The C. elegans glutamate transporters GLT-4 and GLT-5 regulate protein expression, behavior, and lifespan.

The C. elegans Glutamate Transporters GLT-4 and GLT-5 Regulate Protein Expression, Behavior, and Lifespan - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Neurochem Int . Author manuscript; available in PMC: 2026 Jun 1. Published in final edited form as: Neurochem Int. 2025 Mar 25;186:105966. doi: 10.1016/j.neuint.2025.105966 The C. elegans Glutamate Transporters GLT-4 and GLT-5 Regulate Protein Expression, Behavior, and Lifespan Grace J Bronstone 1 , Moriah Harton 1 , Maya Muldowney 1 , James Reigle 2, 3 , Adam J Funk 4 , Sinead M O’Donovan 4 , Robert E McCullumsmith 4, 5 , Deborah E Bauer 1 Author information Article notes Copyright and License information 1. Department of Neuroscience, Wellesley College (Science Center, 106 Central Street, Wellesley MA 02481) 2. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center (3333 Burnet Avenue, Cincinnati OH 45229) 3. Department of Biomedical Informatics, University of Cincinnati College of Medicine (Medical Sciences Building 231 Albert Sabin Way, PO Box 670769, Cincinnati OH 45267) 4. Department of Neuroscience, University of Toledo College of Medicine (179 Block Health Science Building Mail Stop #1007, 3000 Arlington Avenue, Toledo Ohio 43614) 5. Neurosciences Institute, ProMedica (2130 West Central Avenue, Toledo Ohio 43606) ✉ Corresponding Authors: Grace Bronstone ( gbronsto@wellesley.edu ); Deborah Bauer ( dbauer@wellesley.edu ) Issue date 2025 Jun. PMC Copyright notice PMCID: PMC12053503  NIHMSID: NIHMS2069969  PMID: 40147734 The publisher's version of this article is available at Neurochem Int Abstract Glutamate transporters are important for regulating extracellular glutamate levels, impacting neural function and metabolic homeostasis. This study explores the behavioral, lifespan, and proteomic profiles in Caenorhabditis elegans strains with either glt-4 or glt-5 null mutations, highlighting contrasting phenotypes. Δ glt-4 mutants displayed impaired mechanosensory and chemotactic responses, reduced lifespans, and decreased expression levels of ribosomal proteins and chaperonins involved in protein synthesis and folding. In contrast, Δ glt-5 mutants displayed heightened chemorepulsion, extended lifespans, and upregulation of mitochondrial pyruvate carriers and cytoskeletal proteins. Proteomic profiling via mass spectrometry identified 53 differentially expressed proteins in Δ glt-4 mutants and 45 in Δ glt-5 mutants. Δ glt-4 mutants showed disruptions in ribonucleoprotein complex organization and translational processes, including downregulation of glycogen phosphorylase and V-type ATPase subunits, while Δ glt-5 mutants revealed altered metabolic protein expression, such as increased levels of mitochondrial pyruvate carriers and decreased levels of fibrillarin and ribosomal proteins. Gene ontology enrichment analysis highlighted differential regulation of protein biosynthesis and metabolic pathways between the strains. Overall, these findings underscore the distinct, tissue-specific roles of GLT-4 and GLT-5 in C. elegans , with broader implications for glutamate regulation and systemic physiology. The results also reinforce the utility of C. elegans as a model for studying glutamate transporters' impact on behavior, longevity, and proteostasis. Keywords: Caenorhabditis elegans , glutamate transporter, behavioral phenotyping, lifespan analysis, mass spectrometry, proteomic profiling 1. Introduction Glutamate transporters (GluTs) are high-affinity transporters critical for regulating neurotransmission and preventing the toxic accumulation of extracellular glutamate. Although their roles at synapses have been extensively studied in both vertebrate and invertebrate nervous systems, far less is known about how GluTs contribute to systemic processes in non-neuronal tissues ( Mano et al. 2007 ; Malik and Willnow 2019; Moroz et al. 2021). In Caenorhabditis elegans, these transporters exhibit considerable homology to mammalian excitatory amino acid transporters (EAATs), retaining the canonical SLC1 fold—eight transmembrane segments and two re-entrant loops—and sharing approximately 40–50% sequence identity with vertebrate counterparts ( Kanner and Borre 2002 ; Yernool et al. 2004 ; Mano et al. 2007 ; Canul-Tec et al. 2017 ; Katz et al. 2019 ). Such parallels underscore the value of C. elegans as a model for studying glutamate transporter biology beyond the synapse. Six high-affinity GluTs have been identified in C. elegans : GLT-1, GLT-3, GLT-4, GLT-5, GLT-6, and GLT-7 ( Mano et al. 2007 ). GLT-1, an ortholog of mammalian EAAT2, is expressed in cephalic sheath glia, the hypodermis, and muscle, where it is thought to remove extracellular glutamate in a manner analogous to vertebrate astrocytes ( Oikonomou and Shaham 2011 ; Katz et al. 2019 ). GLT-3, GLT-6, and GLT-7 localize primarily to excretory canal cells or the pseudocoelomic fluid, suggesting they may help regulate glutamate levels in circulation ( Mano et al. 2007 ). Despite indications that each transporter plays a unique part in overall glutamate homeostasis, comparatively little is understood about how these roles intersect with systemic metabolism, behavior, longevity, or organism-wide signaling. By contrast, GLT-4 and GLT-5 stand out for their distinct localizations: GLT-4 is found in head muscles and presynaptic neurons, whereas GLT-5 is localized to non-neuronal cells in the pharynx ( Mano et al. 2007 ). Because these two transporters occupy such different tissue contexts, they offer an ideal opportunity to investigate how glutamate transport