino-Saito;S.;Hozumi;Y.;Suwabe;A.;Otake;K.;Sata;M.;Kondo;H.;Sakane;F.;Kanoh;H.;Kubota;I.;Goto;K.
• 通讯作者: K.

Mutant SOD1 linked to familial amyotrophic lateral sclerosis, but not wild-type SOD1,induced ER stress in COS7 cells and transgenic mice.

突变型 SOD1 与家族性肌萎缩侧索硬化症有关，但与野生型 SOD1 无关，可在 COS7 细胞和转基因小鼠中诱导 ER 应激。

• 发表时间: 2003
• 期刊: Biochem.Biophys.Res.Comm. 303
• 作者: Tobisavva;S.;Hozumi;Y.;Arawaka;S.;Koyama.S.;Wasa;M.;Nagai;M.;Aoki;M.;Itoyama;Y.;Goto;K.;Kato;T.
• 通讯作者: T.

Gene expression, cellular localization and enzymatic activity of diacylglycerol kinase isozymes in rat ovary and placenta

大鼠卵巢和胎盘中二酰甘油激酶同工酶的基因表达、细胞定位和酶活性

• 期刊: Cell Tissue Res. (in press)
• 作者: Toya;M.;Hozumi;Y.;Ito;T.;Takeda;M.;Sakane;F.;Kanoh;H.;Saito;H.;Hiroi;M.;Kurachi;H.;Kondo;H.;Goto;K.
• 通讯作者: K.

Ade no virus- me diate d overexpression of diacylglycerol kinase-zeta inhibits, endothelin-1-induced cardiomyocyte hypertrophy.

病毒介导的二酰甘油激酶-zeta 过度表达抑制内皮素-1 诱导的心肌细胞肥大。

• 发表时间: 2005
• 期刊: Circulation 111
• 作者: Yahagi et al.;A.Ishikawa;Toya et al.;Ishikawa A;Katagiri et al.;A.Ishikawa;Takahashi et al.
• 通讯作者: Takahashi et al.

Molecular cloning and characterization of the human diacylglycerol kinase beta gene : alternative splicing generates DGKbeta isotypes with different properties.

人二酰甘油激酶β基因的分子克隆和表征：选择性剪接产生具有不同特性的DGKβ同种型。

• 发表时间: 2003
• 期刊: J. Biol. Chem. 277
• 作者: Caricasole et al.