Interconnected Regulation of Apoptosis and WIPI-Mediated Autophagy

Autophagy is a genetic program that secures the survival of eukaryotic cells to compensate for periods of starvation and cellular stress. Apoptosis, in contrast, is a genetically defined program leading to cell death. Both pathways are interconnected through conserved co-regulatory signaling pathways that are context-dependent. Proper co-regulation of autophagy and apoptosis, whereby autophagy exerts an antiapoptotic function and apoptosis inhibits autophagic survival strategies, critically secures the survival of healthy cells and counteracts genomic instability in eukaryotic organisms. Cancer cells often become resistant to apoptosis and addicted to autophagy, making this scenario highly relevant to define novel therapeutic strategies. The co-regulatory crosstalk between apoptosis and autophagy converges on the production of phosphatidylinositol 3-phosphate (PI3P) essential for the onset of autophagy. WD-repeat protein interacting with phosphoinositides (WIPI) members function as essential PI3P effectors in autophagy and fulfill an important role in health and disease. Here, we summarize details on the regulation of WIPI-mediated autophagy in the context of co-regulatory signals for both apoptosis and autophagy

The menage a trois of autophagy, lipid droplets and liver disease

Autophagic pathways cross with lipid homeostasis and thus provide energy and essential building blocks that are indispensable for liver functions. Energy deficiencies are compensated by breaking down lipid droplets (LDs), intracellular organelles that store neutral lipids, in part by a selective type of autophagy, referred to as lipophagy. The process of lipophagy does not appear to be properly regulated in fatty liver diseases (FLDs), an important risk factor for the development of hepatocellular carcinomas (HCC). Here we provide an overview on our current knowledge of the biogenesis and functions of LDs, and the mechanisms underlying their lysosomal turnover by autophagic processes. This review also focuses on nonalcoholic steatohepatitis (NASH), a specific type of FLD characterized by steatosis, chronic inflammation and cell death. Particular attention is paid to the role of macroautophagy and macrolipophagy in relation to the parenchymal and non-parenchymal cells of the liver in NASH, as this disease has been associated with inappropriate lipophagy in various cell types of the liver. Abbreviations: ACAT: acetyl-CoA acetyltransferase; ACAC/ACC: acetyl-CoA carboxylase; AKT: AKT serine/threonine kinase; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BECN1/Vps30/Atg6: beclin 1; BSCL2/seipin: BSCL2 lipid droplet biogenesis associated, seipin; CMA: chaperone-mediated autophagy; CREB1/CREB: cAMP responsive element binding protein 1; CXCR3: C-X-C motif chemokine receptor 3; DAGs: diacylglycerols; DAMPs: danger/damage-associated molecular patterns; DEN: diethylnitrosamine; DGAT: diacylglycerol O-acyltransferase; DNL: de novo lipogenesis; EHBP1/NACSIN (EH domain binding protein 1); EHD2/PAST2: EH domain containing 2; CoA: coenzyme A; CCL/chemokines: chemokine ligands; CCl(4:) carbon tetrachloride; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FA: fatty acid; FFAs: free fatty acids; FFC: high saturated fats, fructose and cholesterol; FGF21: fibroblast growth factor 21; FITM/FIT: fat storage inducing transmembrane protein; FLD: fatty liver diseases; FOXO: forkhead box O; GABARAP: GABA type A receptor-associated protein; GPAT: glycerol-3-phosphate acyltransferase; HCC: hepatocellular carcinoma; HDAC6: histone deacetylase 6; HECT: homologous to E6-AP C-terminus; HFCD: high fat, choline deficient; HFD: high-fat diet; HSCs: hepatic stellate cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; ITCH/AIP4: itchy E3 ubiquitin protein ligase; KCs: Kupffer cells; LAMP2A: lysosomal associated membrane protein 2A; LDs: lipid droplets; LDL: low density lipoprotein; LEP/OB: leptin; LEPR/OBR: leptin receptor; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIR: LC3-interacting region; LPS: lipopolysaccharide; LSECs: liver sinusoidal endothelial cells; MAGs: monoacylglycerols; MAPK: mitogen-activated protein kinase; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCD: methionine-choline deficient; MGLL/MGL: monoglyceride lipase; MLXIPL/ChREBP: MLX interacting protein like; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver disease; NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; NPC: NPC intracellular cholesterol transporter; NR1H3/LXRα: nuclear receptor subfamily 1 group H member 3; NR1H4/FXR: nuclear receptor subfamily 1 group H member 4; PDGF: platelet derived growth factor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA: patatin like phospholipase domain containing; PNPLA2/ATGL: patatin like phospholipase domain containing 2; PNPLA3/adiponutrin: patatin like phospholipase domain containing 3; PPAR: peroxisome proliferator activated receptor; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARD/PPARδ: peroxisome proliferator activated receptor delta; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SE: sterol esters; SIRT1: sirtuin 1; SPART/SPG20: spartin; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; TAGs: triacylglycerols; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TGFB1/TGFβ: transforming growth factor beta 1; Ub: ubiquitin; UBE2G2/UBC7: ubiquitin conjugating enzyme E2 G2; ULK1/Atg1: unc-51 like autophagy activating kinase 1; USF1: upstream transcription factor 1; VLDL: very-low density lipoprotein; VPS: vacuolar protein sorting; WIPI: WD-repeat domain, phosphoinositide interacting; WDR: WD repeat domai

