Project Outline

Ageing is a complex process that involves a progressive decline in physiological functions of an organism, eventually causing disease and death. During this decline, molecular and cellular damage accumulates such as shortening of telomeres, deleterious mutations, and accumulation of reactive oxygen species and damaged organelles. One of the phenotypic hallmarks of aged cells is the dysregulation of protein homeostasis. The cellular machinery of autophagy along with the proteasome regulates protein homeostasis. Autophagy is an evolutionarily conserved process where the cells degrade their own cellular material. It is involved in protein and organelle degradation and plays an essential role in both cellular and whole-animal homeostasis. During autophagy there is sequestration of cellular material into double-membrane vesicles called autophagosomes. The autophagosomes are subsequently fused with the lysosomes where the sequestered cargoes are degraded by lysosomal hydrolases. The products of degradation are transported back into the cytoplasm through lysosomal membrane permeases and can be reused by the cell (1). Autophagy serves as a cellular response in nutrient starvation but it is also responsible for the removal of aggregated proteins and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Autophagy is implicated in ageing, neurodegeneration, infections, tumorigenesis, heart disease, liver and lung disease and myopathies. Although it was initially believed that autophagy occurs randomly inside the cell, during the last years there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective through receptor and adaptor proteins that contain short linear motifs called LIR motifs (LC3-interacting region motifs)(1). Accumulating evidence now indicates that autophagic degradation declines with age and this gradual reduction of autophagy might have a causative role in the functional impairment of biological systems during ageing. Indeed, loss of autophagy gene function significantly influences longevity (1).

To address the role of selective autophagy during ageing the fruit fly Drosophila melanogaster will be used as a genetically modifiable model organism. The aim of this project is to undertake a systematic analysis of a novel selective autophagy Drosophila CRISPR mutant that was recently created in Nezis’ lab. The main objective of the project is the characterization of the Atg8a LIR and UIM -binding pocket CRISPR mutant in selective autophagy during ageing. Specifically, we will:1) We will define the physiological relevance of Atg8a LIR and UIM point mutations during ageing and neurodegeneration, 2) We identify using quantitative proteomics novel autophagy substrates that accumulate in Atg8a LIR and UIM point mutants, 3) We will re-engineer the autophagic machinery to degrade selected autophagy substrates in vitro and vivo and examine how this could ameliorate disease phenotypes in Drosophila models of human neurodegeneration diseases. We expect to identify novel mechanisms of selective autophagy that regulate ageing.

Techniques

• Basic cell, molecular biology and biochemistry
• Drosophila genetics and cell biology
• Confocal microscopy
• Electron microscopy
• Bioinformatics
• Structural biology modelling