Ghoumari A M, Wehrlé R, Bernard O, Sotelo C, Dusart I
INSERM U106, Hôpital de la Salpétriére, 75651 Paris Cedex 13, France.
Eur J Neurosci. 2000 Aug;12(8):2935-49. doi: 10.1046/j.1460-9568.2000.00186.x.
Neuronal cell death is an essential feature of nervous system development and neurodegenerative diseases. Most Purkinje cells in murine cerebellar organotypic culture die when taken from 1-5-day-old mice (P1-P5), whereas they survive when taken before or after these ages. Using DNA gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) and electron microscopic analyses, we were able to show that this massive Purkinje cell death is apoptotic in nature and reaches a peak at P3. From the several endogenous genes known to be involved in the apoptotic process, we have focused on two: the bcl-2 and the caspase-3 that encode for anti-apoptotic and pro-apoptotic proteins, respectively. Immunostaining for activated Caspase-3 correlated with Purkinje cell death. A better survival of Purkinje cells was observed in P3 slices taken from hu-bcl-2 transgenic mice, and in slices treated with z-DEVD.fmk (an inhibitor of numerous caspases). Thus, these two genes are implicated in the age-related Purkinje cell apoptosis in organotypic culture. As Purkinje cell death in vitro takes place at the same age as Purkinje cells engaged in intense synaptogenesis and dendritic remodeling in vivo, we propose that this apoptosis reflects a naturally occurring Purkinje cell death during this critical period.
神经元细胞死亡是神经系统发育和神经退行性疾病的一个基本特征。在小鼠小脑器官型培养中，大多数浦肯野细胞如果取自1 - 5日龄小鼠（P1 - P5）就会死亡，而在这些年龄之前或之后取材则可以存活。通过DNA凝胶电泳、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）以及电子显微镜分析，我们能够证明这种大规模的浦肯野细胞死亡本质上是凋亡性的，并且在P3时达到峰值。在已知参与凋亡过程的几个内源性基因中，我们重点关注了两个：分别编码抗凋亡蛋白和促凋亡蛋白的bcl - 2和caspase - 3。活化的Caspase - 3免疫染色与浦肯野细胞死亡相关。在取自人bcl - 2转基因小鼠的P3切片以及用z - DEVD.fmk（一种多种半胱天冬酶的抑制剂）处理的切片中，观察到浦肯野细胞有更好的存活情况。因此，这两个基因与器官型培养中与年龄相关的浦肯野细胞凋亡有关。由于体外浦肯野细胞死亡发生的年龄与体内进行强烈突触形成和树突重塑的浦肯野细胞的年龄相同，我们提出这种凋亡反映了在这个关键时期自然发生的浦肯野细胞死亡。
