Huntington’s Disease (HD) is the most common form of strictly inheritable neurodegeneration. Neuronal protein aggregates are closely associated with HD symptoms, and biomolecular pathways that protect against protein aggregation may play a role in mitigating the symptoms of HD or delaying disease onset. Using the model organism C. elegans, we used a heat stressor to exacerbate shortened lifespan phenotypes in order to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened HD worm post-heat stress lifespan, while VD₃ in moderate doses showed trends toward restoring HD post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of the HD pathogenesis mechanism and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/90cb46e0d655f308e82cf01dadc4cbbc.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/6d548d2ec91f91c3360edbaa2097a77f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans strains and maintenance
C. elegans strains used were N2 (Bristol), rmls130 (AM138, expresses Q24 in body-wall muscle cells), and rmls110 (AM101, expresses Q40 under control of a pan-neuronal promoter), purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized in order to identify differences in mean lifespans.
","reagents":"","patternDescription":"Huntington’s disease (HD) is characterized by a CAG expansion in the huntingtin gene, leading to a polyglutamine (polyQ) expansion in the resulting protein, with greater than 35Q leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of HD pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can mimic effects of age with regard to induction of protein misfolding, and proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of stress response can thus be a highly predictive measure of longevity.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer’s disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to heat stress in a C. elegans model of HD (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms, and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
To investigate the effects of exposure to vitamin D3 on early-life heat stress resistance, wild-type (N2) worms, worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter, and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). There was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). We did not see a benefit, however, from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal’s lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the HD pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Kikis EA, Gidalevitz T, Morimoto RI. 2010. Protein Homeostasis in Models of Aging and Age-Related Conformational. Protein Metabolism and Homeostasis in Aging. 694: 138-159. 192.","pubmedId":"","doi":"10.1007/978-1-4419-7002-2_11"},{"reference":"Pradhan S, Gao R, Bush K, Zhang N, Wairkar YP, Sarkar PS. 2022. Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repa. Frontiers in Cellular Neuroscience. 16: 837576. 182.","pubmedId":"","doi":"10.3389/fncel.2022.837576"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"},{"reference":"Gidalevitz T, Ben Zvi A, Ho KH, Brignull HR, Morimoto RI. 2006. Progressive Disruption of Cellular Protein Folding in Models of Polygl. Science. 311: 1471-1474. 151.","pubmedId":"","doi":"10.1126/science.1124514"},{"reference":"Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. 2007. Glucose Restriction Extends Caenorhabditis elegans Life Span by Induci. Cell Metabolism. 6: 280-293. 173.","pubmedId":"","doi":"10.1016/j.cmet.2007.08.011"},{"reference":"Smith L, Owen B, Ibarra P, Blackwell L, Seymour A, Byram M, et al., Farris M. 2024. Osmotic stressors confer age-dependent resistance to heat stress in wi. Translational Medicine of Aging. 8: 25-28. 172.","pubmedId":"","doi":"10.1016/j.tma.2024.06.001"},{"reference":"Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, Kennedy BK, Kaeberlein M. 2008. Dietary restriction suppresses proteotoxicity and enhances longevity b. Aging Cell. 7: 394-404. 186.","pubmedId":"","doi":"10.1111/j.1474-9726.2008.00385.x"},{"reference":"Tauffenberger A, Vaccaro A, Aulas A, Velde CV, Parker JA. 2012. Glucose delays age‐dependent proteotoxicity. Aging Cell. 11: 856-866. 181.","pubmedId":"","doi":"10.1111/j.1474-9726.2012.00855.x"},{"reference":"Gatrell L, Wilkins W, Rana P, Farris M. 2020. Glucose effects on polyglutamine-induced proteotoxic stress in Caenorh. Biochemical and Biophysical Research Communications. 522: 709-715. 180.","pubmedId":"","doi":"10.1016/j.bbrc.2019.11.159"},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature. 464: 504-512. 7.","pubmedId":"","doi":"10.1038/nature08980"},{"reference":"Calderwood SK, Murshid A, Prince T. 2009. The Shock of Aging: Molecular Chaperones and the Heat Shock Response i. Gerontology. 55: 550-558. 179.","pubmedId":"","doi":"10.1159/000225957"},{"reference":"Huggins B, Farris M. 2023. Vitamin D3 promotes longevity in Caenorhabditis elegans. GeroScience. 