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Abstract Age-related macular degeneration (AMD), a complex neurodegenerative disease, is a leading cause of visual impairment worldwide with a strong genetic component. Genetic studies have identified several loci, but few causal genes with functional characterization. Here we highlight multiple lines of evidence which show a causal role in AMD for SLC16A8, which encodes MCT3, a retinal pigment epithelium (RPE) specific lactate transporter. First, in an unbiased, genome-wide analysis of rare coding variants we show multiple SLC16A8 rare variants are associated with AMD risk, corroborating previous borderline significant reports from AMD rare variant studies. Second, we report a novel SLC16A8 mutation in a three-generation family with early onset macular degeneration. Finally, mis-expression in multiple model organisms shows functional and anatomic retinal consequences. This study highlights the important role for SLC16A8 and lactate regulation towards outer retina/RPE health and highlights a potential new therapeutic opportunity for the treatment of AMD.

Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung, and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases. The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation, and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS). Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor (nAchR) subunits. We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD-associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.

Cellular senescence and associated secretory phenotype (SASP) are thought to contribute to aging and tissue dysfunction, though it is unclear how SASP impacts regeneration. Here the authors show that SASP factors impair regeneration, and that Ptk7 is a key secreted protein mediating that dysregulation.

The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.

Stem cell migration is critical during adult tissue regeneration. Here, the authors demonstrate that enteroendocrine cells coordinate stem cell migration towards sites of injury in the Drosophila intestine by activating non-canonical Wnt signaling.

Mitochondrial dysfunction is a fundamental challenge in septic cardiomyopathy. Mitophagy and the mitochondrial unfolded protein response (UPRmt) are the predominant stress-responsive and protective mechanisms involved in repairing damaged mitochondria. Although mitochondrial homeostasis requires the coordinated actions of mitophagy and UPRmt, their molecular basis and interactive actions are poorly understood in sepsis-induced myocardial injury. Our investigations showed that lipopolysaccharide (LPS)-induced sepsis contributed to cardiac dysfunction and mitochondrial damage. Although both mitophagy and UPRmt were slightly activated by LPS in cardiomyocytes, their endogenous activation failed to prevent sepsis-mediated myocardial injury. However, administration of urolithin A, an inducer of mitophagy, obviously reduced sepsis-mediated cardiac depression by normalizing mitochondrial function. Interestingly, this beneficial action was undetectable in cardiomyocyte-specific FUNDC1 knockout (FUNDC1CKO) mice. Notably, supplementation with a mitophagy inducer had no impact on UPRmt, whereas genetic ablation of FUNDC1 significantly upregulated the expression of genes related to UPRmt in LPS-treated hearts. In contrast, enhancement of endogenous UPRmt through oligomycin administration reduced sepsis-mediated mitochondrial injury and myocardial dysfunction; this cardioprotective effect was imperceptible in FUNDC1CKO mice. Lastly, once UPRmt was inhibited, mitophagy-mediated protection of mitochondria and cardiomyocytes was partly blunted. Taken together, it is plausible that endogenous UPRmt and mitophagy are slightly activated by myocardial stress and they work together to sustain mitochondrial performance and cardiac function. Endogenous UPRmt, a downstream signal of mitophagy, played a compensatory role in maintaining mitochondrial homeostasis in the case of mitophagy inhibition. Although UPRmt activation had no negative impact on mitophagy, UPRmt inhibition compromised the partial cardioprotective actions of mitophagy. This study shows how mitophagy modulates UPRmt to attenuate inflammation-related myocardial injury and suggests the potential application of mitophagy and UPRmt targeting in the treatment of myocardial stress.

The molecular mechanisms of mitochondrial dysfunction in the premature ageing Werner syndrome were elusive. Here the authors show that NAD+ depletion-induced impaired mitophagy contributes to this phenomenon, shedding light on potential therapeutics.

Notch signaling mediates intestinal enteroblast specification in Drosophila but the molecular mechanism as to how this is regulated is unclear. Here, the authors show that the transcription factor Klumpfuss ensures enteroblast commitment through repression of enteroendocrine cell fate downstream of Notch.

Regeneration after injury in the Drosophila intestine involves early activation of intestinal stem cells (ISCs) and subsequent return to quiescence. Here the authors show that return to quiescence by ISCs involves BMP Type I receptor Tkv protein stabilization along with AWD mediated internalization into endocytic vesicles.

Protein homeostasis maintenance (proteostasis) is critical for cell function, but declines during aging. Here the authors detail a proteostatic checkpoint in Drosophila intestinal stem cells coordinating cell cycle arrest with protein aggregate clearance, along with its role in aging related intestinal dysfunction.

Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single-cell RNA-seq to explore stem-cell-intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age-related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified intestinal stem cells (ISCs) and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.

Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. Here we identify Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFβ) superfamily, that includes TGFβ and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, we show that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, we find a dynamic response of retinal cells to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFβ/BMP signaling, as supplementation of BMP4 or inhibition of TGFβ using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. Our data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained.

Intestinal stem cells (ISCs) maintain regenerative capacity of the intestinal epithelium. Their function and activity are regulated by transcriptional changes, yet how such changes are coordinated at the genomic level remains unclear. The Cohesin complex regulates transcription globally by generating topologically-associated DNA domains (TADs) that link promotor regions with distant enhancers. We show here that the Cohesin complex prevents premature differentiation of Drosophila ISCs into enterocytes (ECs). Depletion of the Cohesin subunit Rad21 and the loading factor Nipped-B triggers an ISC to EC differentiation program that is independent of Notch signaling, but can be rescued by over-expression of the ISC-specific escargot (esg) transcription factor. Using damID and transcriptomic analysis, we find that Cohesin regulates Esg binding to promoters of differentiation genes, including a group of Notch target genes involved in ISC differentiation. We propose that Cohesin ensures efficient Esg-dependent gene repression to maintain stemness and intestinal homeostasis.

Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance.

Summary: Tissue homeostasis depends on precise yet plastic regulation of stem cell daughter fates. During growth, Drosophila intestinal stem cells (ISCs) adjust fates by switching from asymmetric to symmetric lineages to scale the size of the ISC population. Using a combination of long-term live imaging, lineage tracing, and genetic perturbations, we demonstrate that this switch is executed through the control of mitotic spindle orientation by Jun-N-terminal kinase (JNK) signaling. JNK interacts with the WD40-repeat protein Wdr62 at the spindle and transcriptionally represses the kinesin Kif1a to promote planar spindle orientation. In stress conditions, this function becomes deleterious, resulting in overabundance of symmetric fates and contributing to the loss of tissue homeostasis in the aging animal. Restoring normal ISC spindle orientation by perturbing the JNK/Wdr62/Kif1a axis is sufficient to improve intestinal physiology and extend lifespan. Our findings reveal a critical role for the dynamic control of SC spindle orientation in epithelial maintenance. : Hu and Jasper demonstrate that spindle orientation regulates stem cell fate in Drosophila intestines. JNK activity promotes reorientation from oblique to planar spindles, increasing symmetric outcomes. This switch is required for growth but becomes chronic during stress and age. Restoring oblique spindles in old animals improves tissue physiology, extending lifespan. Keywords: JNK, spindle orientation, cell fate, intestinal stem cell, aging, Wdr62, Kif1a, regeneration, growth, Drosophila

Summary: Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that the IRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan. : A metabolic switch that enhances triglyceride utilization mediates the protective effects of dietary restriction (DR), which extends the health span in multiple species. Luis et al. find that intestinal cells respond to DR by increasing lipid synthesis via the IRE1/XBP1/sugarbabe signaling module, thus extending longevity in Drosophila. Keywords: dietary restriction, aging, IRE1, metabolic adaptation, lipid turnover, longevity, Drosophila

The gastrointestinal tract of complex metazoans is highly compartmentalized. It is lined by a series of specialized epithelia that are regenerated by specific populations of stem cells. To maintain tissue homeostasis, the proliferative activity of stem and/or progenitor cells has to be carefully controlled and coordinated with regionally distinct programs of differentiation. Metaplasias and dysplasias, precancerous lesions that commonly occur in the human gastrointestinal tract, are often associated with the aberrant proliferation and differentiation of stem and/or progenitor cells. The increasingly sophisticated characterization of stem cells in the gastrointestinal tract of mammals and of the fruit fly Drosophila has provided important new insights into these processes and into the mechanisms that drive epithelial dysfunction. In this Review, we discuss recent advances in our understanding of the establishment, maintenance and regulation of diverse intestinal stem cell lineages in the gastrointestinal tract of Drosophila and mice. We also discuss the field's current understanding of the pathogenesis of epithelial dysfunctions.

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.

Regeneration is an important process in multicellular organisms, responsible for homeostatic renewal and repair of different organs after injury. Immune cell activation is observed at early stages of the regenerative response and its regulation is essential for regenerative success. Thus, immune regulators play central roles in optimizing regenerative responses. Neurotrophic factors (NTFs) are secreted molecules, defined by their ability to support neuronal cell types. However, emerging evidence suggests that they can also play important functions in the regulation of immune cell activation and tissue repair. Here we discuss the literature supporting a role of NTFs in the regulation of inflammation and regeneration. We will focus, in particular, in the emerging roles of mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) in the regulation of immune cell function and in the central role that immune modulation plays in their biological activity in vivo. Finally, we will discuss the potential use of these factors to optimize regenerative success in vivo, both within and beyond the nervous system.

The mitochondrial unfolded protein response (UPRmt) has been associated with long lifespan across metazoans. In Caenorhabditis elegans, mild developmental mitochondrial stress activates UPRmt reporters and extends lifespan. We show that similar developmental stress is necessary and sufficient to extend Drosophila lifespan, and identify Phosphoglycerate Mutase 5 (PGAM5) as a mediator of this response. Developmental mitochondrial stress leads to activation of FoxO, via Apoptosis Signal-regulating Kinase 1 (ASK1) and Jun-N-terminal Kinase (JNK). This activation persists into adulthood and induces a select set of chaperones, many of which have been implicated in lifespan extension in flies. Persistent FoxO activation can be reversed by a high-protein diet in adulthood, through mTORC1 and GCN-2 activity. Accordingly, the observed lifespan extension is prevented on a high-protein diet and in FoxO-null flies. The diet-sensitivity of this pathway has important implications for interventions that seek to engage the UPRmt to improve metabolic health and longevity.

In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. Here, we characterize the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila. We find that activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes is required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis.

The gastrointestinal tract is lined by a series of epithelia that share functional requirements but also have distinct, highly specialized roles. Distinct populations of somatic stem cells (SCs) regenerate these epithelia, yet the mechanisms that maintain regional identities of these SCs are not well understood. Here, we identify a role for the BMP-like Dpp signaling pathway in diversifying regenerative processes in the adult gastrointestinal tract of Drosophila. Dpp secreted from enterocytes at the boundary between the posterior midgut and the middle midgut (MM) sets up a morphogen gradient that selectively directs copper cell (CC) regeneration from gastric SCs in the MM and thus determines the size of the CC region. In vertebrates, deregulation of BMP signaling has been associated with Barrett’s metaplasia, wherein the squamous esophageal epithelium is replaced by a columnar epithelium, suggesting that the maintenance of regional SC identities by BMP is conserved.

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.