Effects of Targeted Suppression of Glutaryl-CoA Dehydrogenase by Lentivirus-Mediated shRNA and Excessive Intake of Lysine on Apoptosis in Rat Striatal Neurons
Effects of Targeted Suppression of Glutaryl-CoA Dehydrogenase by Lentivirus-Mediated shRNA and Excessive Intake of Lysine on Apoptosis in Rat Striatal Neurons
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DOI：
10.1371/journal.pone.0063084
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发表时间：
2013-05-02
期刊：
影响因子：
3.7
通讯作者：
Luo, Xiaoping
中科院分区：
文献类型：
--
作者：
Gao, Jinzhi;Zhang, Cai;Luo, Xiaoping
在1型谷氨酸尿症（GA 1）中，戊二酰辅酶A脱氢酶（GCDH）缺乏已被证明是导致谷氨酸蓄积和纹状体变性的原因。然而，GA 1诱导纹状体变性的机制仍不清楚。在这项研究中，我们的目的是建立一个新的神经元模型的GA 1和研究GCDH缺乏和赖氨酸相关代谢产物对大鼠纹状体神经元活力的影响。因此，我们构建了一个慢病毒载体包含短发夹RNA靶向GCDH基因表达（慢病毒-shRNA）在神经元中。含有乱序短发夹RNA构建体的病毒作为对照。添加赖氨酸（5 mmol/L）用于模拟高代谢。使用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物测量细胞活力。采用Hoechst 33342染色和Annexin V-PE/7-AAD染色评估细胞凋亡。线粒体膜电位（MPP）的监测使用四甲基罗丹明甲酯。Western blotting检测caspase 3、8、9的表达水平。我们发现，慢病毒-shRNA诱导神经细胞凋亡和MMP水平降低，并添加5 mmol/L赖氨酸增强这种效果显着。Lentivirus-shRNA上调caspase 3和9的蛋白水平，无论是否存在5 mmol/L赖氨酸。5 mmol/L赖氨酸与慢病毒- shRNA共处理组caspase 8的表达水平高于对照组。半胱天冬酶抑制剂苄氧羰基-Val-Ala-Asp（OMe）-氟甲基酮（fluoromethylketone）在很大程度上阻断了慢病毒-shRNA和5 mmol/L赖氨酸诱导的细胞凋亡。这些结果表明，靶向抑制GCDH的慢病毒介导的shRNA和过量摄入赖氨酸可能是一个有用的细胞模型的GA 1。这些也表明GA 1诱导的纹状体变性是部分半胱天冬酶依赖的。
In glutaric aciduria type 1 (GA1), glutaryl-CoA dehydrogenase (GCDH) deficiency has been shown to be responsible for the accumulation of glutaric acid and striatal degeneration. However, the mechanisms by which GA1 induces striatal degeneration remain unclear. In this study, we aimed to establish a novel neuronal model of GA1 and to investigate the effects of GCDH deficiency and lysine-related metabolites on the viability of rat striatal neurons. Thus we constructed a lentiviral vector containing short hairpin RNA targeted against the GCDH gene expression (lentivirus-shRNA) in neurons. A virus containing a scrambled short hairpin RNA construct served as a control. Addition of lysine (5 mmol/L) was used to mimic hypermetabolism. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis was assessed using Hoechst33342 staining and Annexin V-PE/7-AAD staining. The mitochondrial membrane potential (MPP) was monitored using tetramethylrhodamine methyl ester. The expression levels of caspases 3, 8, and 9 were determined by Western blotting. We found that lentivirus-shRNA induced apoptosis and decreased MMP levels in neurons, and addition of 5 mmol/L lysine enhanced this effect markedly. Lentivirus-shRNA upregulated the protein levels of caspases 3 and 9 regardless of the presence of 5 mmol/L lysine. The expression level of caspase 8 was higher in neurons co-treated with lentivirus- shRNA and 5 mmol/L lysine than in control. Benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and 5 mmol/L lysine to a great extent. These results indicate that the targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine may be a useful cell model of GA1. These also suggest that GA1-induced striatal degeneration is partially caspase-dependent.