Aging is characterized by the accumulation of damage at the cellular level, which leads to tissue malfunction and organismal decline. Autophagy is a cellular, protective mechanism that allows the degradation and recycling of cytoplasmic materials, referred to as cargo; this includes damaged components that, if not eliminated, can compromise cell function. This process is fundamental to maintain cells homoeostasis and longevity, and its malfunctioning has been associated with the onset of age-related diseases, such as neurodegeneration. In turn, evidence suggests that autophagy becomes compromised with age. However, the molecular mechanisms that cause age-dependent autophagy deregulation remain to be identified. Interestingly, key proteins functioning in canonical, degradative autophagy have recently been discovered to play additional non-canonical secretory roles in the cell. However, the regulation of such non-canonical secretory functions is fundamentally unknown; likewise, it is unexplored if such functions are affected with age. The autophagy protein Atg8/LC3B is an example of a protein with roles in both canonical and non-canonical autophagy. LC3B incorporates into degradative and secretory vesicles and is key for the recruitment of protein determinants to them. Our lab recently discovered that LC3B is regulated by a phosphorylation event that is required for autophagy completion (Wilkinson et al., Molecular Cell, 2015); however, the specific step of the autophagy process that is affected by phosphorylation has yet to be determined. In my preliminary studies, I have found that this LC3B phosphorylation dictates the directional transport of LC3B-positive vesicles, and I hypothesize that this could be dictating their degradative or secretory fate. My proposed studies aim to investigate this hypothesis, and will elucidate the relevance of LC3B phosphorylation as a regulatory mechanism important for the bifurcation of degradative and secretory functions of the autophagy machinery. To this end, I will use proteomic and live-imaging approaches to characterize, for the first time, the effect of LC3B phosphorylation in degradative and secretory vesicles formation, cargo composition, and directional transport in cell. Based on the proteomic analysis of the secretory and degradative vesicles, I may identify new components and pathways specifically required for non-canonical, LC3B-mediated secretion, which remain essentially unexplored. Finally, to start probing possible mechanisms underlying age- related autophagy decline, I will investigate how LC3B phosphorylation and its role in the above-described processes are affected in cells and tissues from aged mice. My studies are significant because they will generate new mechanistic insights on the key autophagy protein LC3B. Such knowledge may have important repercussions for our understanding of diseases with an autophagy-deregulation component, such as neurodegeneration, in which the relevance of the secretory functions of the autophagy machinery has yet to be considered.
Autophagy is a protective mechanism that declines during aging. While traditionally studied as a recycling process that degrades cellular components including ?waste?, proteins with functions in degradative autophagy have recently been appreciated to also carry out novel secretory functions of unknown relevance. I will study the molecular mechanisms regulating these divergent functions in the cell, and begin to assess how they progress during aging.