45: 345-358. 142.","pubmedId":"","doi":"10.1007/s11357-022-00637-w"},{"reference":"Leiteritz A, Schmiedl T, Baumanns S, Wenzel U. 2021. Amyloid-beta induced paralysis is reduced by cholecalciferol through i. Nutritional Neuroscience. 24: 82-89. 191.","pubmedId":"","doi":"10.1080/1028415X.2019.1596371"},{"reference":"Mark KA, Dumas KJ, Bhaumik D, Schilling B, Davis S, Oron TR, et al., Lithgow GJ. 2016. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Re. Cell Reports. 17: 1227-1237. 188.","pubmedId":"","doi":"10.1016/j.celrep.2016.09.086"},{"reference":"Messing J, Heuberger R, Schisa J. 2013. Effect of Vitamin D3 on Lifespan in Caenorhabditis elegans. Current Aging Science. 6: 220-224. 189.","pubmedId":"","doi":"10.2174/18746098113066660038"},{"reference":"Park HEH, Jung Y, Lee SJV. 2017. Survival assays using Caenorhabditis elegans. Molecules and Cells. 40: 90-99. 193.","pubmedId":"","doi":"10.14348/molcells.2017.0017"},{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77: 1-94. 86.","pubmedId":"","doi":""}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Huntington’s Disease
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"f1a7f2b7-cba0-4513-a12c-b495051283f7","decision":"revise","abstract":"Huntington’s Disease (HD) is the most common form of inheritable neurodegeneration. Neuronal protein aggregates strongly correlate with HD symptoms; pathways that protect against protein aggregation may help mitigate the symptoms of HD or delay disease onset. We used a C. elegans HD model, exposed to a heat stressor, to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened post-heat stress lifespan, while VD₃ in moderate doses trended toward restoring HD post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of HD pathogenesis mechanisms and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection, and to J.T. Roscoe for assistance with media preparation, worm transfer, and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/63fbce3983c74610defc77832ca28f9e.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/3ef2751a0307d8ceff63b64e17bc648f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans maintenance
C. elegans strains (see below) were purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored when identified, but censored worms were included in the population up to this day. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized to identify differences in mean lifespans.
","reagents":"C. elegans strains
Strain | Genotype | Source |
N2 | wild-type | CGC |
AM138 (Q24) | rmls130 [unc-54p::Q24::YFP], expresses in body-wall muscle cells | CGC |
AM101 (Q40) | rmls110 [F25B3.3p::Q40::YFP], pan-neuronal expression | CGC |
Huntington’s disease (HD) is characterized by a CAG expansion in the huntingtin gene, leading to a polyglutamine (polyQ) expansion in the resulting protein, with greater than 35Q leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of HD pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat. (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can exacerbate shortened lifespan phenotypes and mimic effects of age with regard to induction of protein misfolding; proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of response to prolonged heat can thus be a highly predictive measure of longevity, particularly in an expanded polyglutamine (Q40) background.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer’s disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to a heat stressor in a C. elegans model of HD (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
We investigated the effects of exposure to vitamin D3 on early-life heat stress resistance for wild-type (N2) worms and worms with pan-neuronal Q40 expression. We also examined VD3 effects on worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter. As the Q24 strain is under the poly-Q threshold for HD symptoms, at least in humans, it served as another control strain. Polyglutamine stretches lead to length-dependent aggregation and cellular dysfunction in both neurons and body-wall muscle cells (Teschendorf and Link, 2009), although the different tissue of expression could certainly affect pathogenesis as well. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). There was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). While we did find that high glucose conferred a benefit to young adult wild-type worms enduring heat stress exposure, as has been documented previously (Smith et al., 2024), we did not see a benefit to HD worms from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal’s lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the HD pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Kikis EA, Gidalevitz T, Morimoto RI. 2010. Protein Homeostasis in Models of Aging and Age-Related Conformational. Protein Metabolism and Homeostasis in Aging. 694: 138-159. 