Kyuhou Shin-ichi, Kato Nobuo, Gemba Hisae
Department Physiology (II), Kansai Medical University, 10-15 Fumizono-cho, Moriguchi 570 8506, Japan.
Neurosci Lett. 2006 Mar 27;396(2):91-6. doi: 10.1016/j.neulet.2005.11.023. Epub 2005 Dec 13.
In the current studies, we characterized the molecular and cellular mechanism of cell death in Purkinje cell degeneration (pcd) mice using real-time quantitative PCR, immunohistochemistry, and Western blotting. It appears that endoplasmic reticulum (ER) stress is involved in this degeneration of Purkinje cells because ER stress-related substrates, such as CHOP and caspase 12, were strongly activated in Purkinje cells of pcd mice during the third postnatal (P) week. A significant increase in the expression of the ER-specific chaperone BiP suggested that unfolded protein responses were induced. We also found that Purkinje cells underwent apoptosis via the activation of caspase 3 and subsequent fragmentation of DNA. In addition to the activation of apoptosis in Purkinje cells, many activated microglial cells are found to be present in the molecular layer of the cerebellar cortex. In the later phase of degeneration, there was conspicuous expression of inducible nitric oxide synthase (iNOS), and some Purkinje cells were strongly labeled with an antibody to nitrotyrosine, suggesting that Purkinje cells in pcd mice are damaged by nitric oxide released from microglial cells. Administration of minocycline, which may inhibit iNOS expression, delayed the death of Purkinje cells in pcd mice and mildly improved their motor abilities. These findings suggest that ER stress participates in the degeneration of Purkinje cells and that activation of microglia accelerates Purkinje cell death in pcd mice.
在当前的研究中，我们运用实时定量PCR、免疫组织化学和蛋白质印迹法，对浦肯野细胞变性（pcd）小鼠细胞死亡的分子和细胞机制进行了表征。内质网（ER）应激似乎参与了浦肯野细胞的这种变性，因为在出生后第三周（P），pcd小鼠的浦肯野细胞中，与ER应激相关的底物，如CHOP和半胱天冬酶12被强烈激活。ER特异性伴侣蛋白BiP表达的显著增加表明诱导了未折叠蛋白反应。我们还发现浦肯野细胞通过半胱天冬酶3的激活和随后的DNA片段化而发生凋亡。除了浦肯野细胞凋亡的激活外，还发现许多活化的小胶质细胞存在于小脑皮质的分子层中。在变性后期，诱导型一氧化氮合酶（iNOS）有明显表达，一些浦肯野细胞被硝基酪氨酸抗体强烈标记，这表明pcd小鼠的浦肯野细胞受到小胶质细胞释放的一氧化氮的损伤。给予可能抑制iNOS表达的米诺环素，可延缓pcd小鼠浦肯野细胞的死亡，并轻微改善其运动能力。这些发现表明，ER应激参与了浦肯野细胞的变性，并且小胶质细胞的激活加速了pcd小鼠浦肯野细胞的死亡。
