Neuronal Mechanisms that Drive Organismal Aging Through the Lens of Perception.

Sensory neurons provide organisms with data about the world in which they live, for the purpose of successfully exploiting their environment. The consequences of sensory perception are not simply limited to decision-making behaviors; evidence suggests that sensory perception directly influences physiology and aging, a phenomenon that has been observed in animals across taxa. Therefore, understanding the neural mechanisms by which sensory input influences aging may uncover novel therapeutic targets for aging-related physiologies. In this review, we examine different perceptive experiences that have been most clearly linked to aging or age-related disease: food perception, social perception, time perception, and threat perception. For each, the sensory cues, receptors, and/or pathways that influence aging as well as the individual or groups of neurons involved, if known, are discussed. We conclude with general thoughts about the potential impact of this line of research on human health and aging.

The metabolome as a link in the genotype-phenotype map for peroxide resistance in the fruit fly, Drosophila melanogaster.

BACKGROUND: Genetic association studies that seek to explain the inheritance of complex traits typically fail to explain a majority of the heritability of the trait under study. Thus, we are left with a gap in the map from genotype to phenotype. Several approaches have been used to fill this gap, including those that attempt to map endophenotype such as the transcriptome, proteome or metabolome, that underlie complex traits. Here we used metabolomics to explore the nature of genetic variation for hydrogen peroxide (H2O2) resistance in the sequenced inbred Drosophila Genetic Reference Panel (DGRP). RESULTS: We first studied genetic variation for H2O2 resistance in 179 DGRP lines and along with identifying the insulin signaling modulator u-shaped and several regulators of feeding behavior, we estimate that a substantial amount of phenotypic variation can be explained by a polygenic model of genetic variation. We then profiled a portion of the aqueous metabolome in subsets of eight 'high resistance' lines and eight 'low resistance' lines. We used these lines to represent collections of genotypes that were either resistant or sensitive to the stressor, effectively modeling a discrete trait. Across the range of genotypes in both populations, flies exhibited surprising consistency in their metabolomic signature of resistance. Importantly, the resistance phenotype of these flies was more easily distinguished by their metabolome profiles than by their genotypes. Furthermore, we found a metabolic response to H2O2 in sensitive, but not in resistant genotypes. Metabolomic data further implicated at least two pathways, glycogen and folate metabolism, as determinants of sensitivity to H2O2. We also discovered a confounding effect of feeding behavior on assays involving supplemented food. CONCLUSIONS: This work suggests that the metabolome can be a point of convergence for genetic variation influencing complex traits, and can efficiently elucidate mechanisms underlying trait variation.

Gustatory and metabolic perception of nutrient stress in Drosophila.

Sleep loss is an adaptive response to nutrient deprivation that alters behavior to maximize the chances of feeding before imminent death. Organisms must maintain systems for detecting the quality of the food source to resume healthy levels of sleep when the stress is alleviated. We determined that gustatory perception of sweetness is both necessary and sufficient to suppress starvation-induced sleep loss when animals encounter nutrient-poor food sources. We further find that blocking specific dopaminergic neurons phenocopies the absence of gustatory stimulation, suggesting a specific role for these neurons in transducing taste information to sleep centers in the brain. Finally, we show that gustatory perception is required for survival, specifically in a low nutrient environment. Overall, these results demonstrate an important role for gustatory perception when environmental food availability approaches zero and illustrate the interplay between sensory and metabolic perception of nutrient availability in regulating behavioral state.

Water sensor ppk28 modulates Drosophila lifespan and physiology through AKH signaling.

