QUANTITATIVE ANALYSIS OF AUTOPHAGIC ACTIVITY IN DROSOPHILA NEURAL TISSUES BY MEASURING THE TURNOVER RATES OF PATHWAY SUBSTRATES

The process of macroautophagy occurs in most eukaryotic cells and serves as the main recycling mechanism for the elimination of excess cytoplasmic components. The pathway is upregulated under a wide range of stress-related conditions and basal levels of autophagy are critical for the clearance of age-associated cellular damage, which can accumulate in tong-lived, nondividing cells such as neurons. Traditionally, activation of autophagy has been measured by the microscopic observation of newly formed autophagosomes or by monitoring the further modification of the LC3-I protein to the LC3-II isoform by Western blot analysis. However, using these methods to quantitatively determine autophagic activity that occurs in complex tissues over an entire life span has been a technical challenge and difficult to consistently reproduce. We have shown that Western analysis of protein substrates normally cleared by the pathway can be used to make quantitative estimates of autophagy occurring in tissues such as the adult Drosophila nervous system. By examining the profile of insoluble ubiquitinated proteins (aggregated proteins) we have found that an age-dependent decline in pathway flux or genetic defects in critical autophagic genes can result in the concomitant buildup of substrates that are normally targeted by autophagy to the lysosome. Further, we have found that increasing Atg8a expression (a key rate-limiting component of the pathway) during the time in which autophagy is normally suppressed prevents the age-dependent accumulation of insoluble ubiquitinated proteins in neurons. This technique, as well as the detection of proteins damaged by reactive carbonyl groups, can also be used to measure autophagic activity in both normal and genetically altered flies during the aging process or following their acute exposure to oxidants.