Department of Medical Sciences, Sections of Medical Genetics, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.
Neurochem Int. 2013 Aug;63(2):112-20. doi: 10.1016/j.neuint.2013.05.005. Epub 2013 May 27.
Primary cortical neurons exposed to the mitochondrial toxin NaN3 (0.1-3 mM) were submitted to oxidative stress with H2O2 (30-150 μM), to mimic conditions observed in neurodegenerative disorders. The effects of such treatment on a series of parameters useful in characterizing neuronal damage were investigated: (i) the basal release of glutamate, evaluated as (3)H-d-Aspartate efflux, was sharply, concentration-dependently, increased; (ii) the phosphorylation status of intracellular markers known to be involved in the neurodegenerative processes, in particular in Alzheimer disease: tau and GSK3β were increased, as well as the protein level of β-secretase (BACE1) and p35/25 evaluated by Western blotting, while (iii) the cell metabolic activity, measured with the MTT method, was reduced, in a concentration- and time-dependent manner. The latter effect, as well as tau hyperphosphorylation, was prevented both by a mixture of antioxidant drugs (100 μM ascorbic acid, 10 μM trolox, 100 μM glutathione) and by the anti-Alzheimer drug, memantine, 20 μM. Since it is well known that hippocampal cholinergic neurons are particularly affected in Alzheimer disease, the effects of NaN3 and H2O2 were also studied in electrically stimulated rat hippocampal slices, evaluating the (3)H-Choline efflux, as an index of acetylcholine release. The neurotoxic treatment depressed the neurosecretory function and the mixture of antioxidant drugs, as well as memantine, were able to restore it. The neuronal damage induced by the in vitro protocol adopted in the present work displays peculiarities of neurodegenerative disorders, e.g. Alzheimer disease, underlining the role of mitochondrial failure and oxidative stress, which appear to occur upstream the neurodegenerative process; such protocol could be utilized to test the efficacy of neuroprotective treatments.
原代皮质神经元暴露于线粒体毒素 NaN3（0.1-3mM）中，并用 H2O2（30-150μM）引发氧化应激，以模拟神经退行性疾病中观察到的情况。研究了这种处理对一系列用于描述神经元损伤的有用参数的影响：（i）谷氨酸的基础释放，以（3）H-d-天冬氨酸外排来评估，呈浓度依赖性急剧增加；（ii）参与神经退行性过程的细胞内标志物的磷酸化状态增加，特别是在阿尔茨海默病中：tau 和 GSK3β 增加，以及通过 Western blot 评估的β-分泌酶（BACE1）和 p35/25 的蛋白水平增加，而（iii）细胞代谢活性，用 MTT 法测量，呈浓度和时间依赖性降低。后一种效应以及 tau 过度磷酸化，通过抗氧化药物混合物（100μM 抗坏血酸、10μM trolox、100μM 谷胱甘肽）和抗阿尔茨海默病药物美金刚（20μM）预防。由于众所周知，在阿尔茨海默病中，海马胆碱能神经元特别受到影响，因此还研究了 NaN3 和 H2O2 在电刺激大鼠海马切片中的作用，评估（3）H-胆碱的外排，作为乙酰胆碱释放的指标。神经毒性处理抑制了神经分泌功能，抗氧化药物混合物以及美金刚能够恢复其功能。本工作中采用的体外方案诱导的神经元损伤具有神经退行性疾病的特点，例如阿尔茨海默病，强调了线粒体衰竭和氧化应激的作用，它们似乎发生在神经退行性过程之前；该方案可用于测试神经保护治疗的疗效。
