The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. 59apically and are in contact with the intestinal lumen (Fig. 1A, Supplementary Fig. 1A). 60Therefore, we hypothesized that age-related changes in SJ could directly contribute to 74and an enriched analysis of the dataset revealed that the "cell adhesion" gene ontology 75(GO) category was one of the most representative GO categories that change with age 76(Supplementary Table1). The expression level of the majority of these genes (16 of 18) 77was up-regulated in old flies, indicating that decreased transcription is not a primary 78 mechanism contributing to age-related changes in SJs in the midgut. 79In Drosophila, SJ are divided into two classes based on morphological 95One striking and consistent age-related change in SJs was observed at tricellular 96 junctions (TCJ) (arrowheads, Fig. 1D-I, L-M, P-T), the specialized junction at the 101In the adult posterior midgut, Gli co-localized with Dlg ( Fig. 2A-B'; Supplementary 102 Fig. 2A-A''), as described previously in wing discs 16, 17 . Gli protein was clearly detected 103at EC-EC and EC-EE cell TCJ (Fig. 2 A,C,F); however, no Gli protein was detected in 104ISCs/EBs (Fig. 2D). In midguts from aged flies, Gli localization was largely absent from 105 the TCJ, and protein levels were increased in the cytoplasm (Fig. 2F-H). In hindguts, no 106 changes in Gli localization or protein levels were observed, similar to our observation for 107 other SJ proteins ( Supplementary Fig. 1P-Q). Interestingly, Dlg appeared cytoplasmic, 108rather than membrane-localized, in ISC/EB 'nests', suggesting that definitive SJ may be 109 absent between ISC/EBs and that formation of SJ is coordinated with differentiation. 110Consistent with this hypothesis, SJ were not apparent between ISCs and EBs via EM 111( Fig. 2E). 112Given the significant changes in TCJ (Fig. 1 D-M, P-T) and the striking loss of Gli 113from TCJ in older animals ( Fig. 2G-H age was a significant factor contributing to changes in TCJ (Fig. 2I, J) 118To determine whether compromised TCJ function could contribute to age-related 119 changes in the intestine, Gli was depleted from TCJs using a drug-inducible version of 120 the GAL4-UAS system 19, 20 . Targeted gene expression using the 5966 GS GAL4 "driver" 125Depletion of Gli from ECs resulted in an accelerated loss of barrier integrity (Fig. 1263A ; Supplementary Fig. 2E). Integrity of the intestinal barrier can be assayed by feeding 127 flies a non-absorbable blue food dye. When the intestinal barrier is intact, the dye is 128 retaine...

SummaryIntestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. Here, we show that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. Our findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals.

BackgroundMAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. We have analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development.Methodology and Principal FindingsUsing loss-of-function mutants and over-expression conditions we find that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). We also show that MAP4K3 display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor.Conclusions and SignificanceWe suggest that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability.