Rapamycin preserves gut homeostasis during Drosophila aging

Abstract

Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila.

Keywords: Drosophila; Gerotarget; aging; gut homeostasis; intestinal stem cell; rapamycin.

Figure 1. Rapamycin slows ISCs proliferation rates

A., Rapamycin treatment extends the life span of w^Dah females. Compared to flies on control food (0μM of rapamycin), flies on food having 200μM of rapamycin have increased median life spans (T test p < 0.0001). B and C, esg-positive (esg⁺) cells (green) in control B., and rapamycin treated C., young (3 Days old) fly guts. D.-D′, esg⁺cells (green) and ISCs (by Delta) in control aging (20Days old) fly guts. E.-E′, esg⁺cells (green) and ISCs (by Dl) in rapamycin treated aging (20Days old) fly guts.F., Quantification of midgut GFP cells in control and rapamycin treated 20Days old guts. Mean±SD is shown. n = 8-10 guts. **p < 0.01.

Figure 2. Rapamycin slows down the intestinal barrier dysfunction

A., A 10 days old fly after consuming a non-absorbed food dye (FD&C blue dye #1). The dye is restricted to the proboscis and digestive tract. B., A 40 days old aged “Smurf” fly after consuming the same food dye. The blue dye is seen throughout the body due to loss of intestinal integrity. C., Analysis of intestinal integrity as a function of age. In control flies, the percentage of “Smurf” flies in the population increases with age. Rapamycin pretreatment improves intestinal integrity in aged flies. (***p < 0.001, n >60 females for each genotype), Data are represented as mean ± SEM.

Figure 3. Rapamycin activating the autophagy in the aging guts

A., Intestinal expression of antioxidant gene catalase in 30 days old female fly guts. B.-D., Expression levels of autophagy genes in 30 days old female fly guts. (*p < 0.05, ** p < 0.01,***p < 0.001, n = 3 of RNA extracted from 7 intestines/replicate).

Figure 4. Rapamycin treatment impacts FOXO associated genes in the intestine

A., Expression levels of gene Foxo in intestines of 30 days old flies at control and rapamycin induction. B., Expression levels of insulin receptor (InR) in intestines of 30 days old flies at control and rapamycin induction. C., Expression levels of AMP gene Dpt in intestines of 30 days old flies at control and rapamycin induction. D., Duox mRNA expression were quantified by RT-PCR in intestines of 30 days old flies at control and rapamycin induction. (***p < 0.001, *p < 0.05).

Figure 5. Rapamycin treatment delayed the commensal intestinal dysplasia by up-regulating negative regulators of IMD/Rel pathway

A., CFUs in intestinal extracts of control and rapamycin treated flies. Midgut homogenates from flies at 7 days or 40 days of age were plated on nutrient-rich medium (NR) or on selective plates allowing growth of Lactobacilli (LMRS), Acetobacteria (ACE), or Enterobacteria (ENT). B., qRT-PCR for rel expression C.-E., qRT-PCR for pgrp-sc1, pgrp-sc2, caudal and usp36 expression (n = 3; *p < 0.05,**p < 0.01,***p < 0.001).