Popescu B O, Cedazo-Minguez A, Popescu L M, Winblad B, Cowburn R F, Ankarcrona M
Karolinska Institute, NEUROTEC, Division of Geriatric Medicine, KFC, NOVUM, Huddinge, Sweden.
J Neurosci Res. 2001 Oct 1;66(1):122-34. doi: 10.1002/jnr.1204.
Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating gamma-secretase activity, a protease involved in beta-amyloid precursor protein processing to the neurotoxic beta-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis.
早老素（PSs）在大多数家族性阿尔茨海默病（FAD）病例中发生突变。突变的PSs可能通过多种促凋亡机制导致FAD，或通过调节γ-分泌酶活性来实现，γ-分泌酶是一种参与β-淀粉样前体蛋白加工成神经毒性β-淀粉样肽的蛋白酶。除了正常的内切蛋白水解加工外，PSs还是半胱天冬酶的底物，可被切割成不同的N端和C端片段。到目前为止，关于PSs切割在凋亡机制中的作用知之甚少。在此，我们使用稳定转染野生型或9号外显子缺失的早老素1（PS1）的SH-SY5Y神经母细胞瘤细胞，在暴露于钙离子载体A23187后的时间进程研究中进行观察。在暴露于A23187期间及之后，与未转染和野生型PS1转染的细胞相比，9号外显子缺失的PS1细胞内的钙水平更高。与对照细胞系相比，9号外显子缺失的PS1过表达增加了A23187暴露后的细胞死亡和凋亡细胞的富集。将野生型PS1细胞与9号外显子缺失的PS1细胞进行比较，并分析PS1与其他半胱天冬酶底物切割之间的时间关系。与野生型PS1细胞相比，9号外显子缺失的PS1细胞表现出更高的半胱天冬酶-3激活以及PS1和聚（ADP-核糖）聚合酶（PARP）的更大程度切割。9号外显子缺失的PS1切割比其他半胱天冬酶底物切割（即PARP和凝溶胶蛋白）更早发生，与可检测到的最低限度半胱天冬酶-3激活同时出现。因此，PS1的选择性切割可能在凋亡过程中激活的蛋白水解级联反应调节中起重要作用。
