Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark.
J Biol Chem. 2013 Jun 14;288(24):17313-35. doi: 10.1074/jbc.M112.401174. Epub 2013 Apr 29.
Aggregation of α-synuclein can be promoted by the tubulin polymerization-promoting protein/p25α, which we have used here as a tool to study the role of autophagy in the clearance of α-synuclein. In NGF-differentiated PC12 catecholaminergic nerve cells, we show that de novo expressed p25α co-localizes with α-synuclein and causes its aggregation and distribution into autophagosomes. However, p25α also lowered the mobility of autophagosomes and hindered the final maturation of autophagosomes by preventing their fusion with lysosomes for the final degradation of α-synuclein. Instead, p25α caused a 4-fold increase in the basal level of α-synuclein secreted into the medium. Secretion was strictly dependent on autophagy and could be up-regulated (trehalose and Rab1A) or down-regulated (3-methyladenine and ATG5 shRNA) by enhancers or inhibitors of autophagy or by modulating minus-end-directed (HDAC6 shRNA) or plus-end-directed (Rab8) trafficking of autophagosomes along microtubules. Finally, we show in the absence of tubulin polymerization-promoting protein/p25α that α-synuclein release was modulated by dominant mutants of Rab27A, known to regulate exocytosis of late endosomal (and amphisomal) elements, and that both lysosomal fusion block and secretion of α-synuclein could be replicated by knockdown of the p25α target, HDAC6, the predominant cytosolic deacetylase in neurons. Our data indicate that unconventional secretion of α-synuclein can be mediated through exophagy and that factors, which increase the pool of autophagosomes/amphisomes (e.g. lysosomal disturbance) or alter the polarity of vesicular transport of autophagosomes on microtubules, can result in an increased release of α-synuclein monomer and aggregates to the surroundings.
α-突触核蛋白的聚集可以被微管蛋白聚合促进蛋白/p25α 促进，我们在这里将其用作研究自噬在清除 α-突触核蛋白中的作用的工具。在 NGF 分化的 PC12 儿茶酚胺能神经细胞中，我们表明新表达的 p25α 与 α-突触核蛋白共定位，并导致其聚集和分布到自噬体中。然而，p25α 也降低了自噬体的流动性，并通过阻止其与溶酶体融合来阻止自噬体的最终成熟，从而最终降解 α-突触核蛋白，从而阻碍了自噬体的最终成熟。相反，p25α 导致基础水平的 α-突触核蛋白分泌到培养基中增加了 4 倍。分泌严格依赖于自噬作用，可以通过自噬的增强剂（海藻糖和 Rab1A）或抑制剂（3-甲基腺嘌呤和 ATG5 shRNA）或通过调节微管上自噬体的负向（HDAC6 shRNA）或正向（Rab8）运输来上调或下调。最后，我们在没有微管蛋白聚合促进蛋白/p25α 的情况下表明，已知调节晚期内体（和胞内体）元件胞吐作用的 Rab27A 的显性突变体调节 α-突触核蛋白的释放，并且溶酶体融合阻断和 α-突触核蛋白的分泌可以通过 p25α 靶标，即主要存在于神经元中的细胞质脱乙酰酶 HDAC6 的敲低来复制。我们的数据表明，α-突触核蛋白的非典型分泌可以通过外噬作用介导，并且增加自噬体/胞内体池的因素（例如溶酶体干扰）或改变微管上自噬体囊泡运输的极性，会导致α-突触核蛋白单体和聚集体向周围环境的释放增加。
