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，已知运动神经元（MN）的两种类型的程序性细胞死亡（PCD）发生，一种发生在分化的相对晚期，另一种发生在分化的早期。在第二种类型中，MN的PCD仅发生在E4和E5之间的非肢体支配颈髓。为了阐明caspase-3在颈髓MN早期PCD中的作用，我们在体内检测了caspase-3的激活和caspase-3抑制剂的作用，在st 24（E4.5），当观察到许多固缩的死亡神经元时，在颈段腹侧含MN区域中Caspase-3样活性增加。免疫组织化学观察与抗体对活化形式的caspase-3的激活表明，caspase-3的激活之前的DNA片段化和形态学表现的PCD在宫颈MN。为了进一步研究caspase-3样活性的作用，我们用抑制caspase-3样活性的Ac-DEVD-CHO处理胚胎， ...更多信息 y.在Ac-DEVD-CHO处理的胚胎中，固缩细胞的细胞核仅显示中度浓缩。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处理的胚胎之间无显著差异。TUNEL染色显示，DNA片段没有发生在这些中度浓缩的细胞核，即使电子显微镜观察清楚地表明，退行性变化发生在细胞质中。这些结果表明，caspase-3样活性在细胞凋亡的核变化中起作用。为了研究参与细胞质凋亡变化的其他caspase，我们用Boc-Asp-FMK（BAF）处理胚胎，抑制广谱caspase。BAF处理12小时后，固缩细胞数明显少于对照组。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，其细胞质略嗜酸性。这些细胞缺乏DNA片段化并保留MN特异性标记。电镜下可见许多较小的细胞，细胞核形状不规则，胞质电子密度略高，细胞器正常。在用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。电镜下可见大量异常变性的细胞。存活MN的总数不超过对照和Ac-DEVD-CHO处理的胚胎。这些结果表明，其他半胱天冬酶，可以被BAF抑制，但不能被Ac-DEVD-CHO在垂死的MN的凋亡变化的执行中发挥作用。虽然这种半胱天冬酶的抑制大大延迟PCD的速率，MN不能最终通过抑制半胱天冬酶被拯救。少

项目成果

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