Dataset Information

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

ABSTRACT: Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.

SUBMITTER: Humbert M

PROVIDER: S-EPMC7150830 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

Humbert Magali M Morán María M de la Cruz-Ojeda Patricia P Muntané Jordi J Wiedmer Tabea T Apostolova Nadezda N McKenna Sharon L SL Velasco Guillermo G Balduini Walter W Eckhart Leopold L Janji Bassam B Sampaio-Marques Belém B Ludovico Paula P Žerovnik Eva E Langer Rupert R Perren Aurel A Engedal Nikolai N Tschan Mario P MP

Biology 20200321 3

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of s ...[more]

PMID: 32245178

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Project description:The proton pump vacuolar (V)-ATPase is the driving force that mediates the concentration of cationic drugs (weak bases) in the late endosome-lysosome continuum; secondary cell reactions include the protracted transformation of enlarged vacuoles into autophagosomes. We used the inherently fluorescent tertiary amine quinacrine in murine models to further assess the accumulation and signaling associated with cation trapping. Primary fibroblasts concentrate quinacrine ?5,000-fold from their culture medium (KM 9.8 µM; transport studies). The drug is present in perinuclear granules that are mostly positive for Rab7 and LAMP1 (microscopy). Both drug uptake and retention are extensively inhibited by treatments with the V-ATPase inhibitor bafilomycin A1. The H(+) ionophore monensin also prevented quinacrine concentration by fibroblasts. However, inhibition of plasma membrane transporters or of the autophagic process with spautin-1 did not alter quinacrine transport parameters. Ancillary experiments did not support that low micromolar concentrations of quinacrine are substrates for organic cation transporters-1 to -3 or P-glycoprotein. The secondary autophagy induced by quinacrine in cells may derive from the accumulation of incompetent autophagolysosomes, as judged from the accumulation of p62/SQSTM1 and LC3 II (immunoblots). Accordingly, protracted lysosomogenesis is evidenced by increased expression of LAMP1 and LAMP2 in quinacrine-treated fibroblasts (48 h, immunoblots), a response that follows the nuclear translocation of the lysosomal genesis transcription factor TFEB and upregulation of LAMP1 and -2 mRNAs (24 h). Quinacrine administration to live mice evidenced variable distribution to various organs and heterogeneous accumulation within the lung (stereo-microscopy, extraction). Dose-dependent in vivo autophagic and lysosomal accumulation was observed in the lung (immunoblots). No evidence has been found for transport or extrusion mechanisms modulating the cellular uptake of micromolar quinacrine at the plasma membrane level. As shown in vitro and in vivo, V-ATPase-mediated cation sequestration is associated, above a certain threshold, to autophagic flux inhibition and feed-back lysosomogenesis.

Dataset Information

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

Publications

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot.

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