Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
Neurobiol Dis. 2010 Apr;38(1):104-15. doi: 10.1016/j.nbd.2010.01.004. Epub 2010 Jan 18.
Using APP(NLh)/APP(NLh) x PS-1(P246L)/PS-1(P246L) human double knock-in (APP/PS-1) mice, we examined whether phosphatidylserine (PtdSer) asymmetry is significantly altered in brain of this familial Alzheimer disease mouse model in an age-dependent manner as a result of oxidative stress, toxic Abeta(1-42) oligomer production, and/or apoptosis. Annexin V (AV) and NBD-PS fluorescence in synaptosomes of wild-type (WT) and APP/PS-1 mice were used to determine PtdSer exposure with age, while Mg(2+) ATPase activity was determined to correlate PtdSer asymmetry changes with PtdSer translocase, flippase, activity. AV and NBD-PS results demonstrated significant PtdSer exposure beginning at 9 months compared to 1-month-old WT controls for both assays, a trend that was exacerbated in synaptosomes of APP/PS-1 mice. Decreasing Mg(2+) ATPase activity confirms that the age-related loss of PtdSer asymmetry is likely due to loss of flippase activity, more prominent in APP/PS-1 brain. Two-site sandwich ELISA on SDS- and FA-soluble APP/PS-1 brain fractions were conducted to correlate Abeta(1-40) and Abeta(1-42) levels with age-related trends determined from the AV, NBD-PS, and Mg(2+) ATPase assays. ELISA revealed a significant increase in both SDS- and FA-soluble Abeta(1-40) and Abeta(1-42) with age, consistent with PtdSer and flippase assay trends. Lastly, because PtdSer exposure is affected by pro-apoptotic caspase-3, levels of both latent and active forms were measured. Western blotting results demonstrated an increase in both active fragments of caspase-3 with age, while levels of pro-caspase-3 decrease. These results are discussed with relevance to loss of lipid asymmetry and consequent neurotoxicity in brain of subjects with Alzheimer disease.
使用 APP(NLh)/APP(NLh) x PS-1(P246L)/PS-1(P246L) 人双敲入 (APP/PS-1) 小鼠，我们研究了氧化应激、有毒 Abeta(1-42) 寡聚体产生和/或细胞凋亡是否会导致这种家族性阿尔茨海默病小鼠模型的大脑中的磷脂酰丝氨酸 (PtdSer) 不对称性随着年龄的增长而发生显著改变。用膜联蛋白 V (AV) 和 NBD-PS 荧光来测定野生型 (WT) 和 APP/PS-1 小鼠突触小体中的 PtdSer 暴露情况随年龄的变化，同时用 Mg(2+) ATP 酶活性来关联 PtdSer 不对称性变化与 PtdSer 转位酶、翻转酶的活性。AV 和 NBD-PS 的结果表明，与 1 个月龄的 WT 对照组相比，两种检测方法中，9 个月龄的 APP/PS-1 小鼠的 PtdSer 暴露均显著增加，这种趋势在 APP/PS-1 小鼠的突触小体中更为明显。Mg(2+) ATP 酶活性的降低证实，与年龄相关的 PtdSer 不对称性的丧失可能是由于翻转酶活性的丧失，而在 APP/PS-1 脑中更为明显。对 SDS 和 FA 可溶性 APP/PS-1 脑部分进行双位点夹心 ELISA，以将 Abeta(1-40) 和 Abeta(1-42) 水平与从 AV、NBD-PS 和 Mg(2+) ATP 酶测定中确定的与年龄相关的趋势相关联。ELISA 显示 SDS 和 FA 可溶性 Abeta(1-40) 和 Abeta(1-42) 随年龄的增长呈显著增加，与 PtdSer 和翻转酶测定趋势一致。最后，由于 PtdSer 暴露受促凋亡半胱天冬酶-3 的影响，因此测量了两种潜伏和活性形式的半胱天冬酶-3 水平。Western blot 结果表明，随着年龄的增长，半胱天冬酶-3 的两种活性片段都增加，而前半胱天冬酶-3 的水平下降。这些结果与阿尔茨海默病患者大脑中脂质不对称性丧失和随之而来的神经毒性的相关讨论。
