ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells.

Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers.

CD44 facilitates adhesive interactions in airineme-mediated intercellular signaling.

Specialized cellular protrusions facilitate local intercellular communications in various species, including mammals. Among these, airinemes play a crucial role in pigment pattern formation in zebrafish by mediating long-distance Notch signaling between pigment cells. Remarkably, airinemes exhibit large vesicle-like structure at their tips, which are pulled by a macrophage subpopulation and delivered to target cells. The interaction between macrophages and Delta-ligand carrying airineme vesicles is essential for initiating airineme-mediated signaling, yet the molecular detail of this interaction remains elusive. Through high-resolution live imaging and genetic in vivo manipulations, we found that adhesive interactions via the extracellular domain of CD44, a class I transmembrane glycoprotein, between macrophages and airineme vesicles are critical for airineme signaling. Mutants lacking the extracellular domain of CD44 lose their adhesiveness, resulting in a significant reduction in airineme extension and pigment pattern defects. Our findings provide valuable insights into the role of adhesive interactions between signal-sending cells and macrophages in long-range intercellular signaling.

Feasibility of Virtual Shopping Budget-Management Training on Executive Functions in Healthy Young Adults: A Pilot Study.

To date, budget management in virtual shopping training has not been given much importance. The main objective of this study was to investigate the effects of virtual shopping budget-management training on executive functions and brain activation. Sixteen participants were randomly assigned to the experimental group that received virtual shopping budget-management training or the waitlist control group for a total of 16 sessions. To examine the effects of virtual shopping budget-management training on brain activation, HbO2 was measured in the prefrontal cortex via functional near-infrared spectroscopy (fNIRS) during the Trail Making Test Part B (TMT-B) and Stroop test. Mann-Whitney and Wilcoxon signed-rank tests were used to compare outcomes between and within the two groups. The virtual shopping budget-management training showed no significant difference in all outcomes between both groups (p > 0.05). No significant differences were observed in HbO2 levels during both TMT-B (p > 0.05) and the Stroop test (p > 0.05). However, in the pre-post comparisons, there was a significant difference in the TMT-B (p < 0.05) and Stroop test (p < 0.05) in the experimental group. In this study, although we did not find a distinct advantage in training, it confirmed its potential for clinical benefits in healthy young adults through training.

Cerebellar Gray Matter Volume and its Role in Executive Function, and Attention: Sex Differences by Age in Adolescents.

Objective: This research measures the regional GMV (rGMV) of the cerebellum, attention, Executive Function (EF) and we aimed to identify their correlation and sex differences in children and adolescents. Methods: Subjects comprised 114 children (male = 62, female = 52, 12.44 ± 2.99 years old) from South Korea. Participants were divided into three groups by age (age 6-9, 10-13, and 14-17). The Stroop Color and Word Test (SCWT), Wisconsin Card Sorting Test (WCST), and Advanced Test of Attention (ATA) were used to estimate executive function. Magnetic resonance imaging (MRI) images were analyzed with Regional Voxel-Based Morphometry Analysis. Results: The correlations between cerebellar rGMV and SCWT, WCST, and ATA subcategories showed difference by age and sex. In 6-9 age group, girls showed more overall correlations with cerebellar regions than boys, in WCST Categories Completed and ATA results. In age 10-13 group, more regions of cerebellum corresponded to SCWT subcategories in girls. Nevertheless, more correlation between cerebellar rGMV, WCST subcategories and some ATA subtests were observed in boys in the same age group. In the adolescent group, aged 14-17, boys showed more correlation with cerebellar rGMV, while girls showed little correlation. Conclusion: This study highlights that sex-different cerebellum maturation in adolescence might be correlated with EF and attention. These results provides evidence that cerebellum modulates higher cognitive functioning during child development.

Association of Extensive Video Gaming and Cognitive Function Changes in Brain-Imaging Studies of Pro Gamers and Individuals With Gaming Disorder: Systematic Literature Review.

