Molecular and cellular biological study about the abnormal neurite outgrowth and protein phosphorylation in temporal lobe epilepsy

颞叶癫痫的一个形态学特征是海马齿状回颗粒细胞突起的异常生长。Ca^2+/钙调蛋白依赖性蛋白激酶II（CaM激酶II）的某些亚型可能与这种异常的神经突生长有关。在CaM激酶II中，已经从脑中鉴定出四个亚基和几种亚型。为了探讨钙调素激酶Ⅱ各亚型在颞叶癫痫发病中的作用，我们对钙调素激酶Ⅱ各亚型的结构和功能进行了研究，主要研究了钙调素激酶Ⅱ δ亚基（CaM kinase Ⅱ δ）的结构和功能。1)我们制备了抗CaM激酶II δ的抗体，并检测了该亚基在脑中的定位。2)我们从大鼠脑组织中获得了钙调素激酶Ⅱ δ 6种亚型的cDNA。在这些亚型中，delta 3定位于转染的NG 108 -15细胞的细胞核中。3)NG 108 - 15细胞中delta 3的过表达诱导脑源性神经营养因子（BDNF）的基因表达，其促进脑中特定神经元群体的存活和分化。4)我们发现，在PC 12细胞和NG 108 -15细胞中的CaM激酶II的主要亚型是delta 2。δ 2主要定位于高尔基体。5）其他亚型在NG 108-15细胞中定位于细胞质。因此，这些异构体可能通过磷酸化微管相关蛋白2等细胞骨架蛋白参与神经突起生长的调控。这些结果表明，每种异构体都定位于神经元的特定部位，在神经元功能中发挥重要作用。

A morphological characteristic of temporal lobe epilepsy consists of the abnormal neurite outgrowth of the hippocampal dentate granule cells. Some isoforms of Ca^<2+>/calmodulin-dependent protein kinase II (CaM kinase II) may be related to this abnormal neurite outgrowth. Among CaM kinase II, four subunits and several isoforms have been identified from brain. In order to investigate the pathophysiological roles of each isoform of CaM kinase II in temporal lobe epilepsy, we studied about the structures and functions of CaM kinase II isoforms mainly about the delta subunit of CaM kinase II (CaM kinase IIdelta). 1) We prepared the antibody against CaM kinase IIdelta and examined the localization of the subunit in brain. 2) We obtained the cDNAs of 6 isoforms of CaM kinase IIdelta from rat brain. Among these isoforms, delta3 was localized in the nuclei in transfected NG1O8-15 cells. 3) Overexpression of delta3 in NG1O8- 15 cells induced the gene expression of brain-derived neurotrophic factor (BDNF) which promotes survival and differentiation of specific populations of neurons in the brain. 4) We found that the major isoform of CaM kinase II in PC12 cells and NG108-15 cells was delta2. And delta2 appeared to be mainly localized in Golgi's apparatus.5) Other isoforms were localized in the cytoplasm in NG 108-15 cells. Therefore, these isoforms may be involved in the regulation of neurite outgrowth via phosphorylation of cytoskeletal proteins such as microtubule-associated protein 2.These results suggest that each isoform is localized in the specific site in neurons to play some important roles in neuronal functions.