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Loss of gut integrity is linked to various human diseases including inflammatory bowel disease. However, the mechanisms that lead to loss of barrier function remain poorly understood. Using D. melanogaster, we demonstrate that dietary restriction (DR) slows the age-related decline in intestinal integrity by enhancing enterocyte cellular fitness through up-regulation of dMyc in the intestinal epithelium. Reduction of dMyc in enterocytes induced cell death, which leads to increased gut permeability and reduced lifespan upon DR. Genetic mosaic and epistasis analyses suggest that cell competition, whereby neighboring cells eliminate unfit cells by apoptosis, mediates cell death in enterocytes with reduced levels of dMyc. We observed that enterocyte apoptosis was necessary for the increased gut permeability and shortened lifespan upon loss of dMyc. Furthermore, moderate activation of dMyc in the post-mitotic enteroblasts and enterocytes was sufficient to extend health-span on rich nutrient diets. We propose that dMyc acts as a barometer of enterocyte cell fitness impacting intestinal barrier function in response to changes in diet and age., Author summary Dietary restriction (DR) is a robust environmental method to slow aging and age-related diseases in diverse organisms. Age-related disruption of gut integrity has been observed in both mammals and fruit flies and is a determinant of lifespan. In Drosophila, DR is able to slow the age-related decline in gut integrity. Although commensal dysbiosis has been proposed as a leading cause of gut barrier dysfunction, antibiotic treatment does not prevent the age-related increase in gut permeability. We identify that an intrinsic mechanism regulates gut barrier function through regulation of enterocyte apoptosis by ‘cell competition’. We show DR up-regulates dMyc expression in the gut which enhances enterocyte cellular fitness, prevents the age-related decline in gut integrity, and contributes to DR-induced lifespan extension. Conversely, on a rich diet, inhibition of dMyc in the enterocytes leads to cell death that enhances gut permeability and leads to systemic inflammation and shortened lifespan.

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., Author Summary The long-term maintenance of tissue homeostasis in barrier epithelia requires precise coordination of cellular stress and inflammatory responses with regenerative processes. This coordination is lost with age, resulting in degenerative and proliferative diseases. The Unfolded Protein Response of the Endoplasmic Reticulum (UPRER) is emerging as a central regulator of tissue homeostasis in barrier epithelia. The UPRER adjusts the protein folding capacity of the ER in response to protein stress in stem cells and differentiated cells, and thus influences proliferative homeostasis, cell differentiation and epithelial inflammatory responses. How these responses are coordinated to maintain epithelial homeostasis in aging organisms remains unclear. In a previous study, we have found that the UPRER controls intestinal stem cell (ISC) proliferation in the Drosophila intestinal epithelium by influencing the intracellular redox state. How signaling through the canonical ER stress sensor PERK (PKR-like ER kinase) is integrated into this signaling network remained unclear. Here we show that PERK serves as a central regulator of ISC proliferation and tissue homeostasis in response ER stress. Strikingly, we find that within the intestinal epithelium, PERK is activated specifically in ISCs in response to both systemic and local ER stress, and is required for ISC proliferation under both homeostatic and stress conditions. We identify JAK/Stat signaling as an activator of PERK in ISCs in response to ER stress in neighboring cells, and find that the wide-spread age-associated increase in PERK activity in ISCs is a cause of age-related dysplasia in this tissue. Accordingly, limiting PERK activity in ISCs promotes homeostasis of the intestinal epithelium in old flies and extends lifespan.

Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs) and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK) signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan., Author Summary Somatic stem cells are critical for regeneration of many tissues, thus ensuring long-term maintenance of tissue function. Proliferation of stem and progenitor cells has to be limited, however, to prevent hyperproliferative diseases and cancer in aging animals. This conflict between the need for stem cell proliferative potential and cancer prevention compromises regeneration in many high-turnover tissues of aging animals, including humans. It remains to be established whether and how proliferative homeostasis can be optimized to positively influence lifespan. Our work addresses this question using fruitflies as a model, taking advantage of the recent discovery of regenerative processes in adult flies. In old flies, intestinal stem cells (ISCs) hyperproliferate, causing an accumulation of mis-differentiated daughter cells (a phenotype termed intestinal dysplasia). We show that the balance between regeneration and dysplasia in this tissue significantly influences lifespan. When ISC proliferation rates are reduced, but not completely inhibited, dysplasia is limited and lifespan is increased. This can be achieved by moderately reducing insulin and stress signaling activities, as well as by expressing protective proteins in somatic stem cell lineages. Our results show that optimizing proliferative homeostasis (i.e. limiting dysplasia, but allowing sufficient regeneration) in high-turnover tissues is an efficient strategy to extend lifespan.

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., Author Summary Loss of proper protein homeostasis (proteostasis) as well as increased production of reactive oxygen species (ROS) is a hallmark of aging. In complex metazoans, these processes can result in proliferative diseases and cancers. The protein folding capacity of the endoplasmic reticulum (ER) is monitored and maintained by the unfolded protein response of the ER (UPRER). In this study, we identify a coordinated role of UPRER and oxidative stress signaling in regulating the proliferation of intestinal stem cells (ISCs). We find that the ER-stress responsive transcription factor Xbp1 and the ER-associated degradation pathway component Hrd1 are sufficient and required cell autonomously in ISCs to limit their proliferative activity. This function is dependent on the activities of the stress sensor JNK and the redox-responsive transcription factor CncC, which we have previously identified as regulators of ISC proliferation. We further show here that promoting ER homeostasis in aging ISCs is sufficient to limit age-associated epithelial dysplasia. Our results establish the integration of UPRER and oxidative stress signaling as a central mechanism promoting regenerative homeostasis in the intestinal epithelium.