bims-apauto 2022-01-02 papers

Issue of 2022‒01‒02
nine papers selected by
Su Hyun Lee
Seoul National University

Autophagy. 2021 Dec 29. 1-17

Negative pressure wound therapy improves bone regeneration by promoting osteogenic differentiation via the AMPK-ULK1-autophagy axis.

Sheng Zhang, Yuanlong Xie, Feifei Yan, Yufeng Zhang, Zhiqiang Yang, Zhe Chen, Yong Zhao, Zan Huang, Lin Cai, Zhouming Deng.

Deficient bone regeneration causes bone defects or nonunion in a substantial proportion of trauma patients that urges for novel therapies. To develop a reliable therapy, we investigated the effect of negative pressure wound therapy (NPWT) on bone regeneration in vivo in a rat calvarial defect model. Negative pressure (NP) treatment in vitro was mimicked to test its effect on osteoblast differentiation in rat mesenchymal stem cells (MSCs) and MC3T3-E1 cells. Transcriptomic analyses, pharmaceutical interventions, and shRNA knockdowns were conducted to explore the underlying mechanism and their clinical relevance was investigated in samples from patients with nonunion. The potential application of a combined therapy of MSCs in hydrogels with negative pressure was tested in the rat critical-size calvarial defect model. We found that NPWT promoted bone regeneration in vivo and NP treatment induced osteoblast differentiation in vitro. NP induced osteogenesis via activating macroautophagy/autophagy by AMPK-ULK1 signaling that was impaired in clinical samples from patients with nonunion. More importantly, the combined therapy involving MSCs in hydrogels with negative pressure significantly improved bone regeneration in rat critical-size calvarial defect model. Thus, our study identifies a novel AMPK-ULK1-autophagy axis by which negative pressure promotes osteoblast differentiation of MSCs and bone regeneration. NPWT treatment can potentially be adopted for therapy of bone defects.Abbreviations: ADP, adenosine diphosphate; AICAR/Aic, acadesine; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, biomineralization associated; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ARS, alizarin red S staining; ATG7, autophagy related 7; ATP, adenosine triphosphate; BA1, bafilomycin A1; BGLAP/OCN, bone gamma-carboxyglutamate protein; BL, BL-918; BS, bone surface; BS/TV, bone surface per tissue volume; BV/TV, bone volume per tissue volume; C.C, compound C; CCN1, cellular communication network factor 1; COL1A1, collagen type I alpha 1 chain; COL4A3, collagen type IV alpha 3 chain; COL4A4, collagen type IV alpha 4 chain; COL18A1, collagen type XVIII alpha 1 chain; CQ, chloroquine; GelMA, gelatin methacryloyl hydrogel; GO, Gene Ontology; GSEA, gene set enrichment analysis; HIF1A, hypoxia inducible factor 1 subunit alpha; HPLC, high-performance liquid chromatography; ITGAM/CD11B, integrin subunit alpha M; ITGAX/CD11C, integrin subunit alpha X; ITGB1/CdD9, integrin subunit beta 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; micro-CT, microcomputed tomography; MSCs, mesenchymal stem cells; MTOR, mechanistic target of rapamycin kinase; NP, negative pressure; NPWT, negative pressure wound therapy; PRKAA1/AMPKα1, protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2; PTPRC/CD45, protein tyrosine phosphatase receptor type C; ROS, reactive oxygen species; RUNX2, RUNX family transcription factor 2; SBI, SBI-0206965; SPP1/OPN, secreted phosphoprotein 1; THY1/CD90, Thy-1 cell surface antigen; SQSTM1, sequestosome 1; TGFB3, transforming growth factor beta 3; ULK1/Atg1, unc-51 like autophagy activating kinase 1.

Keywords: AMPK; ULK1; bone nonunion; bone regeneration; macroautophagy/autophagy; mesenchymal stem cells; negative pressure wound therapy; osteoblast differentiation

DOI: https://doi.org/10.1080/15548627.2021.2016231

Methods Mol Biol. 2022 ;2445 3-24

A Balance Between Autophagy and Other Cell Death Modalities in Cancer.

Anna S Gorbunova, Gelina S Kopeina, Boris Zhivotovsky.