Sensory perception modulates lifespan across taxa, presumably due to alterations in physiological homeostasis after central nervous system integration. The coordinating circuitry of this control, however, remains unknown. Here, we used the Drosophila melanogaster gustatory system to dissect one component of sensory regulation of aging. We found that loss of the critical water sensor, pickpocket 28 (ppk28), altered metabolic homeostasis to promote internal lipid and water stores and extended healthy lifespan. Additionally, loss of ppk28 increased neuronal glucagon-like adipokinetic hormone (AKH) signaling, and the AKH receptor was necessary for ppk28 mutant effects. Furthermore, activation of AKH-producing cells alone was sufficient to enhance longevity, suggesting that a perceived lack of water availability triggers a metabolic shift that promotes the production of metabolic water and increases lifespan via AKH signaling. This work provides an example of how discrete gustatory signals recruit nutrient-dependent endocrine systems to coordinate metabolic homeostasis, thereby influencing long-term health and aging.

Circadian organization of behavior and physiology in Drosophila.

Circadian clocks organize behavior and physiology to adapt to daily environmental cycles. Genetic approaches in the fruit fly, Drosophila melanogaster, have revealed widely conserved molecular gears of these 24-h timers. Yet much less is known about how these cell-autonomous clocks confer temporal information to modulate cellular functions. Here we discuss our current knowledge of circadian clock function in Drosophila, providing an overview of the molecular underpinnings of circadian clocks. We then describe the neural network important for circadian rhythms of locomotor activity, including how these molecular clocks might influence neuronal function. Finally, we address a range of behaviors and physiological systems regulated by circadian clocks, including discussion of specific peripheral oscillators and key molecular effectors where they have been described. These studies reveal a remarkable complexity to circadian pathways in this "simple" model organism.

Targeting Cellular Metabolism With CPI-613 Sensitizes Pancreatic Cancer Cells to Radiation Therapy.

Purpose: Local tumor progression is a cause of significant morbidity and mortality in patients with pancreatic ductal adenocarcinoma (PDAC) with surgically unresectable disease. Novel and effective approaches to accomplish durable local control are urgently needed. We tested whether CPI-613 (devimistat), a first-in-class investigational small molecule inhibitor of mitochondrial metabolism, was capable of altering cancer cell energy metabolism and sensitizing PDAC cells to radiation therapy (RT). Methods and Materials: The effect of a combined treatment of RT with CPI-613 on the viability of, clonogenic potential of, and cell death induction in PDAC cells (MiaPaCa-2 and Panc-1) was determined using a trypan blue dye exclusion assay, a colony formation assay, and a 7-amino-actinomycin D assay, respectively. The synergistic effects of CPI-613-RT and chemotherapeutic agents (gemcitabine or 5-fluorouracil) were measured in MiaPaCa-2 cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and spheroid formation assay. Changes in energy metabolism were determined by profiling metabolites treated with either RT, CPI-613, or both using liquid chromatography-mass spectrometry. Results: This study demonstrates that a combination of single-fraction RT (2 and 10 Gy) with CPI-613 significantly inhibits PDAC cell growth compared with RT alone. Molecular analysis revealed inhibition of α-ketoglutarate dehydrogenase at the protein level. In addition, we demonstrate enhanced cell death of PDAC cells when treated with RT-CPI-613 combination. Targeted metabolomic analysis on PDAC cells post-CPI-613-RT treatment revealed alterations in key mitochondrial metabolites, with broader target engagement by the combination treatment, indicating the sensitization of CPI-613-treated PDAC cells to RT. Furthermore, a combination treatment of CPI-613 with either gemcitabine or 5-fluorouracil in the presence of 2 Gy RT synergistically inhibits PDAC cell proliferation. Conclusions: Our results support a novel combination of CPI-613-RT that warrants further preclinical and early-phase clinical investigations. A phase 1 trial designed to identify the maximum tolerated dose of CPI-613 in combination with chemo-RT in patients with PDAC was recently initiated (NCT05325281).

Patient similarity and other artificial intelligence machine learning algorithms in clinical decision aid for shared decision-making in the Prevention of Cardiovascular Toxicity (PACT): a feasibility trial design.