influences both neuronal and non-neuronal facets of metabolic regulation. The full extent of their expression patterns and broader physiological roles, however, remains unclear. Notably, the unique distribution of GluTs in C. elegans helps the organism maintain ambient glutamate levels and finely control transmitter diffusion beyond the synapse ( Chan et al. 2019 ). Leveraging its genetic tractability, well-mapped nervous system, evolutionary conservation with vertebrates, and tolerance for gene deletions, C. elegans provides a powerful whole-organism framework for dissecting GluT function ( Del Rosario et al. 2015 ; Zhang et al. 2020 ). Guided by the observation that GLT-4 and GLT-5 may exert distinct, tissue-specific effects, we focus on the ways in which their targeted disruptions differentially impact metabolic regulation, behavior, and overall organismal homeostasis. In this study, we examine the behavioral, lifespan, and proteomic consequences of glt-4 and glt-5 null mutations (designated Δ glt-4 and Δ glt-5 ), aiming to clarify how these transporters contribute to systemic metabolic regulation and organismal equilibrium. The mechanisms by which GluTs influence protein signaling pathways remain poorly understood, and to our knowledge, no research has yet explored the relationship between the deletion of specific GluTs and total quantitative protein expression in conjunction with behavioral and longevity outcomes. Understanding these interactions could illuminate previously unrecognized links between glutamate homeostasis and metabolic disease in higher organisms. This study demonstrates that integrating behavioral, longevity, and proteomic analyses is crucial for fully characterizing the impact of glutamate transporter knockouts. We hypothesize that perturbations in cellular glutamate levels arising from Δ glt-4 or Δ glt-5 lead to changes in metabolic enzyme expression via transcriptional, translational, or degradation pathways. Although this work does not distinguish between localized and systemic effects, it lays the groundwork for future research to determine whether proteomic shifts stem from local disruptions, systemic signaling, or both. By melding phenotypic and proteomic approaches, this study provides novel insights into the broader systemic roles of GluTs in C. elegans physiology. 2. Materials and methods 2.1. C. elegans strains: The strains used in this study were the glt-4 (bz69) II, glt-5 (bz70) II, and wildtype N2 Bristol laboratory strains. The glt-4 (bz69) mutation corresponds to a 343 bp deletion with an insertion sequence (TTAATC) in the glt-4 gene (T22E5.2, located on chromosome X: 6403422..6408411), and the glt-5 (bz70) mutation is a targeted disruption in the glt-5 gene (Y53C12A.2, located on chromosome II: 9706481..9708885). Both mutations were generated and provided by the C. elegans Gene Knockout Project at the Oklahoma Medical Research Foundation as part of the International C. elegans Gene Knockout Consortium. Both mutant strains were originally described by Mano et al. (2007) . All strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota; RRID: SCR_007341). Worms were maintained on NGM agar plates seeded with E. coli OP50 at 20°C and were transferred to fresh plates every three days to ensure consistent growth and experimental conditions. 2.2. Behavioral assays Behavior testing was based on methods from prior studies investigating GluT deletion in C. elegans ( Fryer et al. 2024 ). All reagents were sourced within the past 2 years and stored according to manufacturer guidelines. 2.2.1. Nose touch assay The mechanosensory response was evaluated using a nose touch assay. Individual L4-stage worms (n = 30 per strain) were transferred to unseeded 5 cm Petri dishes for a 3 minute acclimation period. Worms were gently poked 10 times at 5-10 second intervals with an eyelash tool, and their forward or reverse movements were recorded. 2.2.2. Chemotaxis Chemosensory behavior was assessed via a quadrant assay. Groups of ~4000 worms per strain (±20, across 12 trials) were placed on 5 cm Petri dishes divided into four quadrants. Test quadrants contained 2 μL of 2% sodium azide mixed with a chemoattractant (NaCl) or chemorepellent (1-octanol in ethanol), while control quadrants contained sodium azide with ethanol alone. Worms were washed with an M9 buffer before being placed on the dish. After 1 hour, quadrant distributions were recorded, and chemotactic indices were calculated. 2.2.3. Associative learning Associative learning was assessed using butanone as the conditioned stimulus across 6-10 trials per strain (wildtype: n = 932; Δ glt-4 : n = 918; Δ glt-5 : n = 896). Worms were starved for 1 hour to enhance learning responses, then conditioned for 1 hour on NGM agar plates streaked with 30% butanone in ethanol alongside a rewarding stimulus (OP50 E. coli ). After conditioning, worms were washed with M9 buffer to remove residual bacteria, centrifuged, and transferred to 5 cm Petri dishes prepared for chemotaxis testing. Test and control cues consisted of sodium azide solutions containing either ethanol (control) or 30% butanone in ethanol (test). Worm distributions across quadrants were recorded after 1 hour, and the chemotactic index was calculated. To assess baseline responses to butanone without conditioning, control experiments were conducted. Worms were starved for 1 hour and exposed to butanone without a rewarding stimulus. After washing to remove residual butanone, worms were placed in chemotaxis assay plates with sodium azide solutions containing ethanol or 30% butanone in ethanol. Responses were recorded after 1 hour (wildtype: n = 591; Δ glt-4 : n = 692; Δ glt-5 : n = 595). This ensured that conditioning, rather than exposure alone, was necessary to elicit chemoattractant behavior. To evaluate the retention of learned responses, a post-conditioning test was performed with a rewarding stimulus. Worms were starved for 1 hour, conditioned on NGM plates seeded with E. coli , and exposed to 4 μL of 30% butanone in ethanol streaked on the lid. Plates were sealed with parafilm to prevent evaporation and incubated for 1 hour. Worms were washed with an M9 buffer and transferred to chemotaxis plates for testing. Distributions were recorded after 1 hour. The post-conditioning assay was repeated across 10 trials per strain (n = 100 ± 20 worms per trial). 