Department of Neurosurgery, First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Eur Rev Med Pharmacol Sci. 2017 Dec;21(24):5717-5728. doi: 10.26355/eurrev_201712_14018.
Apolipoprotein E (APOE) gene polymorphism is correlated closely with resistance to brain damage. This study aims to investigate the effects of APOE4 on oxidative stress damaged cerebral cortical neuron.
Primary cerebral cortical neurons were isolated from APOE gene knock-out mice (APOE-/- mice). Oxidative stress damaged APOE-/- mouse cerebral cortical neuron model was established. Three experimental designs (experiment 1, 2, 3) were conducted by employing several methods. Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) analysis were employed for neurotoxicity assessment. Flow cytometry and transferase-mediated deoxyuridine-triphosphate-biotin nick end labeling (TUNEL) were used to examine neuron apoptosis. Immunohistochemistry and Nissl staining were used to identify neuron morphology. Western blot was used to detect phosphorylated CaMK II (p-CaMK II) and cleaved caspase 3 expression. Ca2+ levels in neurons were also examined by detecting fluorescence intensity.
APOE4 treatment (Vehicle + APOE4) significantly aggravates oxidative stress damaged cerebral cortical neuron by increasing LDH levels and decreasing SOD activities, induces neuron apoptosis compared to Vehicle group (p < 0.05). APOE4 treatment significantly enhanced Ca2+ levels compared to Sham group (p < 0.05), MK801 treatment (Vehicle + APOE4 + MK801) significantly decreased Ca2+ levels compared to the Vehicle+APOE4 group at 12 h and 24 h (p < 0.05). APOE4 triggers CaMK II phosphorylation, caspase 3 activation and neurons apoptosis. Both of MK801 and KN93 inhibit CaMK II phosphorylation, decreases caspase 3 activation, and suppresses neurons apoptosis CONCLUSIONS: APOE triggers Ca2+ overload through NMDAR and CaMK II signaling pathway, both of which cause Ca2+ concentration increasing, CaMK II phosphorylation abnormity, and finally aggravate oxidative stress damaged neurons apoptosis.
载脂蛋白 E（APOE）基因多态性与脑损伤抵抗密切相关。本研究旨在探讨 APOE4 对氧化应激损伤大脑皮质神经元的影响。
从 APOE 基因敲除小鼠（APOE-/- 小鼠）中分离原代大脑皮质神经元。建立 APOE-/- 小鼠大脑皮质神经元氧化应激损伤模型。通过几种方法进行了三个实验设计（实验 1、2、3）。乳酸脱氢酶（LDH）和超氧化物歧化酶（SOD）分析用于评估神经毒性。流式细胞术和转移酶介导的脱氧尿苷三磷酸生物素缺口末端标记（TUNEL）用于检测神经元凋亡。免疫组织化学和尼氏染色用于鉴定神经元形态。Western blot 用于检测磷酸化钙调蛋白激酶 II（p-CaMK II）和裂解的半胱天冬酶 3 的表达。还通过检测荧光强度来检测神经元内的 Ca2+水平。
APOE4 处理（载体+APOE4）通过增加 LDH 水平和降低 SOD 活性，与载体组相比，显著加重氧化应激损伤的大脑皮质神经元，导致神经元凋亡（p<0.05）。与 Sham 组相比，APOE4 处理组 Ca2+水平显著升高（p<0.05），MK801 处理组（载体+APOE4+MK801）在 12 小时和 24 小时时，Ca2+水平显著低于载体+APOE4 组（p<0.05）。APOE4 触发 CaMK II 磷酸化、半胱天冬酶 3 活化和神经元凋亡。MK801 和 KN93 均可抑制 CaMK II 磷酸化，降低半胱天冬酶 3 活化，抑制神经元凋亡。
APOE 通过 NMDA 受体和 CaMK II 信号通路引发 Ca2+过载，导致 Ca2+浓度增加、CaMK II 磷酸化异常，最终加重氧化应激损伤的神经元凋亡。