BACKGROUND: The many improvements in cancer therapies have led to an increased number of survivors, which comes with a greater risk of consequent/subsequent cardiovascular disease. Identifying effective management strategies that can mitigate this risk of cardiovascular complications is vital. Therefore, developing computer-driven and personalized clinical decision aid interventions that can provide early detection of patients at risk, stratify that risk, and recommend specific cardio-oncology management guidelines and expert consensus recommendations is critically important. OBJECTIVES: To assess the feasibility, acceptability, and utility of the use of an artificial intelligence (AI)-powered clinical decision aid tool in shared decision making between the cancer survivor patient and the cardiologist regarding prevention of cardiovascular disease. DESIGN: This is a single-center, double-arm, open-label, randomized interventional feasibility study. Our cardio-oncology cohort of > 4000 individuals from our Clinical Research Data Warehouse will be queried to identify at least 200 adult cancer survivors who meet the eligibility criteria. Study participants will be randomized into either the Clinical Decision Aid Group (where patients will use the clinical decision aid in addition to current practice) or the Control Group (current practice). The primary endpoint of this study is to assess for each patient encounter whether cardiovascular medications and imaging pursued were consistent with current medical society recommendations. Additionally, the perceptions of using the clinical decision tool will be evaluated based on patient and physician feedback through surveys and focus groups. This trial will determine whether a clinical decision aid tool improves cancer survivors' medication use and imaging surveillance recommendations aligned with current medical guidelines. TRIAL REGISTRATION: ClinicalTrials.Gov Identifier: NCT05377320.

Sphingolipid metabolism in T cell responses after allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective immunotherapy against hematopoietic malignancies. The infused donor lymphocytes attack malignant cells and normal tissues, termed a graft-verse-leukemia (GVL) effect and graft-verse-host (GVH) response or disease (GVHD), respectively. Although engineering techniques toward donor graft selection have made HCT more specific and effective, primary tumor relapse and GVHD are still major concerns post allo-HCT. High-dose systemic steroids remain to be the first line of GVHD treatment, which may lead to steroid-refractory GVHD with a dismal outcome. Therefore, identifying novel therapeutic strategies that prevent GVHD while preserving GVL activity is highly warranted. Sphingolipid metabolism and metabolites play pivotal roles in regulating T-cell homeostasis and biological functions. In this review, we summarized the recent research progress in this evolving field of sphingolipids with a focus on alloreactive T-cell responses in the context of allo-HCT. We discussed how sphingolipid metabolism regulates T-cell mediated GVH and GVL responses in allo-HCT and presented the rationale and means to target sphingolipid metabolism for the control of GVHD and leukemia relapse.

Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila.

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.

Measurement of solid food intake in Drosophila via consumption-excretion of a dye tracer.

Although the Drosophila melanogaster (fly) model is a popular platform for investigating diet-related phenomena, it can be challenging to measure the volume of agar-based food media flies consume. We addressed this challenge by developing a dye-based method called Consumption-Excretion (Con-Ex). In Con-Ex studies, flies consume solid food labeled with dye, and the volume of food consumed is reflected by the sum of the dye inside of and excreted by flies. Flies consumed-excreted measurable amounts of FD&C Blue No. 1 (Blue 1) and other dyes in Con-Ex studies, but only Blue 1 was readily detectable at concentrations that had no discernable effect on consumption-excretion. In studies with Blue 1, consumption-excretion (i) increased linearly with feeding duration out to 24 h at two different laboratory sites, (ii) was sensitive to starvation, mating status and strain, and (iii) changed in response to alteration of media composition as expected. Additionally, the volume of liquid Blue 1 consumed from capillary tubes was indistinguishable from the volume of Blue 1 excreted by flies, indicating that excreted Blue 1 reflects consumed Blue 1. Our results demonstrate that Con-Ex with Blue 1 as a food tracer is a useful method for assessing ingestion of agar-based food media in adult flies.

Drosophila Neuropeptide F Signaling Independently Regulates Feeding and Sleep-Wake Behavior.