Autophagy is an intracellular self-digestive process involved in catabolic degradation of damaged proteins, and organelles, and the elimination of cellular pathogens. Initially, autophagy was considered as a prosurvival mechanism, but the following insights shed light on its prodeath function. Nowadays, autophagy is established as a crucial player in the development of various diseases through interaction with other molecular pathways within a cell. Additionally, disturbance in autophagy is one of the main pathological alterations that lead to resistance of cancer cells to treatment. These autophagy-related pathologies gave rise to the development of new therapeutic drugs. Here, we summarize the current knowledge on the autophagic role in disease pathogenesis, particularly in cancer, and the interplay between autophagy and other cell death modalities in order to combat cancer.

Keywords: Apoptosis; Autophagy; Autophagy-dependent cell death; Cancer; Necroptosis

DOI: https://doi.org/10.1007/978-1-0716-2071-7_1

Autophagy. 2021 Dec 29. 1-14

DCN released from ferroptotic cells ignites AGER-dependent immune responses.

Jiao Liu, Shan Zhu, Ling Zeng, Jingbo Li, Daniel J Klionsky, Guido Kroemer, Jianxin Jiang, Daolin Tang, Rui Kang.

Ferroptosis is a form of inflammatory cell death for which key mediators remain obscure. Here, we report that the proteoglycan decorin (DCN) is released by cells that are dying from ferroptosis and then acts as an alarm signal to trigger innate and adaptive immune responses. The early release of DCN during ferroptosis is an active process that involves secretory macroautophagy/autophagy and lysosomal exocytosis. Once released, extracellular DCN binds to the receptor advanced glycosylation end-product-specific receptor (AGER) on macrophages to trigger the production of pro-inflammatory cytokines in an NFKB/NF-κB-dependent manner. Pharmacological and genetic inhibition of the DCN-AGER axis protects against ferroptotic death-related acute pancreatitis and limits the capacity of ferroptotic cancer cells to induce a tumor-protective immune response. Thus, DCN is an essential mediator of the inflammatory and immune consequences of ferroptosis.

Keywords: DAMP; Ferroptosis; autophagy; inflammation; macrophages

DOI: https://doi.org/10.1080/15548627.2021.2008692

Methods Mol Biol. 2022 ;2445 207-226

Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.

Benjamin S Padman, Michael Lazarou.

Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.

Keywords: ImageJ/FIJI; Object-based image analysis (OBIA); PINK1/Parkin mitophagy; Phagophore biogenesis; Regions of interest (ROI)

DOI: https://doi.org/10.1007/978-1-0716-2071-7_13

Methods Mol Biol. 2022 ;2445 139-169

Approaches and Protocols to Analyze Autophagy and Its Role in Death of Apoptosis-Resistant Senescent Tumor Cells.

Elena Y Kochetkova, Valery A Pospelov, Tatiana V Pospelova.

Anticancer therapy is complicated by the ability of malignant cells to activate cytoprotective autophagy that rescues treated cells. This protocol describes methods for analysis of autophagic process in apoptosis-resistant tumor cells treated with damaging agents. Induction of autophagy in these cells can activate apoptotic death. Protocol provides methods for Western blotting, immunofluorescent analysis, and transfection of cells with fluorescent protein-tagged LC3-encoding plasmids to analyze autophagy. Different approaches to change autophagy in tumor cells are suggested. A special approach is connected with induction of cellular senescence. Senescent cells, which are resistant to apoptosis, are vulnerable to certain damaging agents, in particular, to kinase inhibitors. Methods to induce and analyze senescence are considered. They include detection of proliferation arrest by different ways, mTORC1 activity assay and fluorescent analysis of mTORC1 and lysosome localization as a novel senescence hallmark. Incapability of senescent cells to complete autophagy after damage allows to force them to apoptosis. To demonstrate apoptotic cell death, analysis of caspase activity, Annexin V-FITC binding, DNA fragmentation, and mitochondria and lysosome damage are suggested. The methods described can be applied in studies aimed on developing different strategies of tumor cell elimination through changing autophagy.

Keywords: Apoptotic cell death; Autophagic cell death; Autophagy; Cancer; Cytoplasmic compartmentalization; Senescence; mTORC1

DOI: https://doi.org/10.1007/978-1-0716-2071-7_10

Methods Mol Biol. 2022 ;2445 27-38

Isolation of Autophagy Competent Lysosomes from Cancer Cells by Differential Large-Scale Multilayered Density Gradient Centrifugations.

