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Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

ABSTRACT: Perturbations to cellular homeostasis, including reduction of the cholesterol level, induce autophagy, a self-digestion process of cellular constituents through an autophagosomal-lysosomal pathway. In accord with its function as a membrane organizer and metabolic sentinel, the cellular response to cholesterol depletion comprises multiple phenomena, including the activation of transcriptional responses, accumulation of reactive oxygen species (ROS), and activation of stress-related signaling pathways. However, the molecular mechanisms by which cholesterol depletion regulates autophagy and the putative involvement of transcriptional responses, ROS and/or stress-related signaling in autophagy regulation in this biological context are not fully understood. Here, we find that cholesterol depletion regulates autophagy at three different levels. First, employing RNA-seq, we show that cholesterol depletion increases the expression of autophagy-related genes independent of ROS or JNK activity. Second, analysis of LC3 lipidation and intracellular localization, and of p62 levels and degradation kinetics, reveals that cholesterol depletion mediates autophagy induction while interfering with autophagic flux. Of note, only the latter depends on ROS accumulation and JNK activity. In view of the common use of cholesterol-reducing drugs as therapeutic agents, our findings have important implications for multiple cellular settings in which autophagy plays a prominent role.

SUBMITTER: Shapira KE

PROVIDER: S-EPMC8556405 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

Shapira Keren E KE Shapira Guy G Schmukler Eran E Pasmanik-Chor Metsada M Shomron Noam N Pinkas-Kramarski Ronit R Henis Yoav I YI Ehrlich Marcelo M

Cell death discovery 20211029 1

Perturbations to cellular homeostasis, including reduction of the cholesterol level, induce autophagy, a self-digestion process of cellular constituents through an autophagosomal-lysosomal pathway. In accord with its function as a membrane organizer and metabolic sentinel, the cellular response to cholesterol depletion comprises multiple phenomena, including the activation of transcriptional responses, accumulation of reactive oxygen species (ROS), and activation of stress-related signaling path ...[more]

PMID: 34716312

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Project description:The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii-infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing. IMPORTANCE The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite's ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii.

Project description:BackgroundNeuropathic pain (NP) is one of the most common types of chronic pain and significantly compromises the quality of life. Autophagy is an intracellular catabolic process that is required to maintain cellular homeostasis in response to various stresses. The role of autophagy-related genes in the diagnosis and treatment of neuropathic pain remains unclear.MethodsWe identified autophagy-related differentially expressed genes (ARDEGs) and differentially expressed miRNAs (DE-miRNAs) in neuropathic pain by bioinformatics analysis of the GSE145226 and GSE145199 datasets. These ARDEGs and their co-expressed genes were subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, Gene Set Enrichment Analysis (GSEA) and friends analysis. Meanwhile, we constructed TFs-ARDEGs, miRNA-ARDEGs regulatory network through ChIPBase database and the HTFtarget database, multiMir R package. Finally, we performed immune infiltration analysis of ARDEGs by Single Sample Gene Set Enrichment Analysis (ssGSEA).ResultsWe identified 2 potential autophagy-related differentially expressed genes (Sirt2 and ST7) that may be closely associated with the pathogenesis of neuropathic pain. GO, KEGG and GSEA analysis revealed that these two ARDEGs were mainly enriched in pyridine nucleotide metabolic process, nicotinamide nucleotide metabolic process, Nicotinate and nicotinamide metabolism, NF-κB pathway, KRAS signaling, P53 pathway. In the TFs-ARDEGs and miRNA-ARDEGs regulatory network, miR-140-5p and Cebpb were predicted to be as crucial regulators in the progression of NP. For the ssGSEA results, Sirt2 was positively correlated with Eosinophil and Effector memory CD8+ T cell infiltration, which suggested that it may be involved in the regulation of neuroimmune-related signaling.ConclusionTwo autophagy-related differentially expressed genes, especially Sirt2, may be potential biomarkers for NP, providing more evidence about the crucial role of autophagy in neuropathic pain.

Dataset Information

Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

Publications

Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

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