Gandy Sam, Zhang Yun-wu, Ikin Annat, Schmidt Stephen D, Bogush Alexey, Levy Efrat, Sheffield Roxanne, Nixon Ralph A, Liao Francesca-Fang, Mathews Paul M, Xu Huaxi, Ehrlich Michelle E
Farber Institute for Neurosciences and the Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
J Neurochem. 2007 Aug;102(3):619-26. doi: 10.1111/j.1471-4159.2007.04587.x.
Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42.
对连续培养细胞的研究表明，家族性阿尔茨海默病（FAD）突变型早老素1（PS1）会延迟淀粉样前体蛋白（APP）从反式高尔基体网络（TGN）的输出。在此，我们首次报道了PS1调节培养的脑神经元以及体内APP转运的情况。利用来自转基因小鼠的神经元或源自这些神经元TGN的无细胞APP转运囊泡生物发生系统，我们证明敲入与FAD相关的突变型PS1转基因与APP到达细胞表面的动力学延迟有关。显然，这种延迟至少部分归因于APP从TGN的输出受损，这在无细胞APP转运囊泡生物发生试验中得到了证实。为了将研究扩展到体内APP和羧基末端片段（CTF）向脑神经元的转运，我们对APP转基因小鼠的脑进行了亚细胞分级分离，其中一些小鼠携带了第二个编码与FAD相关的突变形式PS1的转基因。FAD突变型PS1的存在与全长APP和APP CTFs的亚细胞定位向富含TGN标志物的碘克沙醇密度梯度级分略有偏移有关。在一组平行实验中，我们使用APP：弗林蛋白酶嵌合蛋白策略来测试人为增加APP：弗林蛋白酶嵌合体的TGN浓度的效果，该嵌合体可能是β-和γ-切割的底物。与野生型APP相比，这种嵌合底物产生了过量的Aβ42。这些数据表明，与FAD相关的突变型人PS1转基因的存在与脑神经元中APP和APP CTFs向富含TGN的级分重新分布有关。嵌合体实验表明，β-/γ-分泌酶底物的TGN富集可能在突变型PS1提高脑内Aβ42水平的作用中起重要作用。
