MOLECULAR CELL BIOLOGICAL STUDY OF CELL DEATH INDUCED CATHEPSIN D

组织蛋白酶 D 诱导细胞死亡的分子细胞生物学研究

• 批准号: 11680738
• 负责人: ISHIHARA Kyoko
• 金额: $ 2.37万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680738/
• 关键词: Lysosome; Proteinase; Cathepsin B; Cathepsin D; CNS; Mitochondria; Ceroid lipofuscin; Knockout mouse; リリソーム; セロイド-リポフスチン; セロイドーリポフスチン

In order to analyze the cell death pathway regulated by lysosomal cathepsins B and D, we used cathepsin D-deficient (CD-/-) mice. CD-/- mice have been shown to manifest seizures and become blind near the terminal stage [approximately postnatal day (P) 26]. We therefore examined the morphological, immunocytochemical, and biochemical features of CNS tissues of these mice. By electron microscopy, autophagosome/autolysosome-like bodies containing part of the cytoplasm, granular osmiophilic deposits, and fingerprint profiles were demostrated in the neuronal perikarya of CD-/- mouse brains after P20. Subunit c of mitochondrial ATP synthase was found to accumulate in the lysosomes of neurons, although the activity of tripeptidyl peptidase-I significantly increased in the brains. These results suggest that the CNS neurons in CD-/- mice show a new form of lysosomal accumulation disease with a phenotype resembling neuronal ceroid lipofuscinosis. Moreover, the retinal layers of CD-/- mice became thinner than those of the control littermates, particularly, the photoreceptor layer was largely abolished, increasing in the number of TUNEL-positive nuclei in the outer nuclear layer. To identify substrates which are specifically cleaved by CD, we used CD-/- mouse embryonic fibroblasts (MEF). Mitochondria! MnSOD was identified as a possible substrate because of its accumulation in the cytoplasm of CD-/-MEF.

为了分析溶酶体组织蛋白B和D对细胞死亡途径的调控，我们以组织蛋白D缺陷(CD-/-)小鼠为研究对象。CD-/-小鼠已被证明表现为癫痫发作，并在末期[大约出生后(P)26]失明。因此，我们检测了这些小鼠中枢神经系统组织的形态、免疫细胞化学和生化特征。经电子显微镜观察，Cd-/-小鼠P20后脑神经元核内可见自噬小体/自溶酶体样小体，内含部分胞浆、颗粒状嗜奥斯粒沉积和指纹图谱。线粒体三磷酸腺苷合成酶的c亚基在神经元的溶酶体中积累，尽管三肽基肽酶-I的活性在大脑中显著增加。这些结果表明，CD-/-小鼠中枢神经系统神经元表现为一种新的溶酶体蓄积性疾病，其表型类似于神经元蜡样脂褐素沉着症。此外，CD-/-小鼠的视网膜层变薄，特别是光感受器层基本消失，外核层TUNEL阳性核增多。为了确定CD特异性切割的底物，我们使用了CD-/-小鼠胚胎成纤维细胞(MEF)。线粒体！由于MnSOD在CD-/-MEF细胞质中的积累，因此被确定为可能的底物。

项目成果

Shibata M. et al.: "Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation"Biochem. Biophys. Res. Commun.. 251・1. 199-203 (1998)

Shibata M.等人：“血清剥夺后组织蛋白酶B和D参与PC12细胞的凋亡”Biochem.Res. 199-203。

Koike M. et al.: "Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons"J Neurosci.. 20・18. 6898-6906 (2000)

小池 M. 等：“组织蛋白酶 D 缺乏诱导小鼠 CNS 神经元中蜡样脂褐质的溶酶体储存”J Neurosci.. 20・18 (2000)。

Ohsawa Y. et al.: "Ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special refference to autophagy and Iysosomal cathepsins"Arch. Histol. Cytol.. 61・5. 395-403 (1998)

Ohsawa Y. 等：“血清剥夺诱导的 PC12 细胞凋亡的超微结构和免疫组织化学研究，特别涉及自噬和溶酶体组织蛋白酶”Cytol.. 61・5。

Isahara K. et al.: "A novel pathway for neuronal cell death regulated by lysosomal cathepsins"ISHIYAKU PUBLISHERS, INC. The regulation of neural cell feath. 5 (1998)

Isahara K.等人：“溶酶体组织蛋白酶调节神经细胞死亡的新途径”ISHIYAKU PUBLISHERS, INC. 神经细胞羽毛的调节。

Kanamori S. et al.: "Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells"Biochem. Biophys. Res. Commun.. 251・1. 204-208 (1998)

Kanamori S.等人：“阳离子依赖性甘露糖6-磷酸受体的过度表达可防止PC12细胞中血清剥夺诱导的细胞死亡”Biochem.Biophys Res.251·208。