A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.

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-ß Transforming growth factor beta; ULK1 unc-51 like autophagy activating kinase 1; XBP1 X-box binding protein 1.