192.","pubmedId":"","doi":"10.1007/978-1-4419-7002-2_11"},{"reference":"Pradhan S, Gao R, Bush K, Zhang N, Wairkar YP, Sarkar PS. 2022. Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repa. Frontiers in Cellular Neuroscience. 16: 837576. 182.","pubmedId":"","doi":"10.3389/fncel.2022.837576"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"},{"reference":"Gidalevitz T, Ben Zvi A, Ho KH, Brignull HR, Morimoto RI. 2006. Progressive Disruption of Cellular Protein Folding in Models of Polygl. Science. 311: 1471-1474. 151.","pubmedId":"","doi":"10.1126/science.1124514"},{"reference":"Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. 2007. Glucose Restriction Extends Caenorhabditis elegans Life Span by Induci. Cell Metabolism. 6: 280-293. 173.","pubmedId":"","doi":"10.1016/j.cmet.2007.08.011"},{"reference":"Smith L, Owen B, Ibarra P, Blackwell L, Seymour A, Byram M, et al., Farris M. 2024. Osmotic stressors confer age-dependent resistance to heat stress in wi. Translational Medicine of Aging. 8: 25-28. 172.","pubmedId":"","doi":"10.1016/j.tma.2024.06.001"},{"reference":"Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, Kennedy BK, Kaeberlein M. 2008. Dietary restriction suppresses proteotoxicity and enhances longevity b. Aging Cell. 7: 394-404. 186.","pubmedId":"","doi":"10.1111/j.1474-9726.2008.00385.x"},{"reference":"Tauffenberger A, Vaccaro A, Aulas A, Velde CV, Parker JA. 2012. Glucose delays age‐dependent proteotoxicity. Aging Cell. 11: 856-866. 181.","pubmedId":"","doi":"10.1111/j.1474-9726.2012.00855.x"},{"reference":"Gatrell L, Wilkins W, Rana P, Farris M. 2020. Glucose effects on polyglutamine-induced proteotoxic stress in Caenorh. Biochemical and Biophysical Research Communications. 522: 709-715. 180.","pubmedId":"","doi":"10.1016/j.bbrc.2019.11.159"},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature. 464: 504-512. 7.","pubmedId":"","doi":"10.1038/nature08980"},{"reference":"Calderwood SK, Murshid A, Prince T. 2009. The Shock of Aging: Molecular Chaperones and the Heat Shock Response i. Gerontology. 55: 550-558. 179.","pubmedId":"","doi":"10.1159/000225957"},{"reference":"Huggins B, Farris M. 2023. Vitamin D3 promotes longevity in Caenorhabditis elegans. GeroScience. 45: 345-358. 142.","pubmedId":"","doi":"10.1007/s11357-022-00637-w"},{"reference":"Leiteritz A, Schmiedl T, Baumanns S, Wenzel U. 2021. Amyloid-beta induced paralysis is reduced by cholecalciferol through i. Nutritional Neuroscience. 24: 82-89. 191.","pubmedId":"","doi":"10.1080/1028415X.2019.1596371"},{"reference":"Mark KA, Dumas KJ, Bhaumik D, Schilling B, Davis S, Oron TR, et al., Lithgow GJ. 2016. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Re. Cell Reports. 17: 1227-1237. 188.","pubmedId":"","doi":"10.1016/j.celrep.2016.09.086"},{"reference":"Messing J, Heuberger R, Schisa J. 2013. Effect of Vitamin D3 on Lifespan in Caenorhabditis elegans. Current Aging Science. 6: 220-224. 189.","pubmedId":"","doi":"10.2174/18746098113066660038"},{"reference":"Park HEH, Jung Y, Lee SJV. 2017. Survival assays using Caenorhabditis elegans. Molecules and Cells. 40: 90-99. 193.","pubmedId":"","doi":"10.14348/molcells.2017.0017"},{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77: 1-94. 86.","pubmedId":"","doi":""},{"reference":"Teschendorf D, Link CD. 2009. What have worm models told us about the mechanisms of neuronal dysfunction in human neurodegenerative diseases?. Molecular Neurodegeneration 4: 38.
","pubmedId":"","doi":"10.1186/1750-1326-4-38"}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Huntington’s Disease
","reviews":[{"reviewer":{"displayName":"Jacob Manjarrez"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"9149fe5c-6ba1-4c6f-8128-c57fcc72cdf8","decision":"revise","abstract":"Polyglutamine (polyQ) diseases are the most common form of inheritable neurodegeneration. Neuronal protein aggregates strongly correlate with polyQ symptoms; pathways that protect against protein aggregation may help mitigate the symptoms of poly-Q or delay disease onset. We used a C. elegans polyQ model, exposed to a heat stressor, to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened post-heat stress lifespan, while VD₃ in moderate doses trended toward restoring polyQ post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of polyQ pathogenesis mechanisms and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection, and to J.T. Roscoe for assistance with media preparation, worm transfer, and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/dbc72b07bec9debecd9c5d7cfcd1303a.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/3ef2751a0307d8ceff63b64e17bc648f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans maintenance
C. elegans strains (see below) were purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored when identified, but censored worms were included in the population up to this day. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized to identify differences in mean lifespans.