2.3. Lifespan analysis: Lifespan measurements for Δ glt-4 and Δ glt-5 strains were conducted using the protocol described by Sutphin and Kaeberlein (2009) , including the use of 5-fluoro-2’-deoxyuridine (FUdR) to prevent reproduction. Related studies have evaluated lifespan differences between the mutant and wildtype strains under controlled conditions with FUdR detail the effectiveness and safety of FUdR in C. elegans lifespan experiments ( Mitchell et al. 1979 ; Van Raamsdonk and Hekimi 2011 ; Rooney et al. 2014 ). 2.3.1. Preparation and synchronization Strains were age-synchronized by isolating eggs from gravid adults using a hypochlorite solution (M9 buffer, 1M NaOH, 5% bleach) and transferring them to freshly seeded NGM plates at 20°C to hatch, designated as Day 0. Under these conditions, worms typically reach the L4 stage (~ 44 – 48 hours post-hatching) by Day 2 of adulthood (Corsi et al., 2015). However, to ensure sufficiently large, synchronized populations, we maintained and expanded mixed-stage cultures prior to bleaching, so in the overall experimental timeline the L4 stage (Day 2 of adulthood) fell on calendar Day 6 – 7. Once L4 worms were identified by the presence of the developing vulva, they were transferred to seeded NGM plates supplemented with 0.1 mg/mL FUdR to inhibit reproduction, and maintained at 20°C for subsequent lifespan assays. 2.3.2. Exclusion criteria Worms were excluded from the lifespan analysis under the following conditions: Physical injury caused during transfer or monitoring. Ruptured vulvae with visible intestinal extrusion. Deaths occurring near the plate edges where accurate determination was compromised. Contaminated plates or worms, with efforts made to transfer worms to uncontaminated plates where possible. Worms censored due to contamination or other unrelated factors were excluded from statistical analysis. If worms were injured during transfer, they were censored on the day following the incident to ensure deaths were not attributed to experimental conditions. 2.3.3. Daily monitoring Fifteen L4-stage worms were plated on each of five FUdR plates per strain (75 worms per strain total). Worms were monitored daily under a dissecting microscope. A worm was declared dead if no response to three gentle prods on the head with a pick was observed. Dead worms were removed and sterilized in a chemical fume hood to prevent contamination. Worms censored due to injury or contamination were recorded and excluded from the final analysis. 2.3.4. Plate preparation and handling NGM agar was prepared at a concentration of 29 g/L and supplemented with FUdR to a final concentration of 0.1 mg/mL. Plates were poured under low-light conditions to minimize FUdR degradation and covered with foil during drying to further protect from light exposure. OP50 cultures were grown overnight at 37°C, vortexed before use, and applied to the center of each plate in seven drops to form a concentrated lawn. Worms were transferred before the OP50 lawn dried. Extra plates were prepared to replace contaminated plates during the experiment. 2.4. Mass spectrometry: Mass spectrometry-based quantitative proteomics (nLC-ESI-MS/MS) was performed to identify differentially expressed proteins in Δ glt-4 and Δ glt-5 strains relative to wildtype. Proteins were ranked by consensus scores derived from peptide abundance profiles. Gene Ontology (GO) enrichment analysis was conducted to categorize the biological processes associated with these proteins, using functional annotations from WormBase and Gene Ontology Consortium ( Ashburner et al. 2000 ; Aleksander et al. 2023 ; Sternberg et al. 2024 ). Enrichment analysis was carried out by combining non-negative matrix factorization (NMF) clustering with statistical rankings (t-test and −log(Q)). Only proteins with combined peptide ranks ≤50 and consensus signature peptides (t ≥10) were included in the analysis. Identified pathways were ranked by significance (−log(Q)) to prioritize biological processes. Hierarchical clustering was used to group the enriched pathways and visualize differences between the Δ glt-4 , Δ glt-5 , and wildtype strains. This analysis allowed for the large-scale identification of proteins with abnormal expression patterns in the GluT mutant strains. The GO enrichment analysis provided insight into the biological roles and functions of these proteins, categorizing the gene products with the most significant positive and negative correlations. The GO database enabled the annotation and visualization of upregulated and downregulated gene sets, grouped into functional categories based on the hierarchical clustering results. 2.4.1. Sample preparation For each genotype, unsynchronized populations of starved worms (80 μL) were collected under identical growth and feeding conditions to ensure that all developmental stages were represented as uniformly as possible. The worms were then treated with protease/phosphatase inhibitors. After centrifugation, pellets were homogenized in Laemmli buffer, denatured at 90°C, and centrifuged to remove insoluble material. The resulting supernatant was stored at −80°C until analysis. 