Iron overload in the brain, defined as excess stores of iron, is known to be associated with neurological disorders. In neurodegeneration accompanied by brain iron accumulation, we reported a specific point mutation, c.974-1G>A in WD Repeat Domain 45 (WDR45), showing iron accumulation in the brain, and autophagy defects in the fibroblasts. In this study, we investigated whether fibroblasts with mutated WDR45 accumulated iron, and other effects on cellular organelles. We first identified the main location of iron accumulation in the mutant fibroblasts and then investigated the effects of this accumulation on cellular organelles, including lipid droplets, mitochondria and lysosomes. Ultrastructure analysis using transmission electron microscopy (TEM) and confocal microscopy showed structural changes in the organelles. Increased numbers of lipid droplets, fragmented mitochondria and increased numbers of lysosomal vesicles with functional disorder due to WDR45 deficiency were observed. Based on correlative light and electron microscopy (CLEM) findings, most of the iron accumulation was noted in the lysosomal vesicles. These changes were associated with defects in autophagy and defective protein and organelle turnover. Gene expression profiling analysis also showed remarkable changes in lipid metabolism, mitochondrial function, and autophagy-related genes. These data suggested that functional and structural changes resulted in impaired lipid metabolism, mitochondrial disorder, and unbalanced autophagy fluxes, caused by iron overload.

Mammals conserve multiple mammalian Atg8-family proteins (mATG8s) consisting of GABARAP (GABA type A receptor-associated protein) and MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) subfamilies that tightly bind to autophagic membranes in a membrane-anchored form. These proteins are crucial for selective autophagy and recruit proteins bearing LC3-interacting region (LIR) motifs. However, because limited research tools are available, information on the specific roles of each membrane-anchored mATG8 in selective autophagy is scarce. In this study, we identified LIR motifs specific to the membrane-anchored form of each mATG8 and characterized the residues critical for their selective interaction using cell-based assays and structural analyses. We then used these selective LIR motifs to develop probes and irreversible deconjugases that targeted selective membrane-anchored mATG8s in the autophagic membrane, revealing that membrane-anchored GABARAP subfamily proteins regulate the aggrephagy of amyotrophic lateral sclerosis-linked protein aggregates. Our tools will be useful for elucidating the functional significance of each mATG8 protein on autophagic membranes in autophagy research.

The autophagy-lysosomal pathway is an essential cellular mechanism that degrades aggregated proteins and damaged cellular components to maintain cellular homeostasis. Here, we identified HEXA-018, a novel compound containing a catechol derivative structure, as a novel inducer of autophagy. HEXA-018 increased the LC3-I/II ratio, which indicates activation of autophagy. Consistent with this result, HEXA-018 effectively increased the numbers of autophagosomes and autolysosomes in neuronal cells. We also found that the activation of autophagy by HEXA-018 is mediated by the AMPK-ULK1 pathway in an mTOR-independent manner. We further showed that ubiquitin proteasome system impairment- or oxidative stress-induced neurotoxicity was significantly reduced by HEXA-018 treatment. Moreover, oxidative stress-induced mitochondrial dysfunction was strongly ameliorated by HEXA-018 treatment. In addition, we investigated the efficacy of HEXA-018 in models of TDP-43 proteinopathy. HEXA-018 treatment mitigated TDP-43 toxicity in cultured neuronal cell lines and Drosophila. Our data indicate that HEXA-018 could be a new drug candidate for TDP-43-associated neurodegenerative diseases.

The process of cellular senescence, which is characterized by stable cell cycle arrest, is strongly associated with dysfunctional cellular metabolism and circadian rhythmicity, both of which are reported to result from and also be causal to cellular senescence. As a result, modifying any of them—senescence, metabolism, or the circadian clock—may affect all three simultaneously. Obesity accelerates aging by disrupting the homeostasis of reactive oxygen species (ROS) via an increased mitochondrial burden of fatty acid oxidation. As a result, if senescence, metabolism, and circadian rhythm are all linked, anti-obesity treatments may improve metabolic regulation while also alleviating senescence and circadian rhythm. Vutiglabridin is a small molecule in clinical trials that improves obesity by enhancing mitochondrial function. We found that chronic treatment of senescent primary human dermal fibroblasts (HDFs) with vutiglabridin alleviates all investigated markers of cellular senescence (SA-β-gal, CDKN1A, CDKN2A) and dysfunctional cellular circadian rhythm (BMAL1) while remarkably preventing the alterations of mitochondrial function and structure that occur during the process of cellular senescence. Our results demonstrate the significant senescence-alleviating effects of vutiglabridin, specifically with the restoration of cellular circadian rhythmicity and metabolic regulation. These data support the potential development of vutiglabridin against aging-associated diseases and corroborate the intricate link between cellular senescence, metabolism, and the circadian clock.