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Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single‐gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age‐related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long‐lived animals with elevated Jun‐N‐terminal Kinase signaling. We find that these animals exhibit a delayed age‐related decline in protein turnover rates, as well as decreased steady‐state neuronal glucose‐6‐phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose‐6‐phosphate dehydrogenase (G6PD). Over‐expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long‐lived mutants.

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.

A decline in protein homeostasis (proteostasis) has been proposed as a hallmark of aging. Somatic stem cells (SCs) uniquely maintain their proteostatic capacity through mechanisms that remain incompletely understood. Here, we describe and characterize a ‘proteostatic checkpoint’ in Drosophila intestinal SCs (ISCs). Following a breakdown of proteostasis, ISCs coordinate cell cycle arrest with protein aggregate clearance by Atg8-mediated activation of the Nrf2-like transcription factor cap-n-collar C (CncC). CncC induces the cell cycle inhibitor Dacapo and proteolytic genes. The capacity to engage this checkpoint is lost in ISCs from aging flies, and we show that it can be restored by treating flies with an Nrf2 activator, or by over-expression of CncC or Atg8a. This limits age-related intestinal barrier dysfunction and can result in lifespan extension. Our findings identify a new mechanism by which somatic SCs preserve proteostasis, and highlight potential intervention strategies to maintain regenerative homeostasis. 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.

In adult epithelial stem cell lineages, the precise differentiation of daughter cells is critical to maintain tissue homeostasis. Notch signaling controls the choice between absorptive and entero-endocrine cell differentiation in both the mammalian small intestine and the Drosophila midgut, yet how Notch promotes lineage restriction remains unclear. Here, we describe a role for the transcription factor Klumpfuss (Klu) in restricting the fate of enteroblasts (EBs) in the Drosophila intestine. Klu is induced in Notch-positive EBs and its activity restricts cell fate towards the enterocyte (EC) lineage. Transcriptomics and DamID profiling show that Klu suppresses enteroendocrine (EE) fate by repressing the action of the proneural gene Scute, which is essential for EE differentiation. Loss of Klu results in differentiation of EBs into EE cells. Our findings provide mechanistic insight into how lineage commitment in progenitor cell differentiation can be ensured downstream of initial specification cues. 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.

While central circuits governing sleep are well-studied, the contribution of signaling from peripheral tissues remains a critical yet less understood aspect of sleep regulation. The highly conserved RNA-binding protein Pumilio (Pum) is a post-transcriptional regulator expressed in multiple tissues that influence systemic physiology, but its role in modulating basal sleep from peripheral tissues has not been established. Although Pumilio’s function in central neurons has been linked to sleep homeostasis following deprivation, whether it regulates sleep through peripheral mechanisms remains unknown. Here, we use conditional genetic tools in the fruit fly Drosophila melanogaster to demonstrate that genetic manipulation of Pumilio targeting the intestinal stem cells (ISCs) and the endocrine corpus allatum (CA) regulates the transition to sleep. Reducing Pumilio function in either the ISCs or the CA independently and significantly accelerates nighttime sleep onset, while overexpression produces the opposite effect. This behavioral change is accompanied by widespread transcriptional alterations in the head, characterized by a robust upregulation of genes involved in cellular stress responses. Our findings reveal a previously unrecognized gut-endocrine-brain signaling axis and identify peripheral post-transcriptional regulation as a key input to the central control of sleep behavior.

Eric Vilain and colleagues identify missense mutations in the imprinted gene CDKN1C , encoding the p57KIP2 cyclin dependent kinase inhibitor, in individuals with IMAGe syndrome. IMAGe syndrome is a developmental disorder characterized by intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies. IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences 1 . An identity-by-descent analysis in a family with IMAGe syndrome 2 identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2 ) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression 3 , and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C , suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome 4 .

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.

Diaprepes abbreviatus is an agricultural pest known to affect around 270 plant species across the Caribbean and the United States, posing significant challenges to pest management. Chemical control dominates management, but environmental and health concerns motivate microbiome-informed alternatives. However, limited information exists on the gut anatomy, physicochemical environment, and microbial composition of D. abbreviatus. In this study, we provide the first comprehensive characterization of the gut morphology, pH, and microbiota of adult D. abbreviatus in both females and males collected in Puerto Rico. Using dye-based gut tracing, we identified foregut, midgut, and hindgut or posterior gut compartments, and confirmed the presence of a muscular, sclerotized gizzard. Colorimetric analysis revealed a mildly acidic gut environment (approximately pH 4–5, based on qualitative ranges), consistent across sexes and regions. Shotgun metagenomic sequencing of dissected guts from males and females revealed microbial communities distinct from the leaf samples microbiota. While alpha and beta diversity did not differ significantly between sexes, co-occurrence analyses identified sex-specific correlation patterns among bacterial taxa. Notably, Enterobacter cloacae , Pantoea vagans , Lactococcus lactis , and Pseudomonas monteilii were repeatedly detected across individuals and generated metagenomic datasets, and some were localized to the hindgut, suggesting possible niche specialization. The presence of taxa, such as Enterobacter cloacae , previously reported as symbionts in other phytophagous insects further supports the hypothesis that certain bacteria may contribute to host digestion or adaptation. These findings establish a framework for understanding the gut environment and microbial community of D. abbreviatus , and highlight candidate taxa for future functional studies. More broadly, this work supports further research into the potential roles of gut microbiota in the ecology and management of this pest.

The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Autism spectrum disorder (ASD) is a lifelong neurological and developmental disorder that has no cure and is often accompanied by gastrointestinal (GI) issues. The bidirectional communication system known as the gut microbiota-brain axis may help explain how GI dysfunction contributes to neurological symptoms. Loss-of-function mutations in the histone demethylases , or are found in patients with intellectual disability and ASD. Previous studies using a Kdm5 loss-of-function ( ) ASD-like model revealed gut microbial dysbiosis, reduced abundance of , and impaired social behavior. While supplementation rescued intestinal abnormalities, it did not restore social behavior. Here, we evaluated multiple microbiota-based interventions, including probiotic supplementation with , , their combination, and fecal microbiota transplantation (FMT), to determine their capacity to modulate gut microbial composition and behavior in flies. Gut bacterial abundance was quantified using colony-forming unit (CFU) assays and full-length 16S rRNA gene sequencing. Social behavior was assessed using the social distance assay, while anxiety-like behavior and locomotion were evaluated using the open field test. Gut-specific knockdown was used to assess tissue-specific contributions to microbiota and behavioral phenotypes. deficiency resulted in reduced abundance of culturable , , and species, accompanied by impaired social behavior. supplementation restored gut microbial abundance in both whole-body and gut-specific knockdown models but did not significantly rescue social behavior. In contrast, significantly improved social interaction in flies despite minimal effects on gut bacterial abundance, revealing a dissociation between microbial restoration and behavioral outcomes. Gut-specific knockdown phenocopied both microbial and social defects observed in mutants. Notably, FMT from healthy donors partially restored abundance, reshaped gut microbial community structure, and partially improved social behavior in recipient flies. Together, these findings identify as a key regulator of gut microbial viability and social behavior and demonstrate that microbiota-based interventions exert strain- and phenotype-specific effects. Our results reveal that restoration of microbial abundance alone is insufficient to rescue social behavior and highlight the importance of functional host-microbe interactions in gut-brain communication. This work establishes as a tractable platform for dissecting epigenetic regulation of microbiota-behavior relationships relevant to ASD and for evaluating targeted probiotic and microbiota-transfer strategies.