2.4.2. Nano liquid chromatography coupled to electrospray tandem mass spectrometry Protein extracts (500 ng/sample) were loaded onto a trap column for desalting, followed by peptide separation using a gradient of formic acid and acetonitrile. The eluted peptides were ionized via electrospray and analyzed using a SCIEX TripleTOF 5600+ mass spectrometer. Data acquisition was performed in triplicate and processed with Analyst-TF software. 2.4.3. Differential proteomic analysis Proteomic signatures were generated using an in-house R pipeline designed to identify differentially expressed proteins. Peptides were filtered based on statistical significance using a two-tailed t-test (p ≤ 0.05) and ranked using NMF clustering, with a consensus threshold rank of ≤ 50. Consensus peptide signatures were then constructed by integrating peptide abundance profiles across replicates, ensuring robust identification of significant expression changes. To interpret the biological relevance of these changes, GO enrichment analysis was performed using functional annotations from WormBase ( https://wormbase.org/ ) and the Gene Ontology Consortium database ( https://geneontology.org/ ). Pathways were prioritized based on their significance scores (−log(Q)) to highlight the most disrupted biological processes. The enriched processes were visualized using hierarchical clustering to delineate functional differences between Δ glt-4 , Δ glt-5 , and wildtype strains. Biological roles of the identified proteins and their closest mammalian homologs were further annotated using cross-referenced databases, including WormBase, GeneCards, and UniProt ( Stelzer et al. 2016 ; The UniProt Consortium 2023 ). This analysis prioritized proteins with conserved functional relevance to emphasize pathways with potential translational significance. While many C. elegans proteins lack direct mammalian homologs or fully characterized functions, this approach provides insight into conserved biological mechanisms impacted by glutamate transporter deletions. The results of this differential proteomic analysis provide a high-resolution overview of proteomic alterations, enabling the identification of key biological pathways dysregulated in Δ glt-4 and Δ glt-5 strains relative to wildtype. 2.5. Statistical analysis: As an exploratory study, no a priori power analysis was performed. Sample sizes for behavioral assays and lifespan analyses were determined based on established methodologies from comparable C. elegans studies ( Pitt et al. 2019 ; Cornwell and Samuelson 2020 ; Fryer et al.2024 ). Post-hoc power analyses were not conducted due to the study’s design complexity and the use of non-parametric statistical methods where appropriate. However, chemotaxis and associative learning assays featured large sample sizes (approximately 4,000 and 900 worms per group, respectively), which provided sufficient power to detect small-to-medium effect sizes. In contrast, the nose touch assay (n = 30 worms per strain) and lifespan analysis (n = 75 worms per strain) involved smaller sample sizes, increasing the risk of Type II errors ( Gruber et al. 2009 ). This limitation is consistent with previous studies ( Scheer and Bargmann 2023 ). All statistical analyses were performed using Python (version 3.11) and R (version 3.5.0). Results were considered significant at p < 0.05, with adjustments for multiple comparisons (Tukey’s HSD or Bonferroni correction, as appropriate). 2.5.1. Behavioral data analysis Behavioral data were analyzed using both parametric and non-parametric statistical methods, depending on the distribution of the dataset. Parametric datasets were analyzed using one-way ANOVA to compare differences across strains, followed by Tukey’s Honest Significant Difference (HSD) test for post-hoc pairwise comparisons. For datasets violating normality assumptions, as determined by the Shapiro-Wilk test (e.g., nose touch assay), non-parametric analyses were conducted using the Kruskal-Wallis test for overall comparisons and Mann-Whitney U tests with Bonferroni correction for pairwise comparisons. Outliers were assessed using Z-scores (∣Z∣ > 3) but were retained in all analyses to preserve data variance for downstream interpretation. No datasets were normalized. Detailed statistical results, including test statistics and post-hoc comparisons, are provided in Supplementary Table 1 . 2.5.2. Lifespan analysis Survival data were analyzed using Kaplan-Meier survival curves, with statistical differences between strains evaluated via the log-rank (G-rho) test in R (version 3.5.0). Censored worms (e.g., due to contamination, injury, or other unrelated factors) were excluded from the final statistical analysis. 2.5.3. Mass spectrometry data analysis Mass spectrometry data were analyzed using a two-tailed Student’s t-test to compare protein expression differences between experimental groups. Degrees of freedom (df) for the t-test were calculated as n –1, where n represents the number of proteins analyzed within each dataset. For the Δ glt-4 strain, 44 upregulated proteins were analyzed in Supplementary Table 2 (df = 43), and 37 downregulated proteins were analyzed in Supplementary Table 3 (df = 36). Similarly, for the Δ glt-5 strain, 41 upregulated proteins were analyzed in Supplementary Table 4 (df = 40), and 41 downregulated proteins were analyzed in Supplementary Table 5 (df = 40). For each protein, the t-test provided a t-statistic and an associated p-value to assess statistical significance. Detailed results, including t-statistics and p-values for individual proteins, are presented in Supplementary Tables 2 - 5 . Statistical significance was defined as p < 0.05. To further identify enriched biological processes, GO enrichment analysis was conducted using statistical thresholds of p ≤ 0.05 and −log(Q) significance rankings. NMF clustering (threshold: rank ≤ 50) was applied to identify consensus peptide signatures. Data were visualized to prioritize pathways with functional relevance to genotype differences. 