Regulatory Mechanisms of Aging Through the Nutritional and Metabolic Control of Amino Acid Signaling in Model Organisms.

Life activities are supported by the intricate metabolic network that is fueled by nutrients. Nutritional and genetic studies in model organisms have determined that dietary restriction and certain mutations in the insulin signaling pathway lead to lifespan extension. Subsequently, the detailed mechanisms of aging as well as various nutrient signaling pathways and their relationships have been investigated in a wide range of organisms, from yeast to mammals. This review summarizes the roles of nutritional and metabolic signaling in aging and lifespan with a focus on amino acids, the building blocks of organisms. We discuss how lifespan is affected by the sensing, transduction, and metabolism of specific amino acids and consider the influences of life stage, sex, and genetic background on the nutritional control of aging. Our goal is to enhance our understanding of how nutrients affect aging and thus contribute to the biology of aging and lifespan.

Epidermal tyrosine catabolism is crucial for metabolic homeostasis and survival against high-protein diets in Drosophila.

The insect epidermis forms the exoskeleton and determines the body size of an organism. How the epidermis acts as a metabolic regulator to adapt to changes in dietary protein availability remains elusive. Here, we show that the Drosophila epidermis regulates tyrosine (Tyr) catabolism in response to dietary protein levels, thereby promoting metabolic homeostasis. The gene expression profile of the Drosophila larval body wall reveals that enzymes involved in the Tyr degradation pathway, including 4-hydroxyphenylpyruvate dioxygenase (Hpd), are upregulated by increased protein intake. Hpd is specifically expressed in the epidermis and is dynamically regulated by the internal Tyr levels. Whereas basal Hpd expression is maintained by insulin/IGF-1 signalling, Hpd induction on high-protein diet requires activation of the AMP-activated protein kinase (AMPK)-forkhead box O subfamily (FoxO) axis. Impairment of the FoxO-mediated Hpd induction in the epidermis leads to aberrant increases in internal Tyr and its metabolites, disrupting larval development on high-protein diets. Taken together, our findings uncover a crucial role of the epidermis as a metabolic regulator in coping with an unfavourable dietary environment.

Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan.

Commensal microbes in animals have a profound impact on tissue homeostasis, stress resistance, and ageing. We previously showed in Drosophila melanogaster that Acetobacter persici is a member of the gut microbiota that promotes ageing and shortens fly lifespan. However, the molecular mechanism by which this specific bacterial species changes lifespan and physiology remains unclear. The difficulty in studying longevity using gnotobiotic flies is the high risk of contamination during ageing. To overcome this technical challenge, we used a bacteria-conditioned diet enriched with bacterial products and cell wall components. Here, we demonstrate that an A. persici-conditioned diet shortens lifespan and increases intestinal stem cell (ISC) proliferation. Feeding adult flies a diet conditioned with A. persici, but not with Lactiplantibacillus plantarum, can decrease lifespan but increase resistance to paraquat or oral infection of Pseudomonas entomophila, indicating that the bacterium alters the trade-off between lifespan and host defence. A transcriptomic analysis using fly intestine revealed that A. persici preferably induces antimicrobial peptides (AMPs), while L. plantarum upregulates amidase peptidoglycan recognition proteins (PGRPs). The specific induction of these Imd target genes by peptidoglycans from two bacterial species is due to the stimulation of the receptor PGRP-LC in the anterior midgut for AMPs or PGRP-LE from the posterior midgut for amidase PGRPs. Heat-killed A. persici also shortens lifespan and increases ISC proliferation via PGRP-LC, but it is not sufficient to alter the stress resistance. Our study emphasizes the significance of peptidoglycan specificity in determining the gut bacterial impact on healthspan. It also unveils the postbiotic effect of specific gut bacterial species, which turns flies into a "live fast, die young" lifestyle.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging.

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect ß-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila.

Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. Herein, we develop a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, we demonstrate that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. Our data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration.

High expression of A-type lamin in the leading front is required for Drosophila thorax closure.

Tissue closure involves the coordinated unidirectional movement of a group of cells without loss of cell-cell contact. However, the molecular mechanisms controlling the tissue closure are not fully understood. Here, we demonstrate that Lamin C, the sole A-type lamin in Drosophila, contributes to the process of thorax closure in pupa. High expression of Lamin C was observed at the leading front of the migrating wing imaginal discs. Live imaging analysis revealed that knockdown of Lamin C in the thorax region affected the coordinated movement of the leading front, resulting in incomplete tissue fusion required for formation of the adult thorax. The closure defect due to knockdown of Lamin C correlated with insufficient accumulation of F-actin at the front. Our study indicates a link between A-type lamin and the cell migration behavior during tissue closure.

Drosophila SETDB1 and caspase cooperatively fine-tune cell fate determination of sensory organ precursor.

Drosophila produce a constant number of mechanosensory bristles called macrochaetae (MC), which develop from sensory organ precursor (SOP) cells within a proneural cluster (PNC). However, what ensures the precise determination of SOP cells remains to be elucidated. In this study, we conducted RNAi screening in PNC for genes involved in epigenetic regulation. We identified a H3K9 histone methyltransferase, SETDB1/eggless, as a regulator of SOP development. Knockdown of SETDB1 in PNC led to additional SOPs. We further tested the relationship between SETDB1 and non-apoptotic function of caspase on SOP development. Reinforcing caspase activation by heterozygous Drosophila inhibitor of apoptosis protein 1 (DIAP1) mutation rescued ectopic SOP development caused by SETDB1 knockdown. Knockdown of SETDB1, however, had little effect on caspase activity. Simultaneous loss of SETDB1 and caspase activity resulted in further increase in MC, indicating that the two components work cooperatively. Our study suggests the fine-tuning mechanisms for SOP development by epigenetic methyltransferase and non-apoptotic caspase function.