","reagents":"C. elegans strains
Strain | Genotype | Source |
N2 | wild-type | CGC |
AM138 (Q24) | rmls130 [unc-54p::Q24::YFP], expresses in body-wall muscle cells | CGC |
AM101 (Q40) | rmls110 [F25B3.3p::Q40::YFP], pan-neuronal expression | CGC |
Huntington’s disease (HD) and eight other polyglutamine diseases are characterized by a CAG expansion in the nine causative genes, leading to a polyglutamine (polyQ) expansion in the respective resulting proteins, with longer poly-Q stretches (greater than 35Q in HD) leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of polyQ pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can exacerbate shortened lifespan phenotypes and mimic effects of age with regard to induction of protein misfolding; proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of response to prolonged heat can thus be a highly predictive measure of longevity, particularly in an expanded polyQ (Q40) background.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer’s disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to a heat stressor in a C. elegans model of polyQ disease (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
We investigated the effects of exposure to vitamin D3 on early-life heat stress resistance for wild-type (N2) worms and worms with pan-neuronal Q40 expression. We also examined VD3 effects on worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter. As the Q24 strain is under the polyQ threshold for symptoms in 8 of the 9 polyQ diseases, at least in humans, the Q24 strain primarily served as another control strain. Polyglutamine stretches lead to length-dependent aggregation and cellular dysfunction in both neurons and body-wall muscle cells (Teschendorf and Link, 2009), although the different tissue of expression could certainly affect pathogenesis as well. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). The sample sizes for the VD3 trials were low, so it is difficult to solidify conclusions; there was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). While we did find that high glucose conferred a benefit to young adult wild-type worms enduring heat stress exposure, as has been documented previously (Smith et al., 2024), we did not see a benefit to polyQ (Q40) worms from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal’s lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the polyglutamine pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Kikis EA, Gidalevitz T, Morimoto RI. 2010. Protein Homeostasis in Models of Aging and Age-Related Conformational. Protein Metabolism and Homeostasis in Aging. 694: 138-159. 192.","pubmedId":"","doi":"10.1007/978-1-4419-7002-2_11"},{"reference":"Pradhan S, Gao R, Bush K, Zhang N, Wairkar YP, Sarkar PS. 2022. Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repa. Frontiers in Cellular Neuroscience. 16: 837576. 182.","pubmedId":"","doi":"10.3389/fncel.2022.837576"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"},{"reference":"Gidalevitz T, Ben Zvi A, Ho KH, Brignull HR, Morimoto RI. 2006. Progressive Disruption of Cellular Protein Folding in Models of Polygl. Science. 311: 1471-1474. 151.","pubmedId":"","doi":"10.1126/science.1124514"},{"reference":"Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. 2007. Glucose Restriction Extends Caenorhabditis elegans Life Span by Induci. Cell Metabolism. 6: 280-293. 173.","pubmedId":"","doi":"10.1016/j.cmet.2007.08.011"},{"reference":"Smith L, Owen B, Ibarra P, Blackwell L, Seymour A, Byram M, et al., Farris M. 2024. Osmotic stressors confer age-dependent resistance to heat stress in wi. Translational Medicine of Aging. 8: 25-28. 172.","pubmedId":"","doi":"10.1016/j.tma.2024.06.001"},{"reference":"Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, Kennedy BK, Kaeberlein M. 2008. Dietary restriction suppresses proteotoxicity and enhances longevity b. Aging Cell. 7: 394-404. 186.","pubmedId":"","doi":"10.1111/j.1474-9726.2008.00385.x"},{"reference":"Tauffenberger A, Vaccaro A, Aulas A, Velde CV, Parker JA. 2012. Glucose delays age‐dependent proteotoxicity. Aging Cell. 11: 856-866. 181.","pubmedId":"","doi":"10.1111/j.1474-9726.2012.00855.x"},{"reference":"Gatrell L, Wilkins W, Rana P, Farris M. 2020. Glucose effects on polyglutamine-induced proteotoxic stress in Caenorh. Biochemical and Biophysical Research Communications. 522: 709-715. 180.","pubmedId":"","doi":"10.1016/j.bbrc.2019.11.159"},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature. 464: 504-512. 