3. Results 3.1. Mechanosensory behavior: nose touch assay Mechanosensory behavior is essential for environmental interaction in C. elegans . The nose touch response assay evaluates mechanosensory circuit integrity and the impact of GluT deletions on behavior. Δ glt-4 mutants exhibited significantly reduced reversal responses to nose touch stimulation compared to wildtype worms, indicating a compromised mechanosensory response. Δ glt-5 mutants showed no significant difference from wildtype in this assay, suggesting preserved mechanosensory function ( Figure 1A ). Figure 1. Behavioral responses of Δglt-4 and Δglt-5 mutants. Open in a new tab (a) Reversal responses to mechanosensory stimulation (nose touch) in wildtype, Δ glt-4 , and Δ glt-5 (n = 10 trials/worm, 30 worms/strain). (b) Chemotactic indices in response to attractive (NaCl) and aversive (1-octanol) odorants (n = 12 trials/stimulus, 166 worms/trial). (c) Chemotactic responses to butanone measured before (Pre) and after (Post) pairing with a food reward (n = 10 trials/strain, 100 worms/trial). 3.2. Chemotaxis assay Chemotaxis assays measure the ability of C. elegans to sense and respond to chemical stimuli, offering insights into sensory processing and behavioral decision-making. Both Δ glt-4 and Δ glt-5 mutants exhibited deficits in chemoattraction relative to wildtype. Δ glt-4 mutants exhibited more severe impairments in chemoattraction and a significant reduction in chemorepulsion to aversive stimuli such as octanol. Conversely, Δ glt-5 mutants demonstrated a hyperactive chemotactic response, indicating differential modulation of sensory behaviors by the two transporters ( Figure 1B ). 3.3. Association learning assay Associative learning tests the ability of C. elegans to pair a sensory cue with a reward, serving as a measure of cognitive function. In pre-association trials, Δ glt-4 and Δ glt-5 mutants displayed weaker chemorepulsion to butanone compared to wildtype. Post-association, wildtype and Δ glt-5 mutants exhibited strong learned chemoattraction, while Δ glt-4 mutants demonstrated significantly diminished learning capability, highlighting potential cognitive deficits associated with the deletion of glt-4 ( Figure 1C ). In each panel, the longer horizontal lines at the top (labeled with asterisks) indicate statistically significant differences based on one-way ANOVA with Tukey’s post-hoc test (p < 0.05). The short horizontal line across each group’s data points shows the mean of that group. See Supplementary Table 1 for the complete statistical data. 3.4. Lifespan analysis Lifespan studies explore the broader physiological effects of GluT deletions on health and metabolic homeostasis. Consistent with prior reports (~25 days at 20°C), wildtype C. elegans had a median lifespan of 23 days. Δ glt-4 mutants exhibited significantly shorter lifespans, aligned with behavioral and cognitive impairments. Conversely, Δ glt-5 mutants showed extended lifespans, suggesting adaptive effects linked to transporter deletion ( Figure 2 ). Figure 2. Kaplan-Meier survival curves for Δ glt-4 and Δ glt-5 . Open in a new tab Lifespan was tracked in L4 worms daily (wildtype n = 143; Δ glt-4 n = 123; Δ glt-5 n = 155). Log-rank analysis indicated significantly reduced lifespan for Δ glt-4 and increased lifespan for Δ glt-5 . Note: Shaded regions denote 95% confidence intervals. Data represents means across three biological replicates of each strain. 3.5. Proteomic analysis Mass spectrometry identified 53 differentially expressed proteins in Δ glt-4 mutants and 45 in Δ glt-5 mutants. Hierarchical clustering revealed distinct proteomic profiles. Δ glt-4 mutants exhibited disruptions in protein synthesis and stress-response pathways, while Δ glt-5 mutants showed upregulation of metabolic proteins associated with lifespan extension ( Figure 3 ). Figure 3. Differential protein expression in Δ glt-4 and Δ glt-5 strains. Open in a new tab (a, b) Mixture coefficient heatmaps depicting hierarchical clustering of upregulated and downregulated proteins in Δ glt-4 (a) and Δ glt-5 (b) strains relative to wildtype. Mixture coefficients represent the averaged abundance of the top 50 differentially expressed proteins for each sample. (c, d) Heatmaps showing hierarchical clustering of the union of top differentially expressed proteins in Δ glt-4 (c) and Δ glt-5 (d) strains. Proteins were selected based on combined peptide ranks (NMF and t-test ranks ≤ 50), reranked by geometric means, and filtered for consensus signature peptides (t ≥ 10). Expression changes are visualized through signal intensities, with red indicating upregulation and blue indicating downregulation. Data clustering was performed using NMF and unsupervised methods. Notably, in Figure 3 , there appears to be a much clearer pattern of protein expression differences between Δ glt-5 and wildtype, while the Δ glt-4 samples show considerable heterogeneity across the six replicates, largely independent of genotype. GO enrichment analysis revealed significant alterations in biological pathways associated