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

在鸡胚胎脊髓中，运动神经元的程序性细胞死亡(PCD)有两种类型，一种发生在较晚的分化阶段，另一种发生在分化的早期阶段。在第二种类型中，MNS的PCD仅发生在E4~E5之间的非肢体支配的颈髓。为了阐明caspase在颈髓MNS早期PCD中的作用，我们在活体内检测了caspase的激活和caspase抑制剂的作用。在24小时(E4.5)，当观察到许多固缩的死亡神经元时，Caspase-3样活性在颈段MN的腹侧区域增强。免疫组织化学染色显示，Caspase-3活化先于DNA断裂和PCD在宫颈MNS中的形态表现。为了进一步研究Casapase-3样活性的作用，我们用ac-devd-CHO处理胚胎，它抑制了caspase-3样活化…。在Ac-DEVD-CHO处理的胚胎中，固缩细胞的核仅表现为中度凝聚。当计算固缩细胞时，包括这些中度固缩细胞，对照组和Ac-DEVD-CHO处理的胚胎之间没有显著差异。TUNEL染色显示，这些中等浓缩的细胞核中没有出现DNA片段化，尽管电子显微镜观察清楚地显示，细胞质正在发生退行性变化。这些结果表明caspase-3样活性在细胞凋亡的核变化中起作用。为了研究其他导致细胞质凋亡变化的caspase的参与，我们用抑制广谱caspase的Boc-Asp-FMK(BAF)处理胚胎。经曝气生物滤池处理12h后，固缩细胞数明显少于对照组。甚至在Ac-DEVD-CHO治疗后也很少观察到中度固缩的细胞。取而代之的是，许多细胞的核更小，细胞质更嗜酸性。这些细胞缺乏DNA片段化，保留了MN特异性标记。电子显微镜下可见许多较小的细胞，核形不规则，胞浆略有电子致密，细胞器明显正常。在BAF处理24小时后，固缩细胞的数量增加到与Ac-DEVD-CHO处理的胚胎相当的水平，但TUNEL阳性细胞的数量仍然少于对照组。电子显微镜观察发现有许多异常变性细胞。存活的MN总数不多于对照组和Ac-DEVD-CHO处理的胚胎。这些结果表明，在垂死的MN中，可以被BAF抑制但不能被Ac-DEVD-CHO抑制的其他caspase在执行凋亡变化中发挥作用。尽管抑制这类caspase可显著延缓PCD的发生，但抑制caspase并不能最终挽救MNS。较少

项目成果

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