7.","pubmedId":"","doi":"10.1038/nature08980"},{"reference":"Calderwood SK, Murshid A, Prince T. 2009. The Shock of Aging: Molecular Chaperones and the Heat Shock Response i. Gerontology. 55: 550-558. 179.","pubmedId":"","doi":"10.1159/000225957"},{"reference":"Huggins B, Farris M. 2023. Vitamin D3 promotes longevity in Caenorhabditis elegans. GeroScience. 45: 345-358. 142.","pubmedId":"","doi":"10.1007/s11357-022-00637-w"},{"reference":"Leiteritz A, Schmiedl T, Baumanns S, Wenzel U. 2021. Amyloid-beta induced paralysis is reduced by cholecalciferol through i. Nutritional Neuroscience. 24: 82-89. 191.","pubmedId":"","doi":"10.1080/1028415X.2019.1596371"},{"reference":"Mark KA, Dumas KJ, Bhaumik D, Schilling B, Davis S, Oron TR, et al., Lithgow GJ. 2016. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Re. Cell Reports. 17: 1227-1237. 188.","pubmedId":"","doi":"10.1016/j.celrep.2016.09.086"},{"reference":"Messing J, Heuberger R, Schisa J. 2013. Effect of Vitamin D3 on Lifespan in Caenorhabditis elegans. Current Aging Science. 6: 220-224. 189.","pubmedId":"","doi":"10.2174/18746098113066660038"},{"reference":"Park HEH, Jung Y, Lee SJV. 2017. Survival assays using Caenorhabditis elegans. Molecules and Cells. 40: 90-99. 193.","pubmedId":"","doi":"10.14348/molcells.2017.0017"},{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77: 1-94. 86.","pubmedId":"","doi":""},{"reference":"Teschendorf D, Link CD. 2009. What have worm models told us about the mechanisms of neuronal dysfunction in human neurodegenerative diseases?. Molecular Neurodegeneration 4: 38.
","pubmedId":"","doi":"10.1186/1750-1326-4-38"}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Polyglutamine Aggregation Disease
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"bcade0b7-e8e2-4a34-8cee-dd055d067d57","decision":"edit","abstract":"Polyglutamine (polyQ) diseases are the most common form of inheritable neurodegeneration. Neuronal protein aggregates strongly correlate with polyQ symptoms; pathways that protect against protein aggregation may help mitigate the symptoms of poly-Q or delay disease onset. We used a C. elegans polyQ model, exposed to a heat stressor, to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened post-heat stress lifespan, while VD₃ in moderate doses trended toward restoring polyQ post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of polyQ pathogenesis mechanisms and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection, and to J.T. Roscoe for assistance with media preparation, worm transfer, and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/dbc72b07bec9debecd9c5d7cfcd1303a.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/3ef2751a0307d8ceff63b64e17bc648f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans maintenance
C. elegans strains (see below) were purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored when identified, but censored worms were included in the population up to this day. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized to identify differences in mean lifespans.
","reagents":"C. elegans strains
Strain | Genotype | Source |
wild-type | CGC | |
AM138 (Q24) | rmls130 [unc-54p::Q24::YFP], expresses in body-wall muscle cells | CGC |
AM101 (Q40) | rmls110 [F25B3.3p::Q40::YFP], pan-neuronal expression | CGC |
Huntington's disease (HD) and eight other polyglutamine diseases are characterized by a CAG expansion in the nine causative genes, leading to a polyglutamine (polyQ) expansion in the respective resulting proteins, with longer poly-Q stretches (greater than 35Q in HD) leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of polyQ pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can exacerbate shortened lifespan phenotypes and mimic effects of age with regard to induction of protein misfolding; proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of response to prolonged heat can thus be a highly predictive measure of longevity, particularly in an expanded polyQ (Q40) background.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer's disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to a heat stressor in a C. elegans model of polyQ disease (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
