MOLECULAR CELL BIOLOGICAL STUDY OF CELL DEATH INDUCED CATHEPSIN D

为了分析由溶酶体组织蛋白酶B和D调节的细胞死亡途径，我们使用组织蛋白酶D缺陷（CD-/-）小鼠。CD-/-小鼠在接近终末阶段[约出生后第26天（P）]表现出癫痫发作并失明。因此，我们研究了这些小鼠中枢神经系统组织的形态学，免疫细胞化学和生化特征。通过电子显微镜，自噬体/自溶小体样体含有部分细胞质，颗粒状嗜锇沉积物，和指纹图谱中的CD-/-小鼠脑神经元胞体P20后demostrated。线粒体ATP合酶C亚单位被发现积累在神经元的溶酶体中，虽然三肽基肽酶-I的活性显着增加的大脑。这些结果表明，在CD-/-小鼠的CNS神经元表现出一种新的形式的溶酶体积累疾病的表型类似神经元蜡样脂褐质沉积症。此外，CD-/-小鼠的视网膜层变得比对照同窝小鼠的视网膜层更薄，特别是，感光细胞层在很大程度上被消除，外核层中TUNEL阳性细胞核的数量增加。为了鉴定CD特异性裂解的底物，我们使用CD-/-小鼠胚胎成纤维细胞（MEF）。线粒体！MnSOD被确定为可能的底物，因为它在CD-/-MEF的细胞质中积累。

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.