APP代谢通路关键基因在非洲爪蛙早期胚胎中的时空表达

Temporal and spatial expression of amyloid β precursor protein(APP) in Xenopus Laevis

目的:Aβ的沉积是形成老年斑的主要原因,同时是阿尔茨海默病的主要病理标志物。Aβ形成来源于该病的主要致病基因淀粉样前体蛋白APP(Amyloid beta precursor protein;XB-GENE-479158;NCBI:Xl.8671),APP在非洲爪蛙早期胚胎时空表达图式目前还不清楚,之前的报道只在某些手术分离的组织中用RT-PCR的方法进行了研究,为进一步在爪蛙中研究这类基因在发育过程中的时空表达及可能的功能,展开了本研究。方法:本研究收集了第2期到第4期非洲爪蛙胚胎包括(卵裂期、囊胚期、原肠胚期、神经轴胚、尾牙期和蝌蚪期),通过整体胚胎原位杂交和半定量RT-PCR检测基因APP在胚胎发育不同时期的时空表达,用双荧光素酶报告基因系统检测APP启动子活性。结果:APP有2种主要的转录产物,短的693~694个氨基酸,长的有750~751个氨基酸,主要差别来自外显子7的可变剪切。其中短的剪切体是主要转录的产物,而长的一个剪切体则表现为持续的低表达。整胚的原位表达结果显示:在神经板阶段,APP主表达在孵化和黏液腺中。神经管闭合后,APP转录水平升高,在体细胞中胚层,前肾和背侧神经管中呈现高表达。在尾芽末期,APP在晶状体、端脑、松果体、间脑和背侧耳泡中表达。在蝌蚪期,APP主要在包括胰腺、胃和十二指肠在内的消化系统中高表达。结论:APP是一个母源性表达的基因,其时空表达分布的特异性预示其在相关部位发育过程中可能发挥重要作用,从而为研究阿尔茨海默病的关键致病基因在胚胎早期的所发挥的作用提供了新的路径。

Objective：Amyloid β protein（AI3） is the central component of neuritic plaques, and also the main neuropathological marker of Alzheimer＇ s Disease （AD）. Aβ is derived from the amyloid β precursor protein （APP, amyloid beta precursor protein; XB-GENE- 479158;NCBI：X1.8671） ,the main pathogenic genes of this disease,and the temporal and partial expression patterns of APP in the embryonic period of Xeponus Laevis （X.laevis） has not been clear yet. This study was designed to examine the temporal and spatial expression pattern of APP in the embryonic period of X.laevis and its possible functions. Methods ：The normal embryonic tissues from frogs （at embryonic stages 2 to 4） were collected,and the expression of APP in different stages of embryonic development was detected by the whole-mount in situ hybridization and RT-PCR. The promoter activity was measured by the dual-reporter assays, and the Western blot analysis was used to determine the protein expression. Results：Our data revealed that the APP gene mainly had two types of mRNA variants：shorter 693-694aa and longer 750-751aa protein products,and the main difference was the alternative splicing of exon 7. All the APP transcripts were maternally ex- pressed, and the shorter variant expressed predominantly, with a steadily increased expression level during the development, while the longer variant expressed weakly,but maintained a constant level during the whole embryonic stage. Results of the whole-mount in situ hybridization showed that the APP mainly expressed in hatching and cement gland at neural plate stage, and after the closure of neural tube, the transcript level of APP elevated, presenting high expression in somatic mes0derm, pronephros and dorsal neural tube. At late tail-bud stage,the APP expressed in lens, telencephalon, epiphysis, diencephalon and dorsal otic vesicle. At tadpole stage,the APP highly expressed in the digestive system,including pancreas, stomach and duodenum. Conclusion ：The speci- ficity in the distribution of temporal and partial expression of APP,a maternally expressed gene,indicates the important role it plays in embryonic development, thus providing a new path to study the role of key pathogenic genes of AD in early embryogenesis.