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Presenilin-1 Asp385 is Indispensable for Neurogenesis,Angiogenesisand β-amyloid Production
Objective:Presenilin(PS)is one of major causative genes of early onset familial Alzheimer's disease(FAD),which accounts for 95％ of genetic factors. Presenilin-1(PS1)is considered as the catalytic subunit of theγ-secretase complex that cleaves type Ⅰ transmembrane proteins,such as amyloid precursor protein(APP),a protein closely related with AD. One conserved aspartate residue (Asp385)in PS1 is thought to constitute the active site of γ-secretase. In vitro,D385A mutation (the aspartate to alanine alteration)leads to the failure of PS1 endoproteolysis and abolishment ofγ-secretase activity. To further investigate the role of Asp385 residue in PS1 in vivo,we generated PS1 D385A knockin(KI)mice,in which an Asp385Ala(D385A)alteration was introduced in the genomic Psen1 locus. Method:The gene-editing method of Cre-LoxP has been used to generate PS1 D385A KI mice;southern blot has been used to detect the genomic DNA from both embryonic stem(ES)cells and mouse tails to confirm its genotype. Northern and western have been used to test the mRNA and protein levels of PS1. Morphology and the proliferation of neural progenitor cells of developing brains has been test by HE staining and BrdU immunohistochemistry. Angiogenesis has been test by both western and immunofluorescence to target the marker of vascular endothelial cells,CD31. In vitro γ-secretase assay has been used to test the activity of γ-secretase. Results:Homozygous D385A KI(KI/KI)mice display severe developmental deficits,including abnormal skeleton development,perinatal lethality,cerebral hemorrhages,enlarged lateral ventricle and massive loss of neural progenitor cells,which is identical to those of Psen1-null mice. D385A mutation dose not interrupt the level of Psen1 mRNA,but disrupt PS1 endoproteolysis. The N- and C-terminal fragments of PS1 are absent,and PS1 holoprotein accumulates(～18-fold)in KI/KI mice relative to the wild type(WT)controls. In addition,D385A mutation abolishesγ-secretase activity in the brain of D385A KI/KI mice,and the activity is significantly reduced in KI/+ mice. Furthermore,compared with WT mice,the protein level of CD31 is significantly increased in KI/+ mice within development and adulthood. In adult,KI/+ mice also show abnormal angiogenesis in both cortex and hippocampus, and cerebrovascular amyloid plaques have been detected in 10 month age. Conclusion:Collectively, these results demonstrate that the Asp385 of PS1 is indispensable for its function in maintaining normal development,neurogenesis,and γ-secretase activity. Furthermore,D385A mutation causes abnormal angiogenesis and amyloid plaques in vessels in adulthood.
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快速老化小鼠胸腺细胞分化与Notch信号相关基因表达关系的研究
目的:研究快速老化小鼠(Senescence-accelerated mice,SAM)增龄过程中胸腺T细胞亚群的变化与Notch 1、Presenilin 1、2(PS1、2)、HES1等Notch信号转导通路相关基因mRNA表达变化及这两种变化的关系.方法:选择出生后1～8周龄SAM,采用流式细胞技术检测胸腺细胞亚群变化、用荧光实时定量PCR的方法检测Notch 1、PS1、PS2、HES1基因mRNA表达的动态变化.结果:SAM出生1～2周,胸腺中CD4+CD8+细胞所占比例较高,4周龄后逐渐降低,各周龄SAMP8与SAMR1之间无显著性差异;在CD4+CD8-细胞比例上,SAM呈增龄性增加,1～6周龄的SAMP8与SAMR1之间无显著性差异,8周龄的SAMP8低于SAMR1;在CD4-CD8+细胞比例上,SAM呈增龄性增加,4周龄后的SAMP8均明显高于SAMR1;Notch 1、PS2、HES1基因mRNA表达量呈增龄性增加,各周龄SAMP8的Notch 1基因表达水平高于SAMR1,4周龄后SAMP8的PS2、HES1基因表达水平高于SAMR1;PS1基因mRNA表达量呈增龄性降低,4周龄后的SAMP8低于SAMR1.结论:Notch 1、PS2、HES1的表达与胸腺CD4-CD8+细胞的分化呈正相关,PS1表达与胸腺CD4-CD8+细胞的分化呈负相关.
