Autophagy is an intracellular catabolic process in which cytoplasmic constituents are delivered to lysosomes for degradation.
Basic levels of autophagy are often low but increase by a wide variety of stimuli such as nutrient deprivation, the presence of damaged or aggregated macromolecules, physiological stress, pharmacological agents or infections. Within the skin, autophagy is considered as a detoxification process able to degrade the macromolecules that accumulate in the cell, thus preventing skin aging.
During autophagy, a cytosolic form of the LC3 protein (microtubule-associated protein light chain 3; LC3-I) is conjugated to form LC-3-phosphatidylethanolamine (LC3-II), which is then recruited at the membranes of autophagosomes. Autophagosomes then fuse with lysosomes to form autolysosomes. The activation of autophagy can therefore be identified by fluorescence visualization of LC3 puncta, corresponding to the formation of autophagosomes. In humans, three isoforms of the LC3 protein (LC3A, LC3B and LC3C) undergo post-translational modifications during autophagy. Of these isoforms, LC3B is the most abundant and well-studied.
Damaged organelles and proteins with a long half-life are engulfed by the newly formed autophagosome. The double membrane structure is marked by LC3-II (conjugated to phosphatidylethanolamine) on the surface. The autophagosome fuses with the lysosome under low pH conditions to form the lytic vesicle, the autolysosome. Within this compartment organelles and proteins are degraded by lysosomal hydrolyses. Schema not drawn to scale (Kotiadis et al., 2014).
Quantification of the autophagosomes is usually performed by fluorescent immunolabeling of the LC3 protein. An increase in the number of LC3s dots or "puncta" is associated with a greater number of autophagosomes. However, as autophagosomes are an intermediate structure in a dynamic process, their number at a specific time is function of the rate of their generation from one side and their conversion to autolysosomes from the other side. Consequently, an accumulation of autophagosomes can originate from an induction of autophagy but also from a blockage of the autophagic process, at a stage subsequent to the formation of autophagosomes. Hence the simple determination of the number of autophagosomes (and therefore the level of LC3-II) is not sufficient to estimate the overall autophagic activity.
The term "autophagic flux" is used to account for the dynamic process of autophagosomes, including synthesis, provision to the lysosomes, and degradation of autophagic substrates. The measurement of this flux is a better indicator of autophagic activity. One of the major methods used to study the autophagic flux is the measurement of LC3 "turnover" which is based on the observation that LC3 is degraded in autolysosomes. If the cells are treated with lysosomotropic agents, such as ammonium chloride, chloroquine or bafilomycin A1, which inhibit acidification in the lysosome or inhibit autophagosome-lysosome fusion, the degradation of LC3-II is blocked, resulting in its accumulation.
Using this approach, the difference in amount of LC3-II between samples in the presence and absence of lysosomal inhibitor represents the amount of LC3 which is delivered to the lysosomes for degradation and therefore corresponds to the autophagic flux. Quantification of LC3-II after a treatment, in the presence and absence of lysosomal inhibitor, can be performed by the Western blot technique. In comparison with a reference, this method makes it possible to demonstrate a possible effect of a treatment on the autophagic activity of the cells.
StratiCELL proposes the two complementary approaches to quantify the effect of a compound on the autophagic activity of cells:
Fluorescent immunostaining of the LC3B protein can be performed to carry out a rapid screening of active ingredients and to select those presenting an effect (inducing or blocking) on the autophagy process. Both epidermal keratinocytes and human dermal fibroblasts can be used as valid models. Visualization of the increased presence of LC3 puncta or dots after treatment with an active reflects an effect of the latter on the autophagic process. At this stage, it may be an induction of autophagy or a blockage of the process.
lc3b immunostaining from normal human epidermal keratinocytes
NHEKs keratinocytes were treated with rapamycin at 5µM used as autophagy inducer and with chloroquine at 20 µM used as autophagic flux blocking agent. A fluorescence immunolabelling was performed with an antibody directed against LC3B protein. The treatment with rapamycin showed a slight increase of LC3B puncta numbers. The treatment with chloroquine showed an important accumulation of LC3B proteins and thus of autophagosomes. The reference, DMSO at 0.1%, was used as solvent for rapamycin and chloroquine preparation.
The Western blot approach is then proposed for the selected actives, as the separation of the two forms of LC3B (LC3B-I and LC3B-II) makes it possible to quantify, in presence and absence of a lysosomal inhibitor such as bafilomycin A1, the levels of LC3B-II present in the protein samples and actually delivered to the lysosomes. These levels are compared to a reference condition in order to assess the effect of a treatment on the autophagic activity of the cells.
Western blot showing LC3-I and LC3-II relative amounts