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

BACKGROUND: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. RESULTS: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. CONCLUSIONS: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.

The LacZ gene, which encodes Escherichia coli ß-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic ß-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

Persephone/Spätzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila.

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as damage-associated molecular patterns (DAMPs) and a noninfectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. In this study, we found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spätzle (Spz). We speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. We demonstrated that Toll signaling activation required the action of Persephone, a CLIP domain serine protease that usually reacts to microbial proteolytic activities. Our findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants.

Interplay of cell proliferation and cell death in Drosophila tissue regeneration.

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death-induced tissue response in the reconstruction of damaged tissues.

Selective ablation of ß-galactosidase-expressing cells with a rationally designed activatable photosensitizer.

We have developed an activatable photosensitizer capable of specifically inducing the death of ß-galactosidase-expressing cells in response to photoirradiation. By using a selenium-substituted rhodol scaffold bearing ß-galactoside as a targeting substituent, we designed and synthesized HMDESeR-ßGal, which has a non-phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, ß-galactosidase efficiently converted HMDESeR-ßGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible-wavelength absorption and the ability to generate singlet oxygen ((1)O2). When HMDESeR-ßGal was applied to larval Drosophila melanogaster wing disks, which express ß-galactosidase only in the posterior region, photoirradiation induced cell death in the ß-galactosidase-expressing region with high specificity.

Microbiota-derived acetylcholine can promote gut motility in Drosophila melanogaster.

The gut microbiota is crucial for intestinal health, including gastrointestinal (GI) motility. How commensal bacterial species influence GI motility has not been fully elucidated. A major factor of GI motility is the gut contraction promoting the propulsive movement of orally ingested materials. Here, we developed a method to monitor and quantify gut contractions in living Drosophila melanogaster larvae. We found that the culture medium of an isolated strain Lactiplantibacillus plantarum Lsi promoted gut contraction in vivo, which was not observed in Leuconostoc sp. Leui nor Acetobacter persici Ai culture medium. To identify bacteria-derived metabolites, we performed metabolome analysis of the culture media by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the 66 metabolites detected, we found that some metabolites changed in a species-specific manner. Among them, acetylcholine was specifically produced by L. plantarum. Feeding exogenous acetylcholine increased the frequency of gut contractions, which was blocked by D-tubocurarine, an inhibitor of nicotinic acetylcholine receptors. In this study, we propose a mechanism by which the gut microbiota influences Drosophila gut motility. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Imaging the uptake of deuterated methionine in Drosophila with stimulated Raman scattering.

Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging. Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo. Results: Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level. Discussion: These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue.

Early-adult methionine restriction reduces methionine sulfoxide and extends lifespan in Drosophila.

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension.

Super-resolution vibrational imaging based on photoswitchable Raman probe.

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.

Sensing of the non-essential amino acid tyrosine governs the response to protein restriction in Drosophila.

The intake of dietary protein regulates growth, metabolism, fecundity and lifespan across various species, which makes amino acid (AA)-sensing vital for adaptation to the nutritional environment. The general control nonderepressible 2 (GCN2)-activating transcription factor 4 (ATF4) pathway and the mechanistic target of rapamycin complex 1 (mTORC1) pathway are involved in AA-sensing. However, it is not fully understood which AAs regulate these two pathways in living animals and how they coordinate responses to protein restriction. Here we show in Drosophila that the non-essential AA tyrosine (Tyr) is a nutritional cue in the fat body necessary and sufficient for promoting adaptive responses to a low-protein diet, which entails reduction of protein synthesis and mTORC1 activity and increased food intake. This adaptation is regulated by dietary Tyr through GCN2-independent induction of ATF4 target genes in the fat body. This study identifies the Tyr-ATF4 axis as a regulator of the physiological response to a low-protein diet and sheds light on the essential function of a non-essential nutrient.

Probing Methionine Uptake in Live Cells by Deuterium Labeling and Stimulated Raman Scattering.

The small biomolecule methionine (Met) is a fundamental amino acid required for a vast range of biological processes such as protein synthesis, cancer metabolism, and epigenetics. However, it is still difficult to visualize the subcellular distribution of small biomolecules including Met in a minimally invasive manner. Here, we demonstrate stimulated Raman scattering (SRS) imaging of cellular uptake of deuterated methionine (d8-Met) in live HeLa cells by way of comparison to the previously used alkyne-labeled Met analogue─homopropargylglycine (Hpg). We show that the solutions of d8-Met and Hpg have similar SRS signal intensities. Furthermore, by careful image analysis with background subtraction, we succeed in the SRS imaging of cellular uptake of d8-Met with a much greater signal intensity than Hpg, possibly reflecting the increased and minimally invasive uptake kinetics of d8-Met compared with Hpg. We anticipate that d8-Met and other deuterated biomolecules will be useful for investigating metabolic processes with subcellular resolution.