with ribonucleoprotein complex organization and peptide biosynthesis processes in both glt-4 and glt-5 knockout strains, with Δ glt-4 displaying broader disruptions ( Figure 4 ). Mass spectrometry-based quantitative proteomics identified 23 significantly enriched biological pathways in total. GO enrichment analysis showed that Δ glt-4 had two upregulated and 17 downregulated biological processes, while Δ glt-5 exhibited five upregulated and two downregulated biological processes. Proteins involved in peptide biosynthetic processes (Δ glt-4 = 3.46, Δ glt-5 = 4.16) and ribonucleoprotein complex organization (Δ glt-4 = 2.41, Δ glt-5 = 2.62) were among the most significantly altered pathways, highlighting differential regulation of translation-related and stress-response processes between the two knockout strains. Figure 4. Gene Ontology pathway analysis of proteins identified by mass spectrometry-based proteomics. Open in a new tab Biological processes are ranked by their significance (−log(Q)), highlighting differences between knockout strains and wildtype. (a) Δ glt-4 displayed two significantly upregulated processes, including peptide biosynthetic processes and ribonucleoprotein complex subunit organization, and 17 downregulated processes, with notable disruptions in muscle system processes, nuclear transport, and metabolic pathways. (b) Δ glt-5 exhibited five upregulated processes, including actin filament-based processes and metabolic pathways, and two downregulated processes, primarily involving peptide biosynthetic processes and ribonucleoprotein complex subunit organization. Supplementary Figures 1 - 3 provide additional GO analysis enrichment data for the cellular components and molecular functions. Analysis of differentially expressed proteins revealed unique patterns of regulation in each strain. In Δ glt-4 , upregulated proteins included components of the ribosome such as rpl-6 and rpl-8 as well as proteins involved in protein folding, including cct-6 ( Supplementary Table 2 ). Downregulated proteins in Δ glt-4 were primarily involved in metabolic processes, such as glycogen phosphorylase ( pyg-1 ) and vha-13 , a subunit of the V-type ATPase ( Supplementary Table 3 ). In Δ glt-5 , upregulated proteins were enriched for mitochondrial functions, including mpc-1 , as well as proteins associated with cytoskeletal dynamics, such as myo-1 ( Supplementary Table 4 ). Downregulated proteins in Δ glt-5 included ribosomal proteins such as rps-12 and stress-response proteins, including fib-1 ( Supplementary Table 5 ). Further analysis of the enriched pathways emphasized functional perturbations in conserved biological processes. In Δ glt-4 , processes associated with muscle system activity and actin filament binding were the most disrupted, with enrichment fold changes of 25 and 24, respectively. In Δ glt-5 , enrichment was observed for peptide biosynthetic processes (fold change = 11) and structural components of the ribosome (fold change = 20), underscoring significant disruptions to translation machinery. The complete lists of differentially expressed proteins and enriched pathways, along with their significance scores, can be found under the file name “GO Analysis Data” at https://doi.org/10.6084/m9.figshare.28188068.v2 . Data are sorted by a combined consensus ranking. 4. Discussion Our findings reveal distinct and contrasting effects of glt-4 and glt-5 null mutations in C. elegans , underscoring the complexity of glutamate transporter functions and their tissue-specific contributions to both neural and systemic processes. The phenotypic differences between the two strains suggest unique roles for GLT-4 and GLT-5 in maintaining organismal homeostasis. Δ glt-4 mutants showed significant impairments in sensory processing, associative learning, and reduced lifespan. While GLT-4 is reported to localize to presynaptic neurons and muscle cells, its full expression profile remains unclear ( Mano et al. 2007 ). Low-level, undetected expression in additional tissues could be one factor contributing to the systemic effects observed in Δ glt-4 , including proteostasis and metabolic alterations. The finding that Δ glt-4 exhibits upregulation of proteins associated with translation and ribonucleoprotein complex organization (e.g., ribosomal proteins) hints at potential links between disrupted neural glutamate regulation and systemic cellular stress. Because glutamate functions as both a neurotransmitter and a metabolic substrate, it requires efficient recycling to prevent excitotoxicity and maintain neuronal function ( Del Rosario et al. 2015 ; Tanaka 2016 ; Sugimoto et al. 2018 ). Consistent with this, Δ glt-4 showed reduced lifespan, which could be aligned with scenarios of excessive extracellular glutamate and neurotoxicity ( Lau and Tymianski 2010 ). This raises questions about whether the observed metabolic changes in Δ glt-4 arise secondarily from neuronal dysfunction or reflect direct, low-level GLT-4 expression in peripheral tissues. Previous work in C. elegans and rodents have shown that glutamate transporters can provide substrates to neuronal mitochondria ( McNair et al. 2019 ), suggesting that diminished transporter function could compromise neuronal energy metabolism and possibly contribute to the observed behavioral and lifespan deficits ( Hyder et al. 2006 ; Du et al. 2008 ; Harris et al. 2012 ). Studies employing high-sensitivity approaches (e.g., single-cell RNA sequencing) or tissue-specific rescue experiments could help clarify whether GLT-4 is essential primarily in neurons, muscle, or other cell types. By contrast, Δ glt-5 mutants demonstrated relatively