Essential Role of Neuron-Enriched Diacylglycerol Kinase (DGK), DGKβ in Neurite Spine Formation, Contributing to Cognitive Function

BackgroundDiacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKβ is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated.
Methodology/Principal FindingsWe, therefore, developed DGKβ KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKβ. In addition, overexpression of DGKβ in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKβ, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKβ but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed.
Conclusions/SignificanceThese results demonstrate that membrane-localized DGKβ regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.

背景
二酰甘油（Diacylglycerol, DG）激酶（DGK）可催化DG磷酸化生成磷脂酸（phosphatidic acid, PA）。在哺乳动物的10种DGK亚型中，DGKβ属于膜定位型亚型，在大脑皮层、海马体与尾壳核（caudate-putamen）中呈高表达状态。然而，其在神经元及高级脑功能中的生理作用尚未得到阐明。

方法学与主要研究结果
为此，我们借助睡美人转座子系统（Sleeping Beauty transposon system）构建了DGKβ敲除（KO）小鼠，发现该小鼠海马CA1区的长时程增强（long-term potentiation, LTP）水平显著降低，进而在Y迷宫（Y-maze）与莫里斯水迷宫（Morris water-maze）实验中表现出空间记忆与长时记忆等认知功能障碍。
对DGKβ敲除小鼠的原代培养海马神经元（primary cultured hippocampal neurons）进行形态学分析，可见其树突分支与棘突数量均少于野生型（wild type）小鼠。这一形态学缺陷可通过过表达（overexpression）DGKβ得以挽救。
此外，在SH-SY5Y细胞与原代培养海马神经元中过表达DGKβ，均可促进树突分支与棘突的形成；而一种DGKβ剪接变体（splice variant）虽保留激酶活性，但丧失膜定位能力，无法诱导树突分支与棘突的形成。
在过表达DGKβ而非该剪接变体的细胞中，其细胞膜（plasma membrane）上的DGK产物PA水平升高，底物DG水平降低。
尤为关键的是，我们在DGKβ敲除小鼠的海马CA1区中证实了棘突密度降低以及PA与DG的含量异常。

结论与意义
本研究结果表明，膜定位型DGKβ可通过调控脂质代谢调节棘突形成，从而维持认知过程（包括记忆）中突触传递相关的神经网络稳态。