Faculty Bibliography

A number of age-dependent degenerative diseases are caused by chronic endoplasmic reticulum (ER) stress in vital cells. In many cases, the afflicted cells suffer from ER stress since birth, but the death of irreplaceable cells occurs only late in life. Although our understanding of ER stress-induced cell death has advanced significantly, most of the known mechanisms involve pathways that signal within hours, and it remains unclear how these pathways regulate cell death that occurs only decades later. Here, I highlight the conceptual issues and suggest ways to make sense of the age-related effect of ER stress-induced cell death in degenerative diseases

Drosophila has served as a particularly attractive model to study cell death due to the vast array of tools for genetic manipulation under defined spatial and temporal conditions in vivo as well as in cultured cells. These genetic methods have been well supplemented by enzymatic assays and a panel of antibodies recognizing cell death markers. This chapter discusses reporters, mutants, and assays used by various laboratories to study cell death in the context of development and in response to external insults.

Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms.

Intestinal homeostasis requires precise control of intestinal stem cell (ISC) proliferation. In Drosophila, this control declines with age largely due to chronic activation of stress signaling and associated chronic inflammatory conditions. An important contributor to this condition is the age-associated increase in endoplasmic reticulum (ER) stress. Here we show that the PKR-like ER kinase (PERK) integrates both cell-autonomous and non-autonomous ER stress stimuli to induce ISC proliferation. In addition to responding to cell-intrinsic ER stress, PERK is also specifically activated in ISCs by JAK/Stat signaling in response to ER stress in neighboring cells. The activation of PERK is required for homeostatic regeneration, as well as for acute regenerative responses, yet the chronic engagement of this response becomes deleterious in aging flies. Accordingly, knocking down PERK in ISCs is sufficient to promote intestinal homeostasis and extend lifespan. Our studies highlight the significance of the PERK branch of the unfolded protein response of the ER (UPRER) in intestinal homeostasis and provide a viable strategy to improve organismal health- and lifespan.

The synthesis of proteins in the endoplasmic reticulum (ER) that exceeds the protein folding capacity of this organelle is a frequent cause of cellular dysfunction and disease. An example of such a disease is alpha-1-antitrypsin (A1AT) deficiency, caused by destabilizing mutations in this glycoprotein. It is considered that the mutant proteins are recognized in the ER by lectins and are subsequently degraded through the proteasome, leading to a deficiency in this enzyme in the afflicted patients. We previously established a Drosophila model of this disease by overexpressing the null Hong Kong (NHK) allele of this gene and found that the Drosophila lectin, ER degradation-enhancing alpha-mannosidase-like protein 2 (EDEM2), can accelerate the degradation of A1AT when overexpressed. NHK is a rare allele, and in this study, we investigated in depth the mechanisms through which Drosophila EDEMs affect the degradation of the Z variant, which is the predominant disease allele. Specifically, we report that the Z allele does not activate ER stress signaling as prominently as the NHK allele, but similarly requires both Drosophila EDEM1 and EDEM2 for the degradation of the protein. We demonstrate that EDEMs are required for their ubiquitination, and without EDEMs, glycosylated A1AT mutants accumulate in cells. These results support the role of the EDEM-mediated ubiquitination of the alpha-1-antitrypsin Z (ATZ) allele, and establish a Drosophila model for the study of this protein and disease.

[This corrects the article DOI: 10.1371/journal.pone.0069147.].

Inhibitors of apoptosis (IAPs) family of genes encode baculovirus IAP-repeat domain-containing proteins with antiapoptotic function. These proteins also contain RING or UBC domains and act by binding to major proapoptotic factors and ubiquitylating them. High levels of IAPs inhibit caspase-mediated apoptosis. For these cells to undergo apoptosis, IAP function must be neutralized by IAP-antagonists. Mammalian IAP knockouts do not exhibit obvious developmental phenotypes, but the cells are more sensitized to apoptosis in response to injury. Loss of the mammalian IAP-antagonist ARTS results in reduced stem cell apoptosis. In addition to the antiapoptotic properties, IAPs regulate the innate immune response, and the loss of IAP function in humans is associated with immunodeficiency. The roles of IAPs in Drosophila apoptosis regulation are more apparent, where the loss of IAP1, or the expression of IAP-antagonists in Drosophila cells, is sufficient to trigger apoptosis. In this organism, apoptosis as a fate is conferred by the transcriptional induction of the IAP-antagonists. Many signaling pathways often converge on shared enhancer regions of IAP-antagonists. Cell death sensitivity is further regulated by posttranscriptional mechanisms, including those regulated by kinases, miRs, and ubiquitin ligases. These mechanisms are employed to eliminate damaged or virus-infected cells, limit neuroblast (neural stem cell) numbers, generate neuronal diversity, and sculpt tissue morphogenesis.

Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5'-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5' UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.

The Unfolded Protein Response of the endoplasmic reticulum (UPRER) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. Here we show that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. We find that ISCs require activation of the UPRER for regenerative responses, but that a tissue-wide increase in ER stress triggers ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. Our results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium.

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.