milder behavioral changes alongside extended lifespan. The localization of GLT-5 to the pharynx ( Mano et al. 2007 ) suggests a tissue-specific role—possible related to feeding mechanics or local glutamate-dependent signaling ( Lee et al. 2008 ). Given glutamate’s inhibitory effects in pharyngeal circuits, partial disruptions in feeding efficiency or local glutamate homeostasis might shape organism-wide metabolic adaptation. This interpretation is in line with previous observations in eat-4 mutants, where vesicular glutamate transporter loss affected pharyngeal dysfunction and led to systemic metabolic and morphological changes ( Lee et al. 2008 ; Serrano-Saiz et al. 2013 ; Leonelli et al. 2020 ). Nevertheless, it remains unknown whether the Δ glt-5 phenotypes result directly from altered pharyngeal glutamate metabolism or indirectly from compensatory responses. Clarifying this would require pharynx-specific manipulations. Notably, Δ glt-5 shows broad upregulation of metabolic proteins involved in TCA cycle activity ( Harrison et al. 2009 ; Williams et al. 2009 ; Chin et al. 2014 ), suggesting a possible metabolic reprogramming that correlates with its extended lifespan. Proteomic analysis likewise revealed molecular features that may help explain the distinct behavioral and lifespan outcomes of the two mutants. In Δ glt-4 , we found upregulation of processes such as “peptide biosynthetic process” and “ribonucleoprotein complex subunit organization,” which could reflect an intensified translational drive that potentially overwhelms proteostasis ( Denzel et al. 2019 ). Downregulation of key metabolic enzymes including glycogen phosphorylase and V-type ATPase subunits might further compromise energy production ( Moriyama et al. 1992 ; Nishi and Forgac 2002 ). By comparison, Δ glt-5 downregulates several translation-related GO categories but upregulates specific metabolic pathways (e.g., actin filament-based process, regulation of pH, purine nucleotide metabolic process), a pattern that could suggest improved energy efficiency ( Edwards et al. 2015 ; da Silveira et al. 2019 ). Overexpression of TCA cycle enzymes in Δ glt-5 could theoretically increase alpha-ketoglutarate levels, a factor associated with longevity ( Chin et al. 2014 ; Chitalia 2014 ). Meanwhile, Δ glt-4 downregulates certain TCA components (e.g., aconitase, isocitrate dehydrogenase), which might reduce alpha-ketoglutarate formation and potentially exacerbate neurotoxic or metabolic stress ( Birsoy et al. 2015 ; Sullivan et al. 2015 ; Zhang et al. 2020 ). These observations are consistent with previous results indicating that lifespan extension through amino acid supplementation in C. elegans involves pathways tied to translation, mitochondrial function, and stress responses ( Edwards et al. 2015 ). One hypothesis to account for the Δ glt-4 phenotype is excitotoxicity following glutamate spillover, similar to the repetitive reversal behaviors observed when GLT-1 is lost in C. elegans ( Katz et al. 2019 ). In such a scenario, excessive extracellular glutamate could make the observed proteomic changes partly downstream of ongoing neurodegeneration ( Mano and Driscoll 2009 ). The paradoxical rise in translational machinery in Δ glt-4 may promote proteotoxicity if protein folding and clearance mechanisms cannot keep pace ( Denzel et al. 2019 ), possibly consistent with the severe sensory and learning deficits ( Mano and Driscoll 2009 ; Ardiel and Rankin 2010 ; Pereira and Kooy 2012 ; Lau et al. 2013 ; Cho et al. 2016 ). By contrast, Δ glt-5 might avoid significant excitotoxic stress and instead exhibit a favorable metabolic reprogramming—downregulating ribosomal components while enhancing pathways linked to energy metabolism ( Hansen et al. 2007 ; Syntichaki et al. 2007 ; Tohyama et al. 2008 )—thereby mitigating some proteostasis burden and extending lifespan. Other proteomic shifts observed in Δ glt-4 and Δ glt-5 , including changes in fatty acid biosynthesis, cytoskeletal composition, and stress-response proteins ( Shamalnasab et al. 2017 ), could further reflect adaptive or maladaptive responses to transporter loss. Although our results do not disentangle direct versus compensatory effects, techniques such as time-course sampling, single-cell transcriptomics, and tissue-specific genetic manipulation may help clarify whether excitotoxicity plays a primary role in shaping these phenotypes or arises secondarily ( Mano and Driscoll 2009 ). In principle, fluorescent reporters for the top proteomic hits could help confirm spatial and temporal expression patterns, while targeted Western blots may validate specific alterations in protein abundance (e.g., alpha-ketoglutarate-related enzymes, ribosomal proteins, or ATP synthase subunits). While detailed follow-up studies of this nature fall outside the scope of our present work, they represent logical next steps to further dissect the interplay between transporter loss, metabolic adaptation, and excitotoxicity. A better understanding of GLT-4 and GLT-5 expression in both neuronal and non-neuronal tissues will also help ascertain whether low-level transporter activity can influence systemic homeostasis ( Tzingounis and Wadiche 2007 ). Additionally, ‘moonlighting proteins’—with multiple roles beyond their canonical enzymatic functions—may contribute to the proteomic profiles we observed ( Paludo et al. 2015 ; Jeffery 2018 ). Finally, carrying out these analyses at multiple time points would help distinguish immediate effects of transporter loss from compensatory or secondary responses. Discriminating between intrinsic consequences of transporter disruption and