We investigated the effects of exposure to vitamin D3 on early-life heat stress resistance for wild-type (N2) worms and worms with pan-neuronal Q40 expression. We also examined VD3 effects on worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter. As the Q24 strain is under the polyQ threshold for symptoms in 8 of the 9 polyQ diseases, at least in humans, the Q24 strain primarily served as another control strain. Polyglutamine stretches lead to length-dependent aggregation and cellular dysfunction in both neurons and body-wall muscle cells (Teschendorf and Link, 2009), although the different tissue of expression could certainly affect pathogenesis as well. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). The sample sizes for the VD3 trials were low, so it is difficult to solidify conclusions; there was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). While we did find that high glucose conferred a benefit to young adult wild-type worms enduring heat stress exposure, as has been documented previously (Smith et al., 2024), we did not see a benefit to polyQ (Q40) worms from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal's lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the polyglutamine pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Kikis EA, Gidalevitz T, Morimoto RI. 2010. Protein Homeostasis in Models of Aging and Age-Related Conformational. Protein Metabolism and Homeostasis in Aging. 694: 138-159. 192.","pubmedId":"","doi":"10.1007/978-1-4419-7002-2_11"},{"reference":"Pradhan S, Gao R, Bush K, Zhang N, Wairkar YP, Sarkar PS. 2022. Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repa. Frontiers in Cellular Neuroscience. 16: 837576. 182.","pubmedId":"","doi":"10.3389/fncel.2022.837576"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"},{"reference":"Gidalevitz T, Ben Zvi A, Ho KH, Brignull HR, Morimoto RI. 2006. Progressive Disruption of Cellular Protein Folding in Models of Polygl. Science. 311: 1471-1474. 151.","pubmedId":"","doi":"10.1126/science.1124514"},{"reference":"Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. 2007. Glucose Restriction Extends Caenorhabditis elegans Life Span by Induci. Cell Metabolism. 6: 280-293. 173.","pubmedId":"","doi":"10.1016/j.cmet.2007.08.011"},{"reference":"Smith L, Owen B, Ibarra P, Blackwell L, Seymour A, Byram M, et al., Farris M. 2024. Osmotic stressors confer age-dependent resistance to heat stress in wi. Translational Medicine of Aging. 8: 25-28. 172.","pubmedId":"","doi":"10.1016/j.tma.2024.06.001"},{"reference":"Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, Kennedy BK, Kaeberlein M. 2008. Dietary restriction suppresses proteotoxicity and enhances longevity b. Aging Cell. 7: 394-404. 186.","pubmedId":"","doi":"10.1111/j.1474-9726.2008.00385.x"},{"reference":"Tauffenberger A, Vaccaro A, Aulas A, Velde CV, Parker JA. 2012. Glucose delays age‐dependent proteotoxicity. Aging Cell. 11: 856-866. 181.","pubmedId":"","doi":"10.1111/j.1474-9726.2012.00855.x"},{"reference":"Gatrell L, Wilkins W, Rana P, Farris M. 2020. Glucose effects on polyglutamine-induced proteotoxic stress in Caenorh. Biochemical and Biophysical Research Communications. 522: 709-715. 180.","pubmedId":"","doi":"10.1016/j.bbrc.2019.11.159"},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature. 464: 504-512. 7.","pubmedId":"","doi":"10.1038/nature08980"},{"reference":"Calderwood SK, Murshid A, Prince T. 2009. The Shock of Aging: Molecular Chaperones and the Heat Shock Response i. Gerontology. 55: 550-558. 179.","pubmedId":"","doi":"10.1159/000225957"},{"reference":"Huggins B, Farris M. 2023. Vitamin D3 promotes longevity in Caenorhabditis elegans. GeroScience. 45: 345-358. 142.","pubmedId":"","doi":"10.1007/s11357-022-00637-w"},{"reference":"Leiteritz A, Schmiedl T, Baumanns S, Wenzel U. 2021. Amyloid-beta induced paralysis is reduced by cholecalciferol through i. Nutritional Neuroscience. 24: 82-89. 191.","pubmedId":"","doi":"10.1080/1028415X.2019.1596371"},{"reference":"Mark KA, Dumas KJ, Bhaumik D, Schilling B, Davis S, Oron TR, et al., Lithgow GJ. 2016. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Re. Cell Reports. 17: 1227-1237. 188.","pubmedId":"","doi":"10.1016/j.celrep.2016.09.086"},{"reference":"Messing J, Heuberger R, Schisa J. 2013. Effect of Vitamin D3 on Lifespan in Caenorhabditis elegans. Current Aging Science. 6: 220-224. 189.","pubmedId":"","doi":"10.2174/18746098113066660038"},{"reference":"Park HEH, Jung Y, Lee SJV. 2017. Survival assays using Caenorhabditis elegans. Molecules and Cells. 40: 90-99. 193.","pubmedId":"","doi":"10.14348/molcells.2017.0017"},{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77: 1-94. 86.","pubmedId":"","doi":""},{"reference":"Teschendorf D, Link CD. 2009. What have worm models told us about the mechanisms of neuronal dysfunction in human neurodegenerative diseases?. Molecular Neurodegeneration 4: 38.
