Targeting mechanisms for calmodulin-dependent protein kinases and their neuronal functions

本项目的目的是阐明钙调素依赖的蛋白激酶，特别是CaM激酶I(CaMKI)的神经元功能。为了更好地了解CaMKI的功能，我们首先用原位杂交组织化学方法检测了四种异构体(a，b2，g，d)在小鼠脑内的发育表达。在发育中的海马区，CaMKI的四种异构体按照出现在海马亚区的时间顺序显示出不同的表达模式。例如，CaMKIδ是海马锥体神经元中含量最丰富的亚型，开始在CA3区表达，其次是CA1区。另一方面，生后2周后，海马锥体细胞层中CaMKIα和CaMKIβ2的表达减少。由于CaMKI的时间表达模式，特别是CaMKIδ，与海马锥体神经元树突形成的时间进程密切相关，我们通过转染各种可能作为显性阴性的CaMKI的激酶死亡突变体，研究了CaMKI在培养的海马神经元树突形成中的功能参与。过表达的CaMKI激酶死亡突变体减少了转基因神经元的平均树突长度，但对初级树突的数量和分支指数没有明显影响。这一发现提示CaMKI可能参与了树突的生长，特别是树突的伸展。

The aim of this project is to clarify the neuronal functions of calmodulin-dependent protein kinases, particularly CaM kinase I (CaMKI). To obtain better understanding of the functions of CaMKI, we first examined the developmental expression of the four isoform (a, b2, g, d) in mouse brain by in situ hybridization histochemistry. In the developing hippocampus, the four isoforms of CaMKI exhibit distinct expression patterns in terms of the chronological order of the appearance in the hippocampal subregions. For example, CaMKIδ, which is the most abundant isoform in the hippocampal pyramidal neurons, started to be expressed in the CA3 regions, followed by the CA1. On the other hand, the expression of CaMKIα and CaMKIβ2 is decreased after the postnatal second week in the hippocampal pyramidal cell layer. Since the temporal expression patterns of CaMKIs, particularly CaMKIδ, correlates well with the temporal schedule of the dendritic formation of the hippocampal pyramidal neurons, we examined the functional involvement of CaMKI in the deridritic formation of cultured hippocampal neurons by transfecting various kinase-dead mutants of CaMKI which were expected to work as dominant negatives. The overexpression of kinase-dead mutants of CaMKI reduced the average dendritic length of the transfected neurons without any significant effects on the number of primary dendrites and branching index. This finding suggest that CaMKI may be involved in the dendritic growth, particularly the extension of dendrites.