Lahiri D K, Lewis S, Farlow M R
Department of Psychiatry and Neurology, Indiana University School of Medicine, Indianapolis 46202-4887.
J Neurosci Res. 1994 Apr 15;37(6):777-87. doi: 10.1002/jnr.490370612.
The characteristic features of Alzheimer's disease (AD) include a high density of beta-amyloid-containing plaques in the cerebral cortex and the loss of basal forebrain cholinergic neurons. Amyloid beta-protein (A beta, Mr. approximately 4.5 kDa) is derived from a family of large (Mr. approximately 110-140 kDa) beta-amyloid precursor proteins (APP) which are integral membrane glycoproteins consisting of a large extracytoplasmic domain, a transmembrane domain, and a short cytoplasmic tail. Secreted derivatives of APP lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated by the proteolytic processing of full length APP by a family of proteolytic enzymes known as APP secretases. Using cell cultures, we investigated the possibility that APP processing can be regulated by a centrally active cholinesterase inhibitor, tacrine, which has recently been shown to improve memory and cognitive functions in patients with AD. We analyzed the level of APP in glial, fibroblast, pheochromocytoma (PC12), and neuroblastoma cells by immunoblotting cell lysates and conditioned media. Normal levels of secretion of soluble APP derivatives by cells into conditioned media were severely inhibited by treating cells with tacrine. A similar decrease after treatment with tacrine was observed when neuroblastoma and PC12 cells were pretreated with either growth factors, phorbol ester, or retinoic acid. To determine whether the effect of tacrine on APP levels was specific or a more general phenomenon affecting other proteins, we measured the level of heat shock protein-70 (HSP-70) and another secretory protein, protease nexin-1 (PN-1). Tacrine treatment did not alter the level of HSP-70 in cell extracts and tacrine affected mildly the secretion of PN-1. Thus, the processing of HSP and PN-1, unlike APP, was not severely affected by treating cells with tacrine. Our results suggest that tacrine may inhibit an acetylcholinesterase-associated proteolytic activity involved in the secretion of APP, which results in less secretion of soluble APP into the conditioned media from tacrine treated cells. These results demonstrate that tacrine regulates APP secretion in cell cultures and suggest the possibility that tacrine therapy of Alzheimer's disease may, in the longer term, have effects on the process of A beta deposition.
阿尔茨海默病（AD）的特征包括大脑皮层中含有β-淀粉样蛋白的斑块密度高以及基底前脑胆碱能神经元的丧失。淀粉样β蛋白（Aβ，分子量约4.5 kDa）源自一大类（分子量约110 - 140 kDa）的β-淀粉样前体蛋白（APP），这些蛋白是整合膜糖蛋白，由一个大的胞外结构域、一个跨膜结构域和一个短的胞质尾巴组成。APP的分泌衍生物缺乏胞质尾巴、跨膜结构域和一小部分胞外结构域，是由一类称为APP分泌酶的蛋白水解酶对全长APP进行蛋白水解加工产生的。利用细胞培养，我们研究了APP加工过程是否可以被一种中枢活性胆碱酯酶抑制剂他克林调节的可能性，最近的研究表明他克林可以改善AD患者的记忆和认知功能。我们通过免疫印迹细胞裂解物和条件培养基分析了胶质细胞、成纤维细胞、嗜铬细胞瘤（PC12）和神经母细胞瘤细胞中APP的水平。用他克林处理细胞会严重抑制细胞向条件培养基中分泌可溶性APP衍生物的正常水平。当神经母细胞瘤和PC12细胞用生长因子、佛波酯或视黄酸预处理后，再用他克林处理也会观察到类似的下降。为了确定他克林对APP水平的影响是特异性的还是影响其他蛋白质的更普遍现象，我们测量了热休克蛋白-70（HSP-70）和另一种分泌蛋白蛋白酶nexin-1（PN-1）的水平。他克林处理不会改变细胞提取物中HSP-70的水平，并且他克林对PN-1的分泌影响较小。因此，与APP不同，HSP和PN-1的加工过程不会受到他克林处理细胞的严重影响。我们的结果表明，他克林可能抑制了与乙酰胆碱酯酶相关的参与APP分泌的蛋白水解活性，这导致从他克林处理的细胞中分泌到条件培养基中的可溶性APP减少。这些结果表明他克林在细胞培养中调节APP分泌，并提示他克林治疗阿尔茨海默病从长远来看可能对Aβ沉积过程产生影响。