BACKGROUND: The World Health Organization announced the inclusion of gaming disorder (GD) in the International Classification of Diseases, 11th Revision, despite some concerns. However, video gaming has been associated with the enhancement of cognitive function. Moreover, despite comparable extensive video gaming, pro gamers have not shown any of the negative symptoms that individuals with GD have reported. It is important to understand the association between extensive video gaming and alterations in brain regions more objectively. OBJECTIVE: This study aimed to systematically explore the association between extensive video gaming and changes in cognitive function by focusing on pro gamers and individuals with GD. METHODS: Studies about pro gamers and individuals with GD were searched for in the PubMed and Web of Science databases using relevant search terms, for example, "pro-gamers" and "(Internet) gaming disorder." While studies for pro gamers were searched for without date restrictions, only studies published since 2013 about individuals with GD were included in search results. Article selection was conducted by following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. RESULTS: By following the PRISMA guidelines, 1903 records with unique titles were identified. Through the screening process of titles and abstracts, 86 full-text articles were accessed to determine their eligibility. A total of 18 studies were included in this systematic review. Among the included 18 studies, six studies included pro gamers as participants, one study included both pro gamers and individuals with GD, and 11 studies included individuals with GD. Pro gamers showed structural and functional alterations in brain regions (eg, the left cingulate cortex, the insula subregions, and the prefrontal regions). Cognitive function (eg, attention and sensorimotor function) and cognitive control improved in pro gamers. Individuals with GD showed structural and functional alterations in brain regions (eg, the striatum, the orbitofrontal cortex, and the amygdala) that were associated with impaired cognitive control and higher levels of craving video game playing. They also showed increased cortical thickness in the middle temporal cortex, which indicated the acquisition of better skills. Moreover, it was suggested that various factors (eg, gaming expertise, duration or severity of GD, and level of self-control) seemed to modulate the association of extensive video game playing with changes in cognitive function. CONCLUSIONS: Although a limited number of studies were identified that included pro gamers and/or individuals who reported showing symptoms of GD for more than 1 year, this review contributed to the objective understanding of the association between extensive video game playing and changes in cognitive function. Conducting studies with a longitudinal design or with various comparison groups in the future would be helpful in deepening the understanding of this association.

GSK3B induces autophagy by phosphorylating ULK1.

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis.

NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter.

In mammals, autophagosome formation is initiated by ULK1 via the posttranslational modification of this protein. However, the precise role of ULK1 ubiquitination in modulating autophagy is unknown. Here, we show that NEDD4L, an E3 ubiquitin ligase, binds ULK1 in pancreatic cancer cells. ULK1 expression was stabilized in NEDD4L knockdown cells compared to that in control cells, suggesting that NEDD4L is involved in ULK1 ubiquitination and its subsequent degradation. Autophagy activity was enhanced in NEDD4L knockdown cells compared to control cells. NEDD4L-depleted cells exhibited an increase in the cellular oxygen consumption rate (OCR) and mitochondrial membrane potential, and maintained mitochondrial fusion status in response to metabolic stress. Enhanced OCR and mitochondrial fusion morphology in NEDD4L knockdown cells were repressed by siRNA targeting ULK1. In addition to ULK1, ASCT2, a glutamine transporter, was accumulated in NEDD4L-depleted cells; this is important for maintaining autophagy activation and mitochondrial metabolic function. Finally, the cellular growth and survival rate increased in NEDD4L knockdown cells compared to control cells. However, the genetic or pharmacological blockade of either ULK1 or ASCT2 in NEDD4L-depleted cells sensitized pancreatic cancer cells, particularly in response to nutrient deprivation. In a mouse xenograft model of pancreatic cancer, the use of autophagy inhibitors suppressed tumor growth more in NEDD4L-depleted cells than in tumors from control cells. NEDD4L and ULK1 levels were inversely correlated in two different pancreatic cancer mouse models-xenograft mouse and KPC mouse models. These results suggest that NEDD4L suppressed autophagy and mitochondrial metabolism by reducing cellular ULK1 or ASCT2 levels, and thus could repress the growth and survival of pancreatic cancer cells. Therefore, ubiquitin ligase-mediated autophagy plays a critical role in regulating mitochondrial metabolism, thereby contributing to the growth and survival of certain cancers with low NEDD4L levels.

The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation.

The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13HORMA complex is responsible for the binding of the PtdIns3K complex. This region adopts a ß-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13HORMA complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13HORMA: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13HORMA: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13HORMA: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.