Liesi P, Stewart R R, Akinshola B E, Wright J M
Laboratory of Molecular and Cellular Neurobiology, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852, USA.
J Neurobiol. 1999 Mar;38(4):441-54.
Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.
采用生化、免疫细胞化学和分子生物学技术，研究N-甲基-D-天冬氨酸（NMDA）受体亚基在迁移缺陷型韦弗小鼠小脑体内以及在有或无维拉帕米挽救情况下的蚓部韦弗颗粒神经元原代培养物中的表达。我们发现，NMDAR1（ζ1）的信使核糖核酸和蛋白在韦弗颗粒神经元原位表达。免疫细胞化学和生化分析表明，韦弗小鼠小脑的颗粒神经元表达R1（ζ1）和R2A（ε1）亚基，但R2B（ε2）亚基表达很少。在韦弗小鼠小脑中，R2B（ε2）亚基主要表达于内颗粒细胞层和白质的神经纤维中。对R1（ζ1）亚基进行逆转录聚合酶链反应并测序分析表明，正常小鼠和韦弗小鼠小脑的ζ1亚基扩增子相同，且可检测到含第22外显子（1 - 2）以及含或不含第5外显子（a/b）或第21外显子（1 - 4）的剪接变体。正常小鼠和韦弗小鼠小脑的R2A（ε1）和R2B（ε2）亚基在正常和韦弗NMDA受体亚基之间或与克隆的小鼠NMDA受体亚基之间未显示出一级结构差异。在蚓部培养物中，正常颗粒神经元表达所有三种NMDA受体亚基（ζ1、ε1和ε2），而韦弗神经元未能表达ε2亚基。维拉帕米挽救韦弗神经元可诱导ε2蛋白沿颗粒神经元表面表达。目前的结果表明，韦弗小鼠小脑中缺乏ε2亚基可能与功能性NMDA受体的缺乏和/或韦弗颗粒神经元的迁移失败有关。我们的数据进一步表明，NMDA受体介导的神经毒性不太可能是韦弗颗粒神经元死亡的介质。事实上，NMDA受体表达和功能的下调可能是韦弗颗粒神经元减少通过这些受体的钙内流的一种保护措施。
