重庆医科大学,中国科学院生物物理研究所,中国科学院生物物理研究所,中国科学院生物物理研究所,中国科学院生物物理研究所,中国科学院生物物理研究所,重庆医科大学
国家重点基础研究计划(973)(2012CB911004)和国家自然科学基金(31270868, 31200601)资助项目
Department of Biochemistry and Molecular Biology,Chongqing Medical University,Chongqing, China,State Key Laboratory of Brain and Cognitive Sciences,Institute of Biophysics,Chinese Academy of Sciences,Beijing, China,State Key Laboratory of Brain and Cognitive Sciences,Institute of Biophysics,Chinese Academy of Sciences,Beijing, China,State Key Laboratory of Brain and Cognitive Sciences,Institute of Biophysics,Chinese Academy of Sciences,Beijing, China,State Key Laboratory of Brain and Cognitive Sciences,Institute of Biophysics,Chinese Academy of Sciences,Beijing, China,State Key Laboratory of Brain and Cognitive Sciences,Institute of Biophysics,Chinese Academy of Sciences,Beijing, China,Department of Biochemistry and Molecular Biology,Chongqing Medical University,Chongqing, China
This work was supported by grants from National Basic Research Program of China(2012CB911004) and The National Natural Science Foundation of China (31270868, 31200601)
衰老相关的认知损伤,如阿尔茨海默病(AD),是一种缓慢、渐进、不可逆的神经退行性疾病.内源(尿)甲醛浓度与AD病人的认知损害程度呈正相关.但是,在以前研究中所采用的甲醛浓度,通常高于AD患者和老年人的病理和生理浓度.因此,研究长时程低浓度(病理浓度)甲醛对神经类细胞的影响,对于揭示甲醛在AD发生发展过程的作用是非常必要的.本文参照AD患者尿甲醛浓度,采用每隔24 h连续传代接种加甲醛(10 μmol/L)的方式,模拟AD病人尿甲醛浓度,观察长时程甲醛的累积对小鼠神经母瘤细胞(N2a)和原代海马神经元的影响.通过高效液相色谱、细胞活力和乳酸脱氢酶释放检测显示,随着孵育时间的延长,培养基内甲醛浓度积累升高,N2a细胞生长受到抑制,在甲醛孵育的后期,细胞死亡显著增加.全息成像显示,长时程病理浓度甲醛的积累,使细胞厚度增加、面积减小、神经突受损、显著削弱细胞的黏附能力.同样条件下,小鼠原代海马神经元的一级神经突的数目显著降低,表明病理浓度甲醛的积累能够削弱神经元之间的连接.免疫印迹和免疫荧光染色显示,甲醛的累积可以使N2a细胞内Tau蛋白的181位苏氨酸(T181)和396位丝氨酸(S396)磷酸化水平显著上升,可能是甲醛累积导致的神经突减少和形态改变的因素之一.以上结果证明,病理浓度甲醛在细胞生存环境中的累积,可以导致神经细胞损伤,尤其是神经突的异常改变.本文为探索甲醛代谢失调与老年认知损害之间关系和机制提供了新的启示.
Age-related cognitive impairment, for instance Alzheimer’s disease (AD), is a chronic, progressive, neurodegeneration disease. The concentration of endogenous formaldehyde (FA) positively correlates with the severity of cognitive impairments in AD patients. However, the FA concentrations used in the previous studies were usually higher than the physiological and pathological levels in aging people. To elucidate the relationship between FA and the pathogenesis of AD, it is necessary to investigate the effect of long-term exposure of neurons to low concentration of FA, which is consistent with the pathological FA concentration. In this study, we established a cell culture method to simulate the chronic low-concentration FA exposure by using a serial passage strategy. Murine neuroblastoma N2a cells and primary murine hippocampal neurons were exposed to a simulated pathological FA concentration referred to AD patients. During the long-term of culture, FA gradually accumulated in the medium and impaired N2a cells. High performance liquid chromatography, cell viability assay and lactate dehydrogenase assay showed that, the FA-elimination capacity of N2a cell decreases with the incubation time, accompanied with inhibition of cell growth and increase in cell death. Holographic microscopy showed that long-term simulated pathological FA exposure attenuated the cells’ adhesive morphology. Cells exposed to FA became thicker, exhibiting impairment of neuronal processes. The number of primary neurites in primary hippocampal neurons were reduced by FA exposure, suggesting a decrease in the connectivity between neurons. Formaldehyde accumulation promoted Tau phosphorylation at its Thr181 and Ser396 epitopes, which may be one of the factors leading to decrease in primary neurites. Our findings indicate that accumulation of simulated pathological concentration of FA impairs neurons, induces Tau hyperphosphrylation and decreases neural connectivity, which would lead to neural dysfunction and eventually contribute to the pathogenesis of age-related cognitive impairment.
王腈,周均,莫炜川,赫英舸,魏艳,赫荣乔,易发平.病理浓度甲醛的累积导致小鼠神经母瘤细胞活力及黏附能力下降[J].生物化学与生物物理进展,2017,44(7):601-614
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