Small molecule regulators of autophagy identified by an image-based high-throughput screen

Abstract

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.A utophagy is a cellular catabolic mechanism mediating the turnover of intracellular organelles and proteins through a lysosome-dependent but proteasome-independent degradative pathway (1, 2). An autophagosome sequesters cytoplasmic constituents, such as mitochondria, endoplasmic reticulum, and ribosomes, by forming a double-membrane vesicle. The outer membrane of the autophagosome then fuses with the lysosome in mammalian cells delivering the sequestered content to the lumen of lysosome for degradation. Autophagy is critical for the survival of yeast and mammalian cells under starvation conditions because it functions to recycle intracellular material for macromolecular synthesis and energy production (3).Autophagy occurs in all cells at low basal levels under normal conditions to perform homeostatic functions, but it can be rapidly up-regulated under starvation or stress conditions (3). Elegant genetic analysis has identified 17 genes that are essential for autophagy in yeast (referred to as the ATG genes) (4, 5). In mammalian cells, mTOR kinase, the target of rapamycin, mediates the major inhibitory signal that shuts off autophagy under nutrient-rich conditions (3). On the other hand, mammalian type III PI3-kinase, the homolog of yeast VPS34 and inhibitable by 3-methyladenine (3-MA) (a nonspecific inhibitor of PI3-kinase), is required for the onset of autophagy. In this regard, rapamycin and 3-MA, the most commonly used chemicals to induce and inhibit autophagy, respectively, provide convenient tools to study autophagy.To explore the mechanism of autophagy and identify additional small molecules that can activate it, we developed a high-throughput image-based screen. This system takes advantage of the local...

Association and dissociation of autophagy, apoptosis and necrosis by systematic chemical study

“…Beyond ultrastructural analyses, one of the most effective methods to detect autophagosomes and autophagolysosomes consists of the transfection-enforced expression of a green fluorescent protein (GFP)-LC3 fusion protein that decorates the membranes of autophago(lyso)somes, both in vivo, in animals , or in vitro, in cultured cells (Kabeya et al, 2000), including in high-content screens (Zhang et al, 2007;Lipinski et al, 2010). GFP-LC3 usually distributes diffusely throughout cells, yet accumulates in so-called 'autophagic puncta' (Kabeya et al, 2000) in the cytoplasm of cells when autophagy is induced.…”

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

“…This observation indicated a reduced proteolysis of DQcollagen IV, which implies altered lysosome functionality ( Figure 9E). Altered lysosome activity has been shown to induce autophagic stress (Zhang et al, 2007). Thus, we investigated whether the enlarged LysoTracker-positive structures found in mhtt cells could be in fact autophagic vacuoles.…”

Eating on the fly: Function and regulation of autophagy during cell growth, survival and death in Drosophila

“…Suppression of neurodegeneration by treatment with rapamycin provided motivation to identify compounds that facilitate autophagy, but act downstream of TOR. Recent screens for such molecules in yeast 34 and mammalian cells 35 identified small molecules that influence autophagy. Significantly, these compounds were able to induce autophagy and clearance of Huntington and alpha-synuclein in mammalian cells, and they also suppressed degeneration in a fly model of Huntington's disease.…”