stress-induced adaptations is thus another key goal for future work. This study utilized technical replicates for mass spectrometry analysis (Δ glt-4 in duplicate, Δ glt-5 in triplicate), and we recognize the importance of including biological replicates to strengthen the robustness of our proteomic findings. While technical replicates provide useful preliminary insights, biological replicates can account for variability in culture conditions and reinforce the significance of observed changes. Differential protein expression may reflect inherent biological variability or environmental factors. Future studies should incorporate biological replicates to enhance reproducibility. In summary, our data indicate that glt-4 and glt-5 null mutants display notably different phenotypic profiles in C. elegans , highlighting the multifaceted roles GluTs play. We propose that Δ glt-4 could primarily affect neuronal glutamate handling and metabolic efficiency, potentially influencing proteostasis and culminating in more severe behavioral impairments and shorter lifespan. Conversely, Δ glt-5 disruption in the pharynx may initiate beneficial metabolic adjustments that conserve energy and support lifespan extension. These inferences draw on the opposing GO enrichment patterns and different regulation of translation machinery and metabolic pathways ( Figure 4 ). Overall, our findings are consistent with the view that glutamate metabolism has significant potential to modulate energy homeostasis through alpha-ketoglutarate and related TCA intermediates, and that mild perturbations in these pathways can correlate with extended lifespan ( Chin et al. 2014 ; Chitalia 2014 ; Onken et al. 2020 ). Future work combining time-course studies, tissue-specific genetic tools, and targeted biochemical validations of proteomic hits will be important for determining how transporter loss, metabolic adaptation, and excitotoxicity might each contribute to the observed outcomes. Supplementary Material 1 NIHMS2069969-supplement-1.xlsx (40.2KB, xlsx) 2 NIHMS2069969-supplement-2.docx (731.1KB, docx) 3 NIHMS2069969-supplement-3.xlsx (9.8KB, xlsx) GLT-4 and GLT-5 mutations in C. elegans reveal distinct phenotypes and proteomic profiles. Δ glt-4 is linked to altered sensory behavior, shorter lifespan, and protein downregulation. Δ glt-5 is linked to enhanced chemorepulsion, longer lifespan, and metabolic protein changes. Proteomics highlights differences in translation, metabolism, and proteostasis pathways. Results underscore the roles of glutamate transporters in systemic and neural regulation. Acknowledgments We thank Joanna Harton, who served as the statistical consultant, for invaluable guidance in data analysis, and Casey Smith for contributions to the lifespan experiments. Funding sources This work was supported by the National Institute of Mental Health [grant numbers R01MH107487, R01MH121102], the National Institute on Aging [R01AG057598]; the Wellesley College Neuroscience Department, and the Department of Neurosciences at the University of Toledo Medical Center. Worm strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs [P40 OD010440]. Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. CRediT authorship contribution statement Grace J. Bronstone: Conceptualization, Formal analysis, Investigation, Visualization, Writing – Original draft Moriah Harton: Formal analysis, Investigation, Methodology Maya Muldowney: Formal analysis, Investigation, Methodology James Reigle: Formal analysis, Investigation, Methodology, Resources, Software Adam J. Funk: Formal analysis, Methodology, Resources Sinead M. O’Donovan: Methodology, Writing – review and editing Robert E. McCullumsmith: Conceptualization, Funding acquisition, Project administration, Resources Deborah E. Bauer: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – Review and editing Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability File S1 contains the raw gene expression data for Δ glt-4 and Δ glt-5 experiments, including detailed annotations and associated metadata. File S2 contains gene ontology pathway analysis of the clustered genes in Δ glt-4 and Δ glt-5 ( Figure S1 ), analysis of the Δ glt-4 consensus sequence and gene products ( Figure S2 ), and analysis of the Δ glt-5 consensus sequence and gene products ( Figure S3 ). Table S1 contains the statistical summaries for behavioral and lifespan experiments, including pairwise comparisons and p-values. 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Front Endocrinol (Lausanne). 11:554994. 10.3389/fendo.2020.554994 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials 1 NIHMS2069969-supplement-1.xlsx (40.2KB, xlsx) 2 NIHMS2069969-supplement-2.docx (731.1KB, docx) 3 NIHMS2069969-supplement-3.xlsx (9.8KB, xlsx) Data Availability Statement File S1 contains the raw gene expression data for Δ glt-4 and Δ glt-5 experiments, including detailed annotations and associated metadata. File S2 contains gene ontology pathway analysis of the clustered genes in Δ glt-4 and Δ glt-5 ( Figure S1 ), analysis of the Δ glt-4 consensus sequence and gene products ( Figure S2 ), and analysis of the Δ glt-5 consensus sequence and gene products ( Figure S3 ). Table S1 contains the statistical summaries for behavioral and lifespan experiments, including pairwise comparisons and p-values. Gene ontology analysis data, pairwise analyses, and the raw data for the behavior and lifespan experiments are publicly available at https://doi.org/10.6084/m9.figshare.28188068.v2 . ACTIONS View on publisher site PDF (1.1 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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