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

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.

Mutation of the plastidial alpha-glucan phosphorylase gene in rice affects the synthesis and structure of starch in the endosperm.

Plastidial phosphorylase (Pho1) accounts for approximately 96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30 degrees C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20 degrees C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.