","pubmedId":"","doi":"10.1186/1750-1326-4-38"}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Polyglutamine Aggregation Disease
","reviews":[{"reviewer":{"displayName":"Jacob Manjarrez"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1791425852997"}]},{"id":"2a3adb56-1ee0-4b4d-8961-5c09f3421364","decision":"accept","abstract":"Polyglutamine (polyQ) diseases are the most common form of inheritable neurodegeneration. Neuronal protein aggregates strongly correlate with polyQ symptoms; pathways that protect against protein aggregation may help mitigate the symptoms of poly-Q or delay disease onset. We used a C. elegans polyQ model, exposed to a heat stressor, to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened post-heat stress lifespan, while VD₃ in moderate doses trended toward restoring polyQ post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of polyQ pathogenesis mechanisms and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection, and to J.T. Roscoe for assistance with media preparation, worm transfer, and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/dbc72b07bec9debecd9c5d7cfcd1303a.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/3ef2751a0307d8ceff63b64e17bc648f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans maintenance
C. elegans strains (see below) were purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored when identified, but censored worms were included in the population up to this day. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized to identify differences in mean lifespans.
","reagents":"C. elegans strains
Strain | Genotype | Source |
wild-type | CGC | |
AM138 (Q24) | rmls130 [unc-54p::Q24::YFP], expresses in body-wall muscle cells | CGC |
AM101 (Q40) | rmls110 [F25B3.3p::Q40::YFP], pan-neuronal expression | CGC |
Huntington's disease (HD) and eight other polyglutamine diseases are characterized by a CAG expansion in the nine causative genes, leading to a polyglutamine (polyQ) expansion in the respective resulting proteins, with longer poly-Q stretches (greater than 35Q in HD) leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of polyQ pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can exacerbate shortened lifespan phenotypes and mimic effects of age with regard to induction of protein misfolding; proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of response to prolonged heat can thus be a highly predictive measure of longevity, particularly in an expanded polyQ (Q40) background.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer's disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to a heat stressor in a C. elegans model of polyQ disease (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
We investigated the effects of exposure to vitamin D3 on early-life heat stress resistance for wild-type (N2) worms and worms with pan-neuronal Q40 expression. We also examined VD3 effects on worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter. As the Q24 strain is under the polyQ threshold for symptoms in 8 of the 9 polyQ diseases, at least in humans, the Q24 strain primarily served as another control strain. Polyglutamine stretches lead to length-dependent aggregation and cellular dysfunction in both neurons and body-wall muscle cells (Teschendorf and Link, 2009), although the different tissue of expression could certainly affect pathogenesis as well. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). The sample sizes for the VD3 trials were low, so it is difficult to solidify conclusions; there was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). While we did find that high glucose conferred a benefit to young adult wild-type worms enduring heat stress exposure, as has been documented previously (Smith et al., 2024), we did not see a benefit to polyQ (Q40) worms from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal's lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the polyglutamine pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Kikis EA, Gidalevitz T, Morimoto RI. 2010. Protein Homeostasis in Models of Aging and Age-Related Conformational. Protein Metabolism and Homeostasis in Aging. 694: 138-159. 192.","pubmedId":"","doi":"10.1007/978-1-4419-7002-2_11"},{"reference":"Pradhan S, Gao R, Bush K, Zhang N, Wairkar YP, Sarkar PS. 2022. Polyglutamine Expansion in Huntingtin and Mechanism of DNA Damage Repa. Frontiers in Cellular Neuroscience. 16: 837576. 