In this study we established a new in vivo assay for measuring LC3b turnover, which is a popular approach for estimating macroautophagic flux.16,22
In our assay, leupeptin is administered to mice via i.p. injection, producing an increase in LC3b-II signal in autolysosomes that can be quantitatively measured. Previously leupeptin has been used to measure LC3b turnover in tissue culture cells16,55
and also to detect the sequestration of cytosolic enzymes in lysosomes in vivo.56,57
Our protocol combines aspects of these earlier reports to enable the quantitative measurement of LC3b turnover via macroautophagy in vivo.
Currently there is a need for new methods to measure macroautophagic flux that are technically simple, and which can complement or refine previously described approaches. For example, the bulk protein degradation assay has been used in vivo,50
but it is cumbersome and has not been extensively employed in animal models. Static levels of p62 have been advanced as a marker for macroautophagic flux in vivo, with reduced levels of this protein indicating increased catabolic activity.35
However static levels of p62 represent a balance between new protein synthesis and clearance, and so this readout requires validation to ensure that changes in p62 quantity are not a function of altered transcription or translation rather than degradation. It may be more straightforward to directly measure p62 turnover using the leupeptin assay rather than trying to infer its rate of turnover based on steady-state levels. Ju et al.32
developed an assay to measure macroautophagic flux in skeletal muscle based on the decay of transfected polyglutamine-luciferase fusion proteins. This assay directly measures macroautophagic flux but relies on the clearance of exogenous substrates that are overexpressed, and has not yet been extended to other organs beyond muscle. The lysosomal inhibitor chloroquine has been used to measure endogenous LC3b turnover in heart muscle.33
While straightforward, the use of chloroquine has not been validated for measuring macroautophagic flux in other organs. Chloroquine also has anti-inflammatory effects,58,59
which make it less desirable for measuring macroautophagic flux in the setting of animal models of inflammation or infection. Finally, Ju et al.34
recently developed an LC3b turnover assay to measure macroautophagic flux in skeletal muscle using colchicine treatment. As with previous efforts their assay has so far been validated for only a single organ (skeletal muscle).34
Moreover their assay requires a two-day treatment with colchicine,34
and so provides a two-day average of LC3b turnover that may miss dynamic changes in macroautophagic flux that occur over shorter time-frames.
The leupeptin assay we describe here offers some advantages over these previously described methods to measure macroautophagic flux. First, our assay detects the turnover of endogenous LC3b, LC3a and p62, as opposed to exogenous polyglutamine repeat-luciferase fusion proteins32
or GFP-LC3b whose rate of decay might differ from endogenous LC3b.27
Second, we validated the leupeptin-based assay in multiple solid organs including liver, heart, lung, kidney and spleen. Third, by utilizing a lysosome and autophagosome enriched fraction for measuring flux, our method adds a level of specificity that is not present when analyzing unfractionated lysates. Fourth, we employed a straightforward means of quantifying macroautophagic flux by extrapolating the intensity of LC3b-II against a standard curve of purified GST-LC3b. Finally, our method offers an alternative to chloroquine and colchicine for the in vivo measurement of LC3 protein turnover. While both of these compounds are effective at increasing LC3b-II levels they are potent anti-inflammatory agents that interfere with TNFα activity.59,60
As such, the leupeptin assay may be advantageous when measuring macroautophagic flux in mouse models of infection or inflammation.
In validating the leupeptin assay we made several novel observations of basal macroautophagic activity. First, we found that the amount LC3b-II accumulation after i.p. injection of leupeptin differed considerably between organs, and was highest and most sustained in the liver ( and
). Potentially, this could reflect a differential potency or bioavailability for leupeptin in different organ types.61,62
An alternate explanation for our data is that liver harbors higher levels of basal macroautophagic activity. Such a conclusion might be supported by the shorter half life of LC3b-II we observed in liver compared to lung after cycloheximide treatment, a compound which is chemically dissimilar to leupeptin (). Irrespective, a direct comparison of flux between organs is potentially misleading, since macroautophagy likely contributes to cellular homeostasis somewhat differently in different specialized tissue types. Nevertheless, the fact that LC3b-II turnover is very robust in the liver may suggest that macroautophagy plays a significant role in the physiologic contributions of this organ even under nutrient-rich conditions. More research is needed to clarify the role of macroautophagy in metabolism.
