ABSTRACT

Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of “autophagy profiles” revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.

Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP⁺: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-β: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

摘要

巨自噬/自噬在包括帕金森病 (PD) 和亨廷顿病 (HD) 等神经退行性疾病在内的各种人类疾病的发病机制中起关键作用。化学自噬诱导剂有望通过消除细胞毒性/受损蛋白质作为疾病调节剂。尽管已经鉴定出许多自噬诱导剂，但由于信号通路之间复杂的串扰，它们的精确分子机制尚不完全清楚。为了解决这个问题，我们进行了几项化学基因组分析，使我们能够理解自噬相关途径之间的优势，然后进行侵袭性清除分析。第一步，评估了 400 多个目标确定的小分子在神经元 PC12D 细胞中激活自噬通量的能力，我们确定了 39 种化合物作为自噬诱导剂。然后，我们通过测试它们对 200 种成熟的信号转导调节剂诱导自噬的影响来分析自噬诱导剂。我们使用“自噬谱”数据集进行的主成分分析 (PCA) 和聚类分析表明，美国食品和药物管理局 (FDA) 批准的两种药物美金刚和氯马斯汀可激活内质网 (ER) 应激反应，从而导致自噬就职。我们还证实，最近发现的自噬诱导剂 SMK-17 通过 PRKC/PKC-TFEB 途径诱导自噬，正如 PCA 所预测的那样。最后，我们表明，在本研究中测试的几乎所有自噬诱导剂都显着增强了在 PD 和 HD 细胞模型中观察到的蛋白质聚集体的清除。这些结果，

缩写： ADK：腺苷激酶；AMPK：AMP激活的蛋白激酶；ATF4：激活转录因子 4；BECN1：beclin-1；DDIT3/CHOP：DNA 损伤诱导转录本 3；EIF2AK3/PERK：真核翻译起始因子 2 α 激酶 3；EIF2S1/eIF2α：真核翻译起始因子 2 亚基 α；ER：内质网；ERN1/IRE1α：内质网到核信号 1；FDA：食品药品监督管理局；GSH：谷胱甘肽；高清：亨廷顿病；HSPA5/GRP78：热休克蛋白家族 A (Hsp70) 成员 5；HTT：亨廷顿；JAK：Janus 激酶，MAP1LC3B/LC3：微管相关蛋白 1 轻链 3 β；MAP2K/MEK：丝裂原活化蛋白激酶激酶；MAP3K8/Tpl2：丝裂原活化蛋白激酶激酶激酶 8；MAPK：丝裂原活化蛋白激酶；国会议员⁺：1-甲基-4-苯基吡啶鎓；MTOR：雷帕霉素激酶的机制靶点；MTORC：MTOR复合体；NAC：N-乙酰半胱氨酸；NGF：神经生长因子2；NMDA：N-甲基-D-天冬氨酸；PCA：主成分分析；PD：帕金森病；PDA：胰腺导管腺癌；PIK3C3：磷脂酰肌醇 3-激酶催化亚基 3 型；PMA：佛波醇 12-肉豆蔻酸酯 13-乙酸酯；PRKC/PKC：蛋白激酶 C；ROCK：Rho 相关的卷曲螺旋蛋白激酶；RR：核糖核苷酸还原酶；SIGMAR1：西格玛非阿片类细胞内受体 1；SQSTM1/p62：隔离体 1；STK11/LKB1：丝氨酸/苏氨酸激酶 11；TFEB：转录因子EB；TGFB/TGF-β：转化生长因子β；ULK1：unc-51 样自噬激活激酶 1；XBP1：X-box 结合蛋白 1。