Trametinib ameliorates aging-associated gut pathology in Drosophila females by reducing Pol III activity in intestinal stem cells.

Pharmacological therapies are promising interventions to slow down aging and reduce multimorbidity in the elderly. Studies in animal models are the first step toward translation of candidate molecules into human therapies, as they aim to elucidate the molecular pathways, cellular mechanisms, and tissue pathologies involved in the anti-aging effects. Trametinib, an allosteric inhibitor of MEK within the Ras/MAPK (Ras/Mitogen-Activated Protein Kinase) pathway and currently used as an anti-cancer treatment, emerged as a geroprotector candidate because it extended lifespan in the fruit fly Drosophila melanogaster. Here, we confirm that trametinib consistently and robustly extends female lifespan, and reduces intestinal stem cell (ISC) proliferation, tumor formation, tissue dysplasia, and barrier disruption in guts in aged flies. In contrast, pro-longevity effects of trametinib are weak and inconsistent in males, and it does not influence gut homeostasis. Inhibition of the Ras/MAPK pathway specifically in ISCs is sufficient to partially recapitulate the effects of trametinib. Moreover, in ISCs, trametinib decreases the activity of the RNA polymerase III (Pol III), a conserved enzyme synthesizing transfer RNAs and other short, non-coding RNAs, and whose inhibition also extends lifespan and reduces gut pathology. Finally, we show that the pro-longevity effect of trametinib in ISCs is partially mediated by Maf1, a repressor of Pol III, suggesting a life-limiting Ras/MAPK-Maf1-Pol III axis in these cells. The mechanism of action described in this work paves the way for further studies on the anti-aging effects of trametinib in mammals and shows its potential for clinical application in humans.

Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease.

Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. We also demonstrate intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, we show that modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. We also reveal that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, we show that pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, is sufficient to stimulate intestinal immune activation. Overall, our data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders.

Sexual identity of enterocytes regulates autophagy to determine intestinal health, lifespan and responses to rapamycin.

Pharmacological attenuation of mTOR presents a promising route for delay of age-related disease. Here we show that treatment of Drosophila with the mTOR inhibitor rapamycin extends lifespan in females, but not in males. Female-specific, age-related gut pathology is markedly slowed by rapamycin treatment, mediated by increased autophagy. Treatment increases enterocyte autophagy in females, via the H3/H4 histone-Bchs axis, whereas males show high basal levels of enterocyte autophagy that are not increased by rapamycin feeding. Enterocyte sexual identity, determined by transformerFemale expression, dictates sexually dimorphic cell size, H3/H4-Bchs expression, basal rates of autophagy, fecundity, intestinal homeostasis and lifespan extension in response to rapamycin. Dimorphism in autophagy is conserved in mice, where intestine, brown adipose tissue and muscle exhibit sex differences in autophagy and response to rapamycin. This study highlights tissue sex as a determining factor in the regulation of metabolic processes by mTOR and the efficacy of mTOR-targeted, anti-aging drug treatments.

Field detection and predicted evolution of spinosad resistance in Ceratitis capitata.

BACKGROUND: The sustainable control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is compromised by the development of resistance to malathion and lambda-cyhalothrin in Spanish field populations. At present, field populations remain susceptible to spinosad. However, the resistant strain JW-100s has been obtained under laboratory selection with spinosad, and resistance has been associated with the presence of different mutations causing truncated transcripts of the α6 subunit of the nicotinic acetylcholine receptor (nAChRα6). RESULTS: An F1 screen assay followed by the molecular characterization of surviving flies has been used to search for spinosad-resistant alleles in field populations. Two different resistant alleles giving rise to truncated isoforms of Ccα6 have been identified, which corresponds to an estimated allelic frequency of at least 0.0023-0.0046. The fitness values of the resistant nAChRα6 alleles found in the laboratory strain JW-100s were estimated to be 0.4 for RR and 0.2 for SR. Mathematical modelling predicted that spinosad-resistant alleles will rapidly decline over time in field populations if their fitness cost was the same as estimated for laboratory-resistant alleles. However, they are predicted to increase in the field if their fitness cost is lower and resistance management strategies are not implemented. CONCLUSION: Spinosad-resistant alleles have been detected in field populations for the first time. Our modelling simulations indicate that the best option to delay the appearance of spinosad resistance would be its rotation with other insecticides without cross-resistance. The integrated F1 screen/molecular genetic analysis presented here can be used for future monitoring studies. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Upregulation of E93 Gene Expression Acts as the Trigger for Metamorphosis Independently of the Threshold Size in the Beetle Tribolium castaneum.

