Macroautophagy, hereafter referred to as autophagy, is a catabolic process involving the degradation of cellular proteins and structures sequestered into a vesicle known as an autophagosome. The initiation of autophagy involves the conversion of a protein microtubule-associated proteins 1A/1B light chain 3B (LC3) from form I to form II allowing interaction with the formation of the autophagosome. Using an LC3-II/I ratio, relative initiation of autophagy can be estimated since higher relative amounts of LC3-II suggests a higher conversion rate of LC3-I to LC3-II, therefore suggesting autophagosomes are being formed at a higher rate. Autophagy’s selectivity, or its ability to degrade specific targets, is dependent on the degradation of ubiquitinated proteins and a protein adaptors, the latter forming a physical bridge between the ubiquitin-tagged cargo and LC3-II present on the forming autophagosome. Without these protein adaptors, autophagy has no selectivity and portions of the cytosol that happen to be near the autophagosome formation site are the only cellular components captured and degraded. Because the entire contents of the autophagosome are degraded following lysosome fusion, the selectivity can be assessed by determining the levels of protein adaptor and ubiquitinated proteins. Autophagy is constitutively active but is strongly stimulated under nutrient deprivation, such as fasting. Impairments of autophagy have been implicated in contractile and/or metabolic deficiencies in muscle diseases, obesity, diabetes, and aging; however, regulation of skeletal muscle autophagy is poorly understood at the molecular level. Here, we examined the role of the two partially homologous unc-51 like autophagy activating kinases 1 and 2 (ULK1 and ULK2) in modulating autophagy and myofiber atrophy during fasting via a microRNA-specific knockdown of these proteins in mouse skeletal muscle and using a non-specific microRNA in the contralateral muscle to allow comparisons of ULK effects within the same animal.
Our results revealed that deficiency of ULK1 caused LC3-I to accumulate in fasted muscle without changes in Lc3b mRNA, indicating an impairment in the step of LC3-I conversion into LC3-II (an essential step in autophagy initiation). Similar trends were observed with other LC3-like proteins (GABL1 and GABL2) suggesting a specific role for ULK1 in regulating autophagy initiation. Deficiency of ULK2 did not affect LC3 or LC3-like proteins suggesting that ULK2 does not regulate autophagy initiation. However, it led to accumulation of ubiquitinated proteins, and the autophagy adaptors p62 and NBR-1, under both basal and fasting conditions. Since autophagy adaptors bind to and are degraded together with ubiquitinated proteins, these findings are consistent with impaired involvement of adaptors and consequent deficient cargo recognition by autophagy. Of note, deficient expression of either ULK1, ULK2, or both ULK1 & ULK2 did not attenuate myofiber atrophy during fasting.
Altogether, these results uncover fundamental divergent roles for ULK1 and ULK2 in modulating autophagy and its selectivity in muscle. Current and future studies in our laboratory will further expand the molecular signature of autophagy activation and selectivity in muscle in order to identify novel targets for therapy in conditions associated with autophagy deficiency.