MOLECULAR CELL BIOLOGICAL STUDY OF CELL DEATH INDUCED CATHEPSIN D

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。

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.