Merve Kacal, Helin Vakifahmetoglu-Norberg.

Accurate isolation of functional and intact lysosomes enables the quantification and analyses of abundances, dynamic changes and enrichment levels of lysosomal content, allowing specific lysosomal investigations induced by autophagy. In this protocol chapter, we describe detailed practical instructions and advices for an efficacious lysosomal enrichment and isolation procedure by differential multilayered density gradient centrifugations using human cancer cell lines. By this method, intact and autophagy competent lysosomes can be isolated from cancer cells based on their distinct density and obtained fractions can further be analyzed for functional lysosomal assays, as well as for protein or metabolic loads to identify select spatiotemporal changes by comparative quantitative measurement. This method has been used to enrich lysosomes from a variety of cancer cells with activated chaperone-mediated autophagy, but can be optimized for other cell lines and tissues for multiple autophagy-induced conditions.

Keywords: Autophagy; Cancer; Chaperone-mediated autophagy; LAMP-2A; Lysosomes

DOI: https://doi.org/10.1007/978-1-0716-2071-7_2

Methods Mol Biol. 2022 ;2445 99-115

Measuring Autophagic Cargo Flux with Keima-Based Probes.

Nikolai Engedal, Tonje Sønstevold, Carsten J Beese, Sarvini Selladurai, Thea Melcher, Julia E Simensen, Lisa B Frankel, Alfonso Urbanucci, Maria L Torgersen.

Autophagy and autophagy-associated genes are implicated in a growing list of cellular, physiological, and pathophysiological processes and conditions. Therefore, it is ever more important to be able to reliably monitor and quantify autophagic activity. Whereas autophagic markers, such as LC3 can provide general indications about autophagy, specific and accurate detection of autophagic activity requires assessment of autophagic cargo flux. Here, we provide protocols on how to monitor bulk and selective autophagy by the use of inducible expression of exogenous probes based on the fluorescent coral protein Keima. To exemplify and demonstrate the power of this system, we provide data obtained by analyses of cytosolic and mitochondrially targeted Keima probes in human retinal epithelial cells treated with the mTOR-inhibitor Torin1 or with the iron chelator deferiprone (DFP). Our data indicate that Torin1 induces autophagic flux of cytosol and mitochondria to a similar degree, that is, compatible with induction of bulk autophagy, whereas DFP induces a highly selective form of mitophagy that efficiently excludes cytosol.

Keywords: Autophagic cargo flux; Autophagy; Bulk autophagy; Deferiprone; LDHB-mKeima; Mito-mKeima; Mitophagy; Selective autophagy; Torin1; mKeima

DOI: https://doi.org/10.1007/978-1-0716-2071-7_7

Methods Mol Biol. 2022 ;2445 65-74

A Quantitative Flow Cytometry-Based Method for Autophagy Detection Across the Cell Cycle.

Vitaliy O Kaminskyy.

Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.

Keywords: Autophagic flux; Autophagosome formation; Autophagy; Cell cycle; Cell permeabilization; DNA staining; Flow cytometry; LC3; Quantification

DOI: https://doi.org/10.1007/978-1-0716-2071-7_5

Methods Mol Biol. 2022 ;2445 329-335

Extraction of Metabolites from Cancer Cells.

Elena Kochetkova, Tina Becirovic, Erik Norberg.

Cancer cells possess an elevated demand for nutrients and metabolites due to their uncontrolled proliferation and need to survive in unfavorable conditions. Autophagy is a conservative degradation pathway that counters lack of nutrients and provides organelle and protein quality control, beyond maintenance of cellular metabolism.Mass spectrometry-based metabolomics is a powerful tool to study the metabolome of a cell. Such analysis requires proper sample preparation including the extraction of metabolites. Here, we provide a protocol for the extraction of metabolites from adherent cancer cells suitable for global metabolome profiling by mass spectrometry.

Keywords: Autophagy; CE-MS; Cancer metabolism; Chaperone-mediated autophagy; GC-MS; LC-MS; Mass spectrometry; Metabolism; Methanol extraction

DOI: https://doi.org/10.1007/978-1-0716-2071-7_20