Body size in holometabolous insects is determined by the size at which the juvenile larva undergoes metamorphosis to the pupal stage. To undergo larva-pupa transition, larva must reach a critical developmental checkpoint, the threshold size (TS); however, the molecular mechanisms through which the TS cues this transition remain to be fully characterized. Here, we use the flour beetle Tribolium castaneum to characterize the molecular mechanisms underlying entry into metamorphosis. We found that T. castaneum reaches a TS at the beginning of the last larval instar, which is associated with the downregulation of TcKr-h1 and the upregulation of TcE93 and TcBr-C. Unexpectedly, we found that while there is an association between TS and TcE93 upregulation, it is the latter that constitutes the molecular trigger for metamorphosis initiation. In light of our results, we evaluate the interactions that control the larva-pupa transition and suggest alternative models.

Multiple mutations in the nicotinic acetylcholine receptor Ccα6 gene associated with resistance to spinosad in medfly.

Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata. Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα63aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα63aAG>AT containing a point mutation in the 5' splicing site of exon 3a (3aAG > AT); and (iii) Ccα63aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

The Occurrence of the Holometabolous Pupal Stage Requires the Interaction between E93, Krüppel-Homolog 1 and Broad-Complex.

Complete metamorphosis (Holometaboly) is a key innovation that underlies the spectacular success of holometabolous insects. Phylogenetic analyses indicate that Holometabola form a monophyletic group that evolved from ancestors exhibiting hemimetabolous development (Hemimetaboly). However, the nature of the changes underlying this crucial transition, including the occurrence of the holometabolan-specific pupal stage, is poorly understood. Using the holometabolous beetle Tribolium castaneum as a model insect, here we show that the transient up-regulation of the anti-metamorphic Krüppel-homolog 1 (TcKr-h1) gene at the end of the last larval instar is critical in the formation of the pupa. We find that depletion of this specific TcKr-h1 peak leads to the precocious up-regulation of the adult-specifier factor TcE93 and, hence, to a direct transformation of the larva into the adult form, bypassing the pupal stage. Moreover, we also find that the TcKr-h1-dependent repression of TcE93 is critical to allow the strong up-regulation of Broad-complex (TcBr-C), a key transcription factor that regulates the correct formation of the pupa in holometabolous insects. Notably, we show that the genetic interaction between Kr-h1 and E93 is also present in the penultimate nymphal instar of the hemimetabolous insect Blattella germanica, suggesting that the evolution of the pupa has been facilitated by the co-option of regulatory mechanisms present in hemimetabolan metamorphosis. Our findings, therefore, contribute to the molecular understanding of insect metamorphosis, and indicate the evolutionary conservation of the genetic circuitry that controls hemimetabolan and holometabolan metamorphosis, thereby shedding light on the evolution of complete metamorphosis.

Evolution of SUMO Function and Chain Formation in Insects.

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.

Transcription factor E93 specifies adult metamorphosis in hemimetabolous and holometabolous insects.

All immature animals undergo remarkable morphological and physiological changes to become mature adults. In winged insects, metamorphic changes either are limited to a few tissues (hemimetaboly) or involve a complete reorganization of most tissues and organs (holometaboly). Despite the differences, the genetic switch between immature and adult forms in both types of insects relies on the disappearance of the antimetamorphic juvenile hormone (JH) and the transcription factors Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) during the last juvenile instar. Here, we show that the transcription factor E93 is the key determinant that promotes adult metamorphosis in both hemimetabolous and holometabolous insects, thus acting as the universal adult specifier. In the hemimetabolous insect Blattella germanica, BgE93 is highly expressed in metamorphic tissues, and RNA interference (RNAi)-mediated knockdown of BgE93 in the nymphal stage prevented the nymphal-adult transition, inducing endless reiteration of nymphal development, even in the absence of JH. We also find that BgE93 down-regulated BgKr-h1 and BgBR-C expression during the last nymphal instar of B. germanica, a key step necessary for proper adult differentiation. This essential role of E93 is conserved in holometabolous insects as TcE93 RNAi in Tribolium castaneum prevented pupal-adult transition and produced a supernumerary second pupa. In this beetle, TcE93 also represses expression of TcKr-h1 and TcBR-C during the pupal stage. Similar results were obtained in the more derived holometabolous insect Drosophila melanogaster, suggesting that winged insects use the same regulatory mechanism to promote adult metamorphosis. This study provides an important insight into the understanding of the molecular basis of adult metamorphosis.

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect.

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.