EXAMINATION OF MECHANISMS OF EARLY PROGRAMMED MOTONEURON DEATH IN THE DEVELOPING CHICK CERVICAL SPINAL CORD

发育中雏鸡颈脊髓早期程序性运动神经元死亡机制的研究

• 批准号: 10680704
• 负责人: YAGINUMA Hiroyuki
• 金额: $ 2.11万
• 依托单位: FUKUSHIMA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680704/
• 关键词: caspase; DEVD; BAF; chick; motoneuron; cervical; apoptosis; programmed cell death; GDNF; RET; 発生

In the chick embryo spinal cord, two types of programmed cell death (PCD) of motoneurons (MNs) are known to occur, one at a relatively late stage and the other at an earlier stage of differentiation. In the second type, the PCD of MNs occurs only in the non-limb innervating cervical spinal cord between E4 and E5. To elucidate the roles for caspases in this early PCD of MNs in the cervical spinal cord, we examined the activation of caspases and the effects of inhibitors of caspases in vivo.Caspase-3 like activity was increased in ventral MN containing region of the cervical segments at st 24 (E4.5) when many pyknotic dying neurons are observed. Immunohistochemical observations with an antibody against activated form of caspase-3 suggested that activation of caspase-3 precedes both DNA fragmentation and the morphological manifestation of PCD in cervical MNs. To further investigate roles for casapase-3 like activity, we treated embryos with Ac-DEVD-CHO that inhibits caspase-3 like activit … More y. In Ac-DEVD-CHO treated embryos, the nuclei of pyknotic cells showed only moderate condensation. When pyknotic cells were counted, including these moderately pyknotic cells, there was no significant difference between controls and Ac-DEVD-CHO treated embryos. TUNEL staining revealed that DNA fragmentation did not occur in these moderately condensed nuclei, even though electron microscopic observations clearly revealed that degenerative changes were occurring in the cytoplasm. These results suggest that caspase-3 like activity plays a role in the apoptotic nuclear changes.To investigate involvement of other caspases that are responsible for apoptotic changes of cytoplasm, we treated embryos with Boc-Asp-FMK (BAF) that inhibits broad spectrum of caspases. After treatment with BAF for 12 hours the number of pyknotic cells remarkably fewer than controls. Even moderately pyknotic cell that were observed after treatment with Ac-DEVD-CHO were rarely observed. Instead, there were many cells whose nuclei are smaller and whose cytoplasm was slightly more eosinophilic. These cells lacked DNA fragmentation and retained MN specific marker. Electron microscopic observation revealed that there are many smaller cells that have irregular shaped nuclei, slightly electron dense cytoplasm and apparently normal organelles. Following treatment with BAF for 24 hours, the number of pyknotic cells increased to the level comparable to embryos treated with Ac-DEVD-CHO, although the number of TUNEL positive cells was still fewer than controls. Electron microscopic observation revealed that there are many aberrantly degenerating cells. The total number of surviving MNs was not more than controls and Ac-DEVD-CHO treated embryos. These results suggest that other caspases that can be inhibited by BAF but not by Ac-DEVD-CHO play roles in execution of apoptotic changes in dying MNs. Although inhibition of such caspases considerably delayed the rate of PCD, MNs can not be finally rescued by inhibition of caspases. Less

在鸡胚脊髓中，已知会发生两种类型的运动神经元（MN）程序性细胞死亡（PCD），一种发生在相对较晚的阶段，另一种发生在分化的早期阶段。在第二种类型中，MN的PCD仅发生在E4和E5之间的非肢体神经支配的颈脊髓中。为了阐明 caspase 在颈髓 MN 早期 PCD 中的作用，我们检查了 caspase 的激活和体内 caspase 抑制剂的作用。在第 24 阶段（E4.5），当观察到许多固缩死亡神经元时，在包含颈段 MN 的腹侧区域中，Caspase-3 样活性增加。使用针对激活形式的 caspase-3 的抗体进行的免疫组织化学观察表明，caspase-3 的激活先于 DNA 片段化和宫颈 MN 中 PCD 的形态表现。为了进一步研究 caspase-3 样活性的作用，我们用 Ac-DEVD-CHO 处理胚胎，Ac-DEVD-CHO 可抑制 caspase-3 样活性……更多 y。在 Ac-DEVD-CHO 处理的胚胎中，固缩细胞的细胞核仅表现出中等程度的凝结。当对固缩细胞（包括这些中度固缩细胞）进行计数时，对照和 Ac-DEVD-CHO 处理的胚胎之间没有显着差异。 TUNEL 染色显示，尽管电子显微镜观察清楚地表明细胞质中发生了退行性变化，但这些中等浓缩的细胞核中并未发生 DNA 断裂。这些结果表明 caspase-3 样活性在细胞凋亡核变化中发挥作用。 为了研究其他负责细胞质凋亡变化的 caspase 的参与，我们用抑制广谱 caspase 的 Boc-Asp-FMK (BAF) 处理胚胎。用 BAF 处理 12 小时后，固缩细胞的数量明显少于对照组。即使在用 Ac-DEVD-CHO 处理后观察到的中度固缩细胞也很少观察到。相反，有许多细胞的细胞核较小，细胞质稍稍嗜酸性。这些细胞缺乏 DNA 碎片并保留了 MN 特异性标记。电镜观察显示，存在许多较小的细胞，其细胞核形状不规则，细胞质电子密度稍高，细胞器明显正常。用 BAF 处理 24 小时后，固缩细胞的数量增加到与用 Ac-DEVD-CHO 处理的胚胎相当的水平，尽管 TUNEL 阳性细胞的数量仍然少于对照。电镜观察发现，存在许多异常退化的细胞。存活的 MN 总数不超过对照和 Ac-DEVD-CHO 处理胚胎。这些结果表明，可以被 BAF 抑制但不能被 Ac-DEVD-CHO 抑制的其他半胱天冬酶在死亡 MN 的凋亡变化的执行中发挥作用。尽管抑制此类半胱天冬酶可显着延缓 PCD 的发生，但抑制半胱天冬酶并不能最终挽救 MN。较少的

项目成果

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