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

我们已经从大鼠脑cDNA文库中分离并鉴定了DGK的几个同工酶。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家族提供了功能启示。有趣的是，大多数同工酶在大脑中以不同的模式表现出丰富的表达，这表明这种酶在神经元功能中的重要性。其中，dgkβ mRNA仅在大脑的受限区域检测到，如尾壳核、伏隔核、嗅结节、嗅球和海马锥体细胞层。在我们之前使用转染的COS-7细胞的实验中，DGKbeta被证明以丝状模式分布在整个细胞质中。然而，该同工酶在神经元中的亚细胞定位尚不清楚。为了解决这一问题，我们进一步研究了用DGKbeta特异性抗体对大鼠脑进行免疫组化检测，并用GFP表达载体将cdna转染原代培养的神经元。在共聚焦激光扫描显微镜下，在相应脑区大量表达DGKbeta mRNA并呈环状结构的神经元中检测到DGKbeta的免疫反应性。为了检测DGKbeta的精确定位，我们采用免疫电镜对免疫标记结构进行了定位。免疫过氧化物酶预包埋电镜下暗免疫反应产物弥散分布于神经元的树突和棘内。此外，在预包埋银强化免疫金电镜下，大多数颗粒金属标记物沉积在神经元树突和棘细胞膜的细胞质面。在培养的转染海马神经元中，GFP-DGKbeta主要被检测为树突突起中的按钮状结构。这些结果提示dgkβ可能参与突触后位点的突触传递和神经元细胞的信号转导。此外，还应该指出的是，DGKzeta和DGKiota这两种同工酶含有核定位信号，这表明这些同工酶可能在核过程中起作用。我们发现短暂性前脑缺血时海马神经元DGKzeta选择性易位。我们发现DGKzeta在海马锥体细胞缺血损伤20分钟后立即从核转移到核周细胞质。先前的研究表明，海马区的神经元死亡发生在48-72小时再灌注后，而不是立即发生，在短暂缺血下，这种现象被称为“延迟神经元死亡”。因此，我们推测海马神经元DGKzeta在缺血损伤早期的核胞质易位可能是延迟神经元死亡的触发事件之一，而不是死亡过程的结果。少

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