Second, we found that LC3b turnover is very rapid, with a half-life of approximately 10 to 40 min, depending on the organ studied and the translation inhibitor used. This data is consistent with the results of Schworer et al.50
who determined the half life of autophagosomes in hepatocytes to be roughly 8 min, based on an EM analysis. Our results are also in line with those of Fass et al. who estimated the average life span of GFP-LC3 puncta to be 35–40 min.
We also found that the LC3b-related proteins LC3a, GABARAP and GATE-16 were turned over with different kinetics in liver. LC3a-II levels were enhanced by leupeptin treatment similar to LC3b and also had a similar half-life after CHX treatment, while GABARAP and GATE-16 were not as rapidly turned over in our assays. This is striking because all three of these proteins have conformational structures that are similar to LC3b, and all have been shown to be targeted to autophagosome membranes.13
Moreover, a recent proteomic screen for ATG8/LC3 interacting proteins found that the known mammalian LC3 homologues largely share the same interacting proteins.64
One explanation for our findings is that LC3b and LC3a might be more abundantly recruited to the interior of developing autophagosomal membranes compared to GABARAP and GATE-16, and so the steady-state levels of LC3a and LC3b are more greatly influenced by macroautophagic turnover. In fact, a recent functional analysis of the LC3 and GABARAP subfamilies in HeLa cells suggested that LC3b is recruited to autophagosome membranes during the elongation phase of their biogenesis.65
In contrast GATE-16 is required for sealing of the nascent autophagosome membrane and dissociation of the ATG5-12-16L complex. This might imply that GATE-16 and its close homologue GABARAP are enveloped by autophagosomes in lower quantity than LC3b and LC3a, thereby explaining the different turnover rates of these proteins in our assays.
Finally, our assay confirmed that beclin 1
heterozygous mice have mildly decreased macroautophagic flux under basal conditions. While this result was intuitively expected, a quantitative measurement of flux in these mice in vivo had not previously been reported. Flux measurements are important in mutant mice, such as beclin 1
heterozygotes, where the defect in macroautophagy is partial and selection pressure over many generations of inbreeding can lead to phenotypic drift. Given the growing availability of genetically altered mice containing complex mutations or deletions in macroautophagy genes it will be important going forward to perform in vivo measurements of macroautophagic flux when using these mice, in order to better understand the contribution of macroautophagy to the observed phenotypes. Moreover, the assays described here provide a way of analyzing potential synergisms between macroautophagy gene mutations and external factors such as diet,66,67
and circadian rhythm.70
Currently, LC3b turnover is one of the most commonly utilized means for inferring macroautophagic flux.22
While our experience confirms the value of LC3b turnover assays for in vivo measurement of flux, it is worth noting some caveats to this approach. LC3b is specifically recruited to autophagosome membranes and as such the quantity of LC3b engulfed by a given autophagosome is a function of its surface area. In contrast, the rate of bulk degradation generated by the macroautophagy system is a function of the volume of cytoplasm being sequestered by autophagosomes. This distinction is important because autophagosomes produced under starvation conditions are roughly 50% larger than those produced under basal conditions,50
and therefore have a lower surface area to volume ratio. As such the quantitative relationship between LC3b turnover and bulk protein turnover can vary to some degree. Utilizing assays based on p62 or NBR-1 turnover27
will not circumvent this issue because the engulfment of these proteins by autophagosomes is dependent on binding to LC3b-II,45,71,72
and so these proteins are themselves indirectly membrane-associated. One additional caveat of LC3b turnover assays is the assumption that LC3b is recruited to autophagosome membranes at similar densities irrespective of the stimuli used to elicit autophagosome production. To our knowledge there is no literature directly addressing this point but, interestingly, autophagosomes produced in response to starvation have higher fusogenic activity in vitro than autophagosomes produced in the basal state.67
Since yeast ATG8 can promote the vesicle tethering and hemifusion in vitro,73
it may be important to investigate the degree to which LC3b density on autophagosome membranes might vary under differing stimuli. Finally, LC3b turnover assays will not detect degradation occurring through the recently described non-canonical macroautophagy pathway because autophagosomes produced by this process do not incorporate LC3b.74
Although these caveats should be kept in mind when interpreting results, we found that the approach of measuring LC3b turnover is clearly capable of detecting physiologically relevant differences in macroautophagic flux between organs and between animals of different genetic backgrounds. As such it represents both a powerful and feasible approach for gauging macroautophagic flux in vivo.
In summary, we demonstrate a useful leupeptin-based assay for estimating macroautophagic flux in vivo. This method should prove valuable for investigating the regulation of macroautophagy in mammalian models of complex diseases.