Migheli A, Piva R, Casolino S, Atzori C, Dlouhy S R, Ghetti B
Department of Neuroscience, Laboratory of Neuropathology, University of Turin, Italy.
Am J Pathol. 1999 Aug;155(2):365-73. doi: 10.1016/S0002-9440(10)65133-4.
A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies.
编码G蛋白激活的内向整流钾通道（GIRK）的基因GIRK2中的一个错义突变，是导致韦弗（wv）小鼠小脑外颗粒层（EGL）细胞凋亡以及中脑多巴胺能神经元非凋亡性死亡的原因。轴突发生和迁移失败被认为是小脑GIRK2通道功能异常的主要后果。我们研究了细胞周期紊乱是否先于迁移和轴突发生失败并导致大量细胞凋亡。为此，我们对P7 - P21的+/+、wv/+和wv/wv小鼠的小脑应用了PCNA、Cdk4、细胞周期蛋白D、细胞周期蛋白A和Cdk抑制剂p27/kip1的免疫组织化学和免疫印迹，以及凋亡DNA片段化的原位末端标记。在+/+和wv/+小鼠中，细胞周期蛋白的表达仅限于EGL的外层、迁移前区。细胞分化标志物p27的抗体呈现相反的染色模式。由于迁移延迟，P21的wv/+小鼠的外层EGL中存在p27阳性细胞斑块。相反，在wv/wv小鼠的所有年龄段，整个EGL均出现明显的细胞周期上调且无p27表达，表明无法关闭细胞周期。有丝分裂指数评估显示细胞周期激活与增殖事件无关。黑质中未表达细胞周期蛋白，这表明成熟多巴胺能神经元的非凋亡性死亡并非由细胞周期重新进入失败所致。我们的数据表明，wv/wv小鼠小脑中细胞周期异常是GIRK2通道功能异常的主要早期后果，可能强烈影响EGL细胞对凋亡的易感性。这些观察结果可能有助于理解人类神经通道病的发病机制。
