Aβ对阿尔茨海默病模型小鼠脑神经元及小胶质细胞内溶酶体的影响

Effects of Aβ on lysosomes in neurons and microglia of Alzheimer’s disease model mice

摘要:

目的： 探讨β-淀粉样蛋白（amyloid β peptide，Aβ）对阿尔茨海默病（Alzheimer’s disease，AD）模型小鼠脑神经元及小胶质细胞内溶酶体的影响。方法： 取6月龄APP/PS1双转基因AD模型小鼠和同窝野生型小鼠各6只，免疫荧光染色观察脑内神经元和小胶质细胞表达以及和溶酶体共表达情况；Western blot检测小鼠脑内自噬相关蛋白表达。用Aβ25-35处理SH-SY5Y细胞后，转录因子EB（transcription factor EB，TFEB）过表达腺病毒干预细胞72 h，Western blot检测TFEB和溶酶体标志物水平。结果： 与野生型组小鼠比较，6月龄AD转基因组小鼠LC3、p62蛋白表达增加（t=4.976，P=0.008；t=4.463，P=0.011），溶酶体标志蛋白LAMP1明显减少（t=14.91，P=0.000 1）。AD模型组小鼠海马区神经元明显丢失，小胶质细胞明显增生，溶酶体表达均明显减少。与野生型组小鼠比较，AD转基因组小鼠磷酸化哺乳动物雷帕霉素靶蛋白（p-mammalian target of rapamycin，p-mTOR）/mTOR蛋白表达量明显升高（t=5.501，P=0.005），TFEB蛋白表达水平明显降低（t=5.899，P=0.004）。Aβ25-35处理使SH-SY5Y细胞TFEB和Lamp1蛋白水平均下调（t=10.94，P<0.001；t=17.22，P<0.001），过表达TFEB后，溶酶体标志物Lamp1蛋白水平明显升高（t=5.136，P<0.01）。结论： Aβ引起AD转基因小鼠脑内神经元和小胶质细胞内溶酶体均减少，可能通过抑制mTOR/TFEB信号通路发挥作用。

Abstract:

Objective: To investigate the effect of Aβ on lysosomes in neurons and microglia of Alzheimer’s disease (AD) model mice. Methods: Six 6-month-old APP/PS1 double transgenic AD mice and six wild type (WT) mice were selected to observe the expression of neurons and microglia, as well as the co-expression of neurons, microglia and lysosomes by immunofluorescence staining. Western blot was used to detect the expression of autophagy related proteins. After SH-SY5Y cells were treated with Aβ25-35, adenovirus overexpressed transcription factor EB (TFEB) was intervened for 72 h. The levels of TFEB and lysosomal markers were detected by Western blot. Results: Compared with wild-type mice, the expression of LC3 and p62 in AD transgenic mice at 6 months old increased (t=4.976, P=0.008; t=4.463, P=0.011), and the lysosomal marker protein LAMP1 decreased significantly (t=14.91, P=0.000 1). In AD model group, neurons were significantly lost, microglia were significantly proliferated and lysosome expression was significantly decreased. Compared with wild-type mice, the expression of p-mammalian target of rapamycin (p-mTOR) /mTOR protein in AD transgenic mice was significantly increased (t=5.501, P=0.005), and the expression of TFEB protein was significantly decreased (t=5.899, P=0.004). Aβ25-35 down regulated the protein levels of TFEB and Lamp1 in SH-SY5Y cells (t=10.94, P<0.001; t=17.22, P<0.001). Overexpression of TFEB significantly increased the protein level of lysosomal marker Lamp1 (t=5.136, P<0.01). Conclusion: Aβ can decrease lysosomes in neurons and microglia of AD model mice, which may act by inhibiting mTOR/TFEB signaling pathway.