2014 Theses Doctoral

The role of phosphatidylinositol 3-kinases in autophagy regulation

Devereaux, Kelly Anne

Autophagy requires the biogenesis of autophagosomes (APs), which are large multilamellar vesicles that sequester cytoplasmic substrates and undergo a maturation process that ultimately leads to their fusion with lysosomes. Previous studies have suggested that local production of phosphatidylinositol-3-phosphate (PI3P) by class III phosphatidylinositol 3-kinase (PI3K) (i.e., Vps34) is required for AP biogenesis at specialized sites of the endoplasmic reticulum called "omegasomes". Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. To address this question, we generated Vps34 knock-out mouse embryonic fibroblasts (MEFs) and analyzed the impact of Vps34 deletion on autophagy in mammalian cells. Using a novel higher affinity 4x-FYVE finger PI3P-binding probe, we found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in the Vps34 knock-out MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
In addition, we used a lipidomic approach to capture the lipid profile of cells in the presence and absence of Vps34 under steady-state and during starvation-induced autophagy. Lipidomics is an emerging powerful tool with the potential to identify new interconnected metabolic lipid networks as well as generate new hypotheses. Here, we identified a new relationship between Vps34 and cholesterol homeostasis. Additionally, we identified specific changes in lysolipids during autophagy.
Lastly, we investigated whether the retromer complex plays a role in autophagy. Retromer is a protein complex that binds PI3P on the endosomal membrane and mediates retrograde trafficking of transmembrane proteins from the endosome to the trans-Golgi network. Recent studies have shown a downregulation of this complex associated with sporadic Alzheimer's disease (AD) and have demonstrated aberrant trafficking and processing of APP, a pathological feature of AD, as a result of retromer deficiency. Because retromer is important for maintaining endo-lysosomal system function, we hypothesized that it promote efficient autophagy and may contribute to the dysfunctional autophagy observed in AD when impaired. Using standard autophagy assays, such as assessing LC3 conjugation and puncta formation, our preliminary studies suggest a negative regulatory role for retromer in autophagy. Additionally, we observed a strong association of retromer with Atg9, an autophagy-related gene transmembrane protein that is believe to traffic lipids to the growing autophagosome membrane and recycle autophagy proteins from this compartment.