González Carolina, Cánovas José, Fresno Javiera, Couve Eduardo, Court Felipe A, Couve Andrés
Program of Physiology and Biophysics, Institute of Biomedical Sciences, Faculty of Medicine, Universidad de Chile, 8380453 Santiago, Chile; Biomedical Neuroscience Institute, Faculty of Medicine, Universidad de Chile, 8380453 Santiago, Chile;
Instituto de Biología, Laboratorio de Microscopía Electrónica, Facultad de Ciencias, Universidad de Valparaíso, 2360102 Valparaíso, Chile;
Proc Natl Acad Sci U S A. 2016 Feb 16;113(7):1823-8. doi: 10.1073/pnas.1514943113. Epub 2016 Feb 2.
The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons.
轴突蛋白质组的调控是产生和维持神经功能的关键。快速和慢速轴浆波已为人所知数十年，但基于局部合成来控制轴突蛋白丰度的其他机制也已被发现。内质网在外周轴突中的存在已有文献记载，但这种局部细胞器是否参与轴突膜蛋白的递送仍不清楚。电压门控钠通道负责动作电位，主要集中在轴突起始段和郎飞结。尽管它们具有重要作用，但关于控制其在成熟轴突中可用性的细胞内运输机制却知之甚少。在这里，我们描述了轴突中的分泌机制及其对钠通道质膜递送的贡献。在体内电穿孔后，评估坐骨神经轴突和脊神经轴突中轴突分泌成分的分布。在体内和体外对细胞内蛋白质运输进行药理学阻断。通过逆转录聚合酶链反应（RT-PCR）和一种保留-释放方法检测轴突电压门控钠通道mRNA和局部运输。我们证明成熟轴突含有内质网和其他生物合成细胞器的成分。轴突细胞器和钠通道定位对内质网到高尔基体运输的局部阻断敏感。更重要的是，分泌细胞器能够在分离的轴突中将钠通道递送至质膜，这表明轴突生物合成途径在调节哺乳动物轴突中的轴突蛋白质组方面具有内在能力。
