Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain.
Facultad de Ciencias Ambientales y Bioquímica, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla La Mancha, 45071, Toledo, Spain; Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IISFJD), 28049, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049, Madrid, Spain.
Neurochem Int. 2019 Dec;131:104541. doi: 10.1016/j.neuint.2019.104541. Epub 2019 Aug 28.
The elevated energy demands in the brain are fulfilled mainly by glucose catabolism. In highly polarized neurons, about 10-50% of mitochondria are transported along microtubules using mitochondrial-born ATP to locations with high energy requirements. In this report, we have investigated the impact of Aralar deficiency on mitochondrial transport in cultured cortical neurons. Aralar/slc25a12/AGC1 is the neuronal isoform of the aspartate-glutamate mitochondrial carrier, a component of the malate-aspartate shuttle (MAS) which plays an important role in redox balance, which is essential to maintain glycolytic pyruvate supply to neuronal mitochondria. Using live imaging microscopy we observed that the lack of Aralar does not affect the number of moving mitochondria nor the Ca-induced stop, the only difference being a 10% increase in mitochondrial velocity in Aralar deficient neurons. Therefore, we evaluated the possible fuels used in each case by studying the relative contribution of oxidative phosphorylation and glycolysis to mitochondrial movement using specific inhibitors. We found that the ATP synthase inhibitor oligomycin caused a smaller inhibition of mitochondrial movement in Aralar-KO than control neurons, whereas the glycolysis inhibitor iodoacetate had similar effects in neurons from both genotypes. In line with these findings, the decrease in cytosolic ATP/ADP ratio caused by oligomycin was more pronounced in control than in Aralar-KO neurons, but no differences were observed with iodoacetate. Oligomycin effect was reverted by aralar re-expression in knock out cultures. As mitochondrial movement is not reduced in Aralar-KO neurons, these results suggest that these neurons may use an additional pathway for mitochondria movement and ATP/ADP ratio maintenance.
大脑的高能量需求主要通过葡萄糖分解代谢来满足。在高度极化的神经元中，约 10-50%的线粒体使用线粒体产生的 ATP 沿着微管被转运到具有高能量需求的位置。在本报告中，我们研究了 Aralar 缺乏对培养的皮质神经元中线粒体转运的影响。Aralar/slc25a12/AGC1 是天冬氨酸-谷氨酸线粒体载体的神经元同工型，是苹果酸-天冬氨酸穿梭（MAS）的组成部分，MAS 在氧化还原平衡中发挥重要作用，对于维持糖酵解丙酮酸向神经元线粒体的供应至关重要。使用活细胞成像显微镜，我们观察到缺乏 Aralar 不会影响移动线粒体的数量或 Ca 诱导的停止，唯一的区别是 Aralar 缺乏神经元中线粒体速度增加了 10%。因此，我们通过使用特定抑制剂研究氧化磷酸化和糖酵解对线粒体运动的相对贡献来评估每种情况下可能使用的燃料。我们发现，与对照神经元相比，ATP 合酶抑制剂寡霉素对 Aralar-KO 神经元中线粒体运动的抑制作用较小，而糖酵解抑制剂碘乙酸对两种基因型神经元的影响相似。与这些发现一致的是，寡霉素引起的胞质溶胶 ATP/ADP 比率降低在对照神经元中比在 Aralar-KO 神经元中更为明显，但碘乙酸没有差异。在敲除培养物中重新表达 Aralar 可逆转寡霉素的作用。由于 Aralar-KO 神经元中线粒体运动没有减少，这些结果表明这些神经元可能使用了另一种线粒体运动和 ATP/ADP 比率维持的途径。
