Effectors and Regulation of Autophagy During Ageing

Ageing is characterized by a decrease in function of cells over time. Eukaryotic cells have evolved numerous surveillance and quality control systems in order to prevent, detect, and remove dysfunctional and damaged components and maintain cellular function. However, despite the existence of these mechanisms, cellular functions decline over cellular lifetime raising the possibility that (1) the capacity of these quality control mechanisms is generally insufficient, (2) damages accumulate existing mechanisms are not equipped to remove or (3) the regulatory systems of cells are connected in a way resulting in an inherently conflicting co-regulation of quality control and other cellular processes.

Autophagy is such a central quality control, homeostatic and stress response pathway conserved in all eukaryotic cells. This process allows cells to counteract stress conditions like nutrient starvation or cellular damage by degrading parts of their own cytoplasm.  Autophagy impairment is associated with the pathophysiology of ageing and a variety of diseases. Evidence indicates that autophagy activity is positively correlated with longevity and that reduced autophagy causes premature ageing and shortened lifespan in species. Conversely, autophagy capacity declines with increased age. We are interested in the interrelationship of this central homeostasis and quality control mechanism and the ageing process on a cellular level. To this end, our three overarching goals are (1) to elucidate the molecular and system-wide mechanisms required for the core process of autophagy and its stress-specific adaptations, (2) to gain a comprehensive understanding of the cellular effectors of autophagy capacity and (3) to determine the causal interrelation of changes in autophagy and in the cell during ageing. To achieve these goals, we have four major research areas: (1) system-wide modulation of the autophagy machinery; (2) spatial organization of autophagosome formation; (3) genome-wide determinants of autophagy capacity; and (4) age-associated effects on the autophagy machinery.

System-wide Modulation of the Autophagy Machinery

A unique feature of autophagy is the de novo formation of double-membrane vesicular structures, termed autophagosomes, that confer an unprecedented degradative capacity. Cargo of various size and nature, such as long-lived proteins, protein aggregates, organelles and invasive pathogens, are encapsulated during the formation of autophagosomes at so-called phagophore assembly sites (PAS) and delivered to vacuoles/lysosomes for degradation. Evolutionarily conserved core autophagy factors, namely autophagy-related genes (Atgs), that are essential for autophagosome biogenesis have been elegantly identified. However, despite recent advances in the field, we still have a very limited understanding of (1) what determines the sites of autophagosome biogenesis, (2) what the membrane sources for forming autophagosomes are, (3) how specific cargoes are selected and (4) how these processes are dynamically adapted to the diverse stresses posing specific and unique challenges to the cell. To address these fundamental questions, we are analysing the protein interaction space of core autophagy components and associated post-translational modifications in S. cerevisiae by proteomics based physical autophagy interaction network (AIN) analysis in response to diverse autophagy inducing and pathologic conditions. The functional significance of these protein interactions are tested by a variety of cytological, e.g. advanced fluorescence microscopy in living cells, and cell biological approaches (see Graef et al., 2013).

Spatial Organization of Autophagosome Formation

The site of autophagosome biogenesis is likely connected to proximal membrane sources and to the selection of cargo for growing autophagosomes. Our analysis of the AIN revealed significant direct and common physical interactions between the autophagy machinery and the early secretory COPII machinery (Graef et al., 2013). Our proteomic, cytological and functional analyses showed that the formation of autophagosomes is spatially, physically and functionally linked to specialized regions of the endoplasmic reticulum (ER), so-called ER exit sites (ERES), which play a role in the biogenesis of COPII vesicles. Specifically, our data show that ERES are core autophagy components required for the assembly of the autophagy machinery during the early stages of autophagosome formation and for the expansion of phagophores, likely by providing a proximal membrane source. Significantly, our experiments in mammalian cells revealed that ERES-associated autophagosome biogenesis is a conserved feature of autophagy. Our future aim is to explore the regulatory and molecular mechanisms that control and establish these hotspots for autophagosome formation.

Genome-wide Determinants of Autophagy Capacity

As indicated by our cell biological analysis, a variety of cellular processes directly or indirectly affect the ability of the cell to perform autophagy and to prevent cellular ageing or disease. However, we are still missing a comprehensive understanding of the cellular processes and factors that determine the autophagy capacity of the cell. We are addressing this deficit using the yeast model system and high content image analysis to identify factors and pathways that modify the autophagy capacity on a genome-wide level. We are analysing the collection of deletions of non-essential genes in S. cerevisiae using automated fluorescence microscopy and machine learning-based image analysis to identify clusters of genes required for the spatial organization and regulation of autophagy.

Age-associated Effects on the Autophagy Machinery

To date, the underlying molecular mechanisms of the interdependence of autophagy and cellular ageing have not been determined. To understand how the autophagy machinery changes with ageing, we are exploring established systems for replicative and chronological ageing in yeast. Careful functional, cytological and protein interaction network analyses of autophagy at different stages of the ageing process will allow us to identify the occurring defects. The combination of these approaches will shed light on the molecular mechanisms underlying the functional interrelation of autophagy and ageing.

Future Research Directions

The combined analysis of autophagy on a molecular as well as system- and genome-wide level will shed light on the fundamental question of how cellular homeostasis and quality control processes are affected by age-associated changes in the cell and may reveal novel and specific targets for therapeutic intervention.