Cell Death and Autophagy

Our laboratory studies the mechanisms that regulate autophagy, cell survival and programmed cell death in the context of normal and abnormal development.  Altered autophagy, cell survival and cell death are associated with a variety of human disorders including cancer.  We use global genomic approaches, including DNA microarrays, proteomics and forward genetic screens, to identify genes and proteins involved in programmed cell death and development.  The Drosophila model system allows us to functionally validate our gene discovery, and also enables the identification of novel genes using forward genetic screens.  Our studies have focused on steroid activation of cell death, and the role of autophagy and other degradation mechanisms during cell survival and death.

Steroid Activation of Programmed Cell Death

At least two morphological forms of programmed cell killing occur during development of all higher animals.  While much is known about apoptotic cell death, little is known about autophagic cell death.  Autophagic cell death in Drosophila salivary glands involves the activation of diverse protein degradation programs that include both caspases and autophagy.  Fly salivary glands provide an in vivo genetic model system for elucidating the complex functions of autophagy in cell survival and cell death during development.

The steroid hormone ecdysone triggers salivary gland cell death by activation of a two-step nuclear hormone receptor-activated gene transcription hierarchy (Figure 1).  The ecdysone receptor, encoded by the nuclear receptors EcR and Usp, in the presence of the competence factor ßFTZ-F1, triggers the first step in this hierarchy by activating transcription of BR-C, E74, and E93.  Mutations in each of these transcriptional regulators prevent the death of salivary glands, and alter transcription of secondary response genes that carry out the cell death program.  By analyzing mutants with phenotypes similar to those seen in BR-C, E74 and E93 mutants, we expect to identify novel cell death regulatory molecules. 

Our microarray analysis demonstrated the robust nature of this steroid-triggered transcriptional hierarchy through the identification of genes known to function in apoptosis but which had not been previously associated with autophagic cell death, as well as many candidate BR-C, E74 and E93 target genes.  Although autophagic cell death is morphologically distinct from apoptosis, our studies have shown that during autophagic death of salivary glands, caspases are activated and caspase substrates are cleaved, suggesting that much of the apoptotic machinery is utilized during autophagic cell death.  Our microarray and proteomics analyses also indicate that several caspase-independent protein degradation mechanisms including matrix metallo-protease, serine protease and autophagy RNAs and proteins are expressed during cell death.  Projects in the Baehrecke lab are aimed toward understanding how steroid-triggered transcription factors regulate secondary response gene transcription, and how these multiple protein degradation programs are coordinated to mediate programmed cell death. 

Degradation of Dying Cells by Autophagy  

Although our studies indicate that much of the apoptotic machinery is utilized during autophagic cell death, dying salivary glands are destroyed without the association of engulfing phagocytes that are necessary for the removal of cell corpses during apoptosis.  This indicates that salivary gland cells contain the machinery needed to dismantle and recycle their own cellular components, and this is supported by the presence of autophagic vacuoles (also known as autophagosomes) in the cytoplasm of salivary gland cells.  Autophagic vacuoles are a hallmark of autophagy, a conserved catabolic process that functions in the degradation of long-lived proteins and in organelle turnover and recycling (Figure 2).  

The role of autophagy during cell death remains controversial. It is important to understand the relationship between autophagy and cell death, as the association of autophagy with cell growth, nutrient utilization, survival and death indicates that this catabolic process is relevant to the treatment of many human disorders including cancer.  Our recent studies indicate that autophagy is required for degradation of dying salivary glands during Drosophila development.  In collaboration with Michael Lenardo at the National Institutes of Health, we have shown that autophagy is required for the caspase-independent death in mammalian cells.  In contrast, our work in collaboration with Paul Taylor at the University of Pennsylvania has shown that autophagy is protective in a fly model of polyglutamine-induced neurodegeneration.  Phosphoinositide 3-kinase (PI3K) pathways regulate cell growth, cell death and autophagy, but the mechanisms that coordinate these cell processes remains uncertain.  We are investigating the role(s) of autophagy in cell growth and cell death, and how steroids and PI3K signaling mechanisms are integrated to regulate autophagy in the context of development (Figure 3).  As in our studies of neurodegeneration, we hope to extend our studies of the genes that are required for autophagy and cell death in fly salivary glands into models of cancer.