"Autophagic flux" in normal mouse tissues: Focus on endogenous LC3A processing

Abstract

Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins, mature ribosomes and even entire organelles. The best-studied autophagic marker is the LC3B protein and it is thought that the amount of LC3B-II correlates with the amount of autophagic membranes. Whether LC3A processing, in addition to LC3B, is a valuable endogenous "autophagic flux" marker is far less clear. The specificity of rabbit polyclonal antibodies to LC3A and LC3B was tested against the commercial available human recombinant proteins LC3A and LC3B. In order to measure "autophagic flux" in mouse liver, lung, kidney and heart we used: (1) a lysosomotropic reagent, chloroquine, which inhibits intralysosomal acidification and ultimately their fusion with autophagosomes, (2) nutrient starvation as an autophagic stimulus and (3) ionizing radiation, which destabilizes lysosomes. According to the immunoblotting work, the LC3A protein follows discrete patterns of LC3A-I and LC3A-II changes in liver, lung, kidney and heart tissues of mice, whereas the LC3B protein did not follow the same pattern under stressor conditions. We conclude that the endogenous LC3A processing is a major marker of autophagy flux in the mouse model. Fractionated samples (soluble vs membrane fractions) should be used in immunoblotting to allow discrimination between the soluble LC3-I and the membrane- associated LC3-II proteins and the kinetics of modification. Furthermore, when dealing with in vivo models it is necessary to check the specificity of the antibodies used against the LC3A and LC3B proteins as their expression and responsiveness is not overlapping. © 2011 Landes Bioscience.