Proper regulation of sleep-wake behavior and feeding is essential for organismal health and survival. While previous studies have isolated discrete neural loci and substrates important for either sleep or feeding, how the brain is organized to coordinate both processes with respect to one another remains poorly understood. Here, we provide evidence that the Drosophila Neuropeptide F (NPF) network forms a critical component of both adult sleep and feeding regulation. Activation of NPF signaling in the brain promotes wakefulness and adult feeding, likely through its cognate receptor NPFR. Flies carrying a loss-of-function NPF allele do not suppress sleep following prolonged starvation conditions, suggesting that NPF acts as a hunger signal to keep the animal awake. NPF-expressing cells, specifically those expressing the circadian photoreceptor cryptochrome, are largely responsible for changes to sleep behavior caused by NPF neuron activation, but not feeding, demonstrating that different NPF neurons separately drive wakefulness and hunger.

The sensory system: More than just a window to the external world.

While the traditional importance of the sensory system lies in its ability to perceive external information about the world, emerging discoveries suggest that sensory perception has a greater impact on health and longevity than was previously appreciated. These effects are conserved across species. In this mini-review, we discuss the specific sensory cues that have been identified to significantly impact organismal physiology and lifespan. Ongoing work in the aging field has begun to identify the downstream molecules that mediate the broad effects of sensory signals. Candidates include FOXO, neuropeptide F (NPF), adipokinetic hormone (AKH), dopamine, serotonin, and octopamine. We then discuss the many implications that arise from our current understanding of the effects of sensory perception on health and longevity.

Drosophila life span and physiology are modulated by sexual perception and reward.

Sensory perception can modulate aging and physiology across taxa. We found that perception of female sexual pheromones through a specific gustatory receptor expressed in a subset of foreleg neurons in male fruit flies, Drosophila melanogaster, rapidly and reversibly decreases fat stores, reduces resistance to starvation, and limits life span. Neurons that express the reward-mediating neuropeptide F are also required for pheromone effects. High-throughput whole-genome RNA sequencing experiments revealed a set of molecular processes that were affected by the activity of the longevity circuit, thereby identifying new candidate cell-nonautonomous aging mechanisms. Mating reversed the effects of pheromone perception; therefore, life span may be modulated through the integrated action of sensory and reward circuits, and healthy aging may be compromised when the expectations defined by sensory perception are discordant with ensuing experience.

DN1(p) circadian neurons coordinate acute light and PDF inputs to produce robust daily behavior in Drosophila.

BACKGROUND: Daily behaviors in animals are determined by the interplay between internal timing signals from circadian clocks and environmental stimuli such as light. How these signals are integrated to produce timely and adaptive behavior is unclear. The fruit fly Drosophila exhibits clock-driven activity increases that anticipate dawn and dusk and free-running rhythms under constant conditions. Flies also respond to the onset of light and dark with acute increases in activity. RESULTS: Mutants of a novel ion channel, narrow abdomen (na), lack a robust increase in activity in response to light and show reduced anticipatory behavior and free-running rhythms, providing a genetic link between photic responses and circadian clock function. We used tissue-specific rescue of na to demonstrate a role for approximately 16-20 circadian pacemaker neurons, a subset of the posterior dorsal neurons 1 (DN1(p)s), in mediating the acute response to the onset of light as well as morning anticipatory behavior. Circadian pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) are especially important for morning anticipation and free-running rhythms and send projections to the DN1(p)s. We also demonstrate that DN1(p)Pdfr expression is sufficient to rescue, at least partially, Pdfr morning anticipation defects as well as defects in free-running rhythms, including those in DN1 molecular clocks. Additionally, these DN1 clocks in wild-type flies are more strongly reset to timing changes in PDF clocks than other pacemaker neurons, suggesting that they are direct targets. CONCLUSIONS: Taking these results together, we demonstrate that the DN1(p)s lie at the nexus of PDF and photic signaling to produce appropriate daily behavior.

The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila.

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

Requirement for ATP by the DNA damage checkpoint clamp loader.

The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.