Department of Immunology and Microbiology, Scripps Florida, The Scripps Research Institute, Jupiter, FL 33458.
Department of Neuroscience, Scripps Florida, The Scripps Research Institute, Jupiter, FL 33458.
Proc Natl Acad Sci U S A. 2018 Mar 13;115(11):E2634-E2643. doi: 10.1073/pnas.1713849115. Epub 2018 Feb 27.
Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.
培养的原代神经元暴露于预先形成的α-突触核蛋白纤维（PFF）中，导致内源性α-突触核蛋白的募集，并将其模板转化为纤维状磷酸化的α-突触核蛋白（pα-synF）聚集体，类似于帕金森病（PD）发病机制中涉及的聚集体。以前曾描述过 Pα-synF 是 PD 患者中的路易体和路易神经突在形态上相似的包含物。我们发现了一种构象上不同的、非纤维状的、磷酸化的α-突触核蛋白物种，我们将其命名为“pα-syn*”。我们独特地描述了 pα-syn* 在 PFF 引发的原代神经元、小鼠大脑和 PD 患者大脑中的存在。通过免疫荧光和药理学操作，我们表明 pα-syn* 是 pα-synF 不完全自噬降解的结果。Pα-synF 被自噬标记物修饰，但 pα-syn* 没有。Western blot 显示 pα-syn* 被 N 和 C 端修剪，导致 12.5 kDa 片段和 SDS 抗性二聚体。溶酶体释放后，pα-syn* 聚集体与线粒体结合，导致线粒体膜去极化、细胞色素 C 释放和线粒体碎片化，通过共聚焦和受激发射损耗纳米显微镜观察到。pα-syn* 招募磷酸化的乙酰辅酶 A 羧化酶 1（ACC1），并与 pα-syn* 显著共定位。ACC1 磷酸化表明低 ATP 水平、AMPK 激活和氧化应激，并通过减少脂酰化诱导线粒体碎片化。pα-syn* 还与 BiP 共定位，BiP 是未折叠蛋白反应的主要调节剂，也是线粒体相关内质网膜的驻留蛋白，线粒体分裂和线粒体自噬的发生部位。通过电子显微镜观察到 pα-syn* 聚集在 Parkin 阳性的噬线粒体空泡中。总之，我们表明 pα-syn* 诱导线粒体毒性和分裂、能量应激和线粒体自噬，表明 pα-syn* 是一种关键的神经毒性α-突触核蛋白物种和治疗靶点。
