Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures
Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures
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胚胎和出生后海马神经元培养物中细胞质和线粒体 ATP 合成的比较分析
DOI：
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发表时间：
2013
影响因子：
4.8
通讯作者：
L. Khiroug
中科院分区：
文献类型：
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作者：
A. Surin;S. Khiroug;L. Gorbacheva;B. Khodorov;V. Pinelis;L. Khiroug
神经元中的ATP通常被认为主要由线粒体通过氧化磷酸化来合成。神经元线粒体主要在培养物中进行研究，即，在从胚胎或新生幼崽中分离的神经元中。虽然一般认为胚胎和出生后培养的神经元都从线粒体氧化磷酸化中获得ATP，但这从未经过实验验证。我们在分离自E17至E18大鼠胚胎或P1至P2大鼠幼仔的培养海马神经元中表达基于FRET的ATP传感器AT1.03，并同时监测[ATP]c与线粒体膜电位（Δ TRM; TMRM）和NAD（P）H自发荧光。在胚胎神经元中，短暂的葡萄糖剥夺诱导[ATP]c几乎完全减少，这是部分可逆的，并通过抑制糖酵解与2-脱氧葡萄糖加速。在没有葡萄糖的情况下，丙酮酸并没有引起84%的胚胎神经元[ATP]c的任何显着增加，并且用寡霉素抑制线粒体ATP合酶也没有降低[ATP]c。此外，寡霉素显著降低了Δ Δ Δ m，表明线粒体在胚胎神经元中充当ATP的消费者而不是生产者。与此形成鲜明对比的是，在出生后的神经元中，在葡萄糖剥夺过程中加入丙酮酸显著增加[ATP]c（54 ± 8%），而寡霉素诱导[ATP]c急剧下降并增加Δ Δ Km。在所有测试的P1-P2神经元中观察到氧化磷酸化的这些迹象。用JC-1电位敏感探针测量Δ m表明，与出生后相比，胚胎培养的神经元线粒体膜电位显著降低，可能是由于线粒体内膜质子渗透性增加。我们的结论是，在胚胎，但不是出生后的神经元培养，ATP的合成主要是糖酵解和线粒体F1 Fo-ATPase的ATP的氧化磷酸化介导的合成是微不足道的。
ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential (ΔΨm; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ΔΨm was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ΔΨm. These signs of oxidative phosphorylation were observed in all tested P1–P2 neurons. Measurement of ΔΨm with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by mitochondrial F1Fo-ATPase is insignificant.
影响因子：
3.4
作者：
Cortassa, S;Aon, MA;O'Rourke, B
通讯作者：
O'Rourke, B