WIPI-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus aureus for Lysosomal Degradation

Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently entrapped in WIPI-1 positive autophagosome-like vesicles when compared to agr-negative cells (SA113). By confocal and electron microscopy we provide evidence that single- and multiple-Staphylococci entrapped undergo cell division. Moreover, the number of WIPI-1 positive autophagosome-like vesicles entrapping Staphylococci significantly increased upon (i) lysosomal inhibition by bafilomycin A1 and (ii) blocking PIKfyve-mediated PtdIns(3,5)P2 generation by YM201636. In summary, our results provide evidence that the PtdIns(3)P effector function of WIPI-1 is utilized during xenophagy of Staphylococcus aureus. We suggest that invading S. aureus cells become entrapped in autophagosome-like WIPI-1 positive vesicles targeted for lysosomal degradation in nonprofessional host cells

Driving next-generation autophagy researchers towards translation (DRIVE), an international PhD training program on autophagy

The European autophagy consortium Driving next-generation autophagy researchers towards translation (DRIVE) held its kick-off meeting in Groningen on the 14(th) and 15(th) of June 2018. This Marie Sklodowska-Curie Early Training Network was approved under the European Union's Horizon 2020 Research and Innovation Program and is funded for 4 years. Within DRIVE, 14 European research teams from academia and industry will train 15 PhD students through applied, cross-disciplinary and collaborative macroautophagy/autophagy research. The goal of DRIVE is to stimulate applied approaches in autophagy research and provide training towards translation, while advancing our knowledge on autophagy in specific physiological and pathological states. The strong focus on translation will prepare the PhD students to be at the forefront to exploit autophagy for the development of therapies directly benefitting patients. Thereby, DRIVE will contribute to filling the educational gap that currently exists between academia and industry, and will prepare its PhD students for alternative and highly flexible professional paths.Non peer reviewe

Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease

PIKfyve Regulation of Endosome-Linked Pathways

The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5)P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5)P2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies

Driving next-generation autophagy researchers towards translation (DRIVE), an international PhD training program on autophagy

The European autophagy consortium Driving next-generation autophagy researchers towards translation (DRIVE) held its kick-off meeting in Groningen on the 14th and 15th of June 2018. This Marie Skłodowska-Curie Early Training Network was approved under the European Union's Horizon 2020 Research and Innovation Program and is funded for 4 years. Within DRIVE, 14 European research teams from academia and industry will train 15 PhD students through applied, cross-disciplinary and collaborative macroautophagy/autophagy research. The goal of DRIVE is to stimulate applied approaches in autophagy research and provide training towards translation, while advancing our knowledge on autophagy in specific physiological and pathological states. The strong focus on translation will prepare the PhD students to be at the forefront to exploit autophagy for the development of therapies directly benefitting patients. Thereby, DRIVE will contribute to filling the educational gap that currently exists between academia and industry, and will prepare its PhD students for alternative and highly flexible professional path

The mechanism of macroautophagy: The movie

This animated movie presents the mechanism of macroautophagy, hereafter autophagy, by showing the molecular features of the formation of autophagosomes, the hallmark organelle of this intracellular catabolic pathway. It is based on our current knowledge and it also illustrates how autophagosomes can recognize and eliminate selected cargoes.</p

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Generation of PI3P in the normally PI3P-deficient ER membrane makes the organelle a platform for autophagosome formation