182.","pubmedId":"","doi":"10.3389/fncel.2022.837576"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"},{"reference":"Gidalevitz T, Ben Zvi A, Ho KH, Brignull HR, Morimoto RI. 2006. Progressive Disruption of Cellular Protein Folding in Models of Polygl. Science. 311: 1471-1474. 151.","pubmedId":"","doi":"10.1126/science.1124514"},{"reference":"Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M. 2007. Glucose Restriction Extends Caenorhabditis elegans Life Span by Induci. Cell Metabolism. 6: 280-293. 173.","pubmedId":"","doi":"10.1016/j.cmet.2007.08.011"},{"reference":"Smith L, Owen B, Ibarra P, Blackwell L, Seymour A, Byram M, et al., Farris M. 2024. Osmotic stressors confer age-dependent resistance to heat stress in wi. Translational Medicine of Aging. 8: 25-28. 172.","pubmedId":"","doi":"10.1016/j.tma.2024.06.001"},{"reference":"Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, Kennedy BK, Kaeberlein M. 2008. Dietary restriction suppresses proteotoxicity and enhances longevity b. Aging Cell. 7: 394-404. 186.","pubmedId":"","doi":"10.1111/j.1474-9726.2008.00385.x"},{"reference":"Tauffenberger A, Vaccaro A, Aulas A, Velde CV, Parker JA. 2012. Glucose delays age‐dependent proteotoxicity. Aging Cell. 11: 856-866. 181.","pubmedId":"","doi":"10.1111/j.1474-9726.2012.00855.x"},{"reference":"Gatrell L, Wilkins W, Rana P, Farris M. 2020. Glucose effects on polyglutamine-induced proteotoxic stress in Caenorh. Biochemical and Biophysical Research Communications. 522: 709-715. 180.","pubmedId":"","doi":"10.1016/j.bbrc.2019.11.159"},{"reference":"Kenyon CJ. 2010. The genetics of ageing. Nature. 464: 504-512. 7.","pubmedId":"","doi":"10.1038/nature08980"},{"reference":"Calderwood SK, Murshid A, Prince T. 2009. The Shock of Aging: Molecular Chaperones and the Heat Shock Response i. Gerontology. 55: 550-558. 179.","pubmedId":"","doi":"10.1159/000225957"},{"reference":"Huggins B, Farris M. 2023. Vitamin D3 promotes longevity in Caenorhabditis elegans. GeroScience. 45: 345-358. 142.","pubmedId":"","doi":"10.1007/s11357-022-00637-w"},{"reference":"Leiteritz A, Schmiedl T, Baumanns S, Wenzel U. 2021. Amyloid-beta induced paralysis is reduced by cholecalciferol through i. Nutritional Neuroscience. 24: 82-89. 191.","pubmedId":"","doi":"10.1080/1028415X.2019.1596371"},{"reference":"Mark KA, Dumas KJ, Bhaumik D, Schilling B, Davis S, Oron TR, et al., Lithgow GJ. 2016. Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Re. Cell Reports. 17: 1227-1237. 188.","pubmedId":"","doi":"10.1016/j.celrep.2016.09.086"},{"reference":"Messing J, Heuberger R, Schisa J. 2013. Effect of Vitamin D3 on Lifespan in Caenorhabditis elegans. Current Aging Science. 6: 220-224. 189.","pubmedId":"","doi":"10.2174/18746098113066660038"},{"reference":"Park HEH, Jung Y, Lee SJV. 2017. Survival assays using Caenorhabditis elegans. Molecules and Cells. 40: 90-99. 193.","pubmedId":"","doi":"10.14348/molcells.2017.0017"},{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
","pubmedId":"4366476","doi":""},{"reference":"Teschendorf D, Link CD. 2009. What have worm models told us about the mechanisms of neuronal dysfunction in human neurodegenerative diseases?. Molecular Neurodegeneration 4: 38.
","pubmedId":"","doi":"10.1186/1750-1326-4-38"}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Polyglutamine Aggregation Disease
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"31380f17-bd47-4b29-af1d-3656348c64e8","decision":"publish","abstract":"Polyglutamine (polyQ) diseases are the most common form of inheritable neurodegeneration. Neuronal protein aggregates strongly correlate with polyQ symptoms; pathways that protect against protein aggregation may help mitigate the symptoms of poly-Q or delay disease onset. We used a C. elegans polyQ model, exposed to a heat stressor, to test potential protective mechanisms: glucose and vitamin D₃ (VD₃) supplementation. Glucose enrichment further shortened post-heat stress lifespan, while VD₃ in moderate doses trended toward restoring polyQ post-stress lifespan back to wild-type levels. These results show effects of early-life interventions that could indicate aspects of polyQ pathogenesis mechanisms and treatment options.
","acknowledgements":"Thank you to the Fall 2023 UCA Department of Biology Molecular Mechanisms of Aging class for assistance with worm transfer and data collection, and to J.T. Roscoe for assistance with media preparation, worm transfer, and data collection.
","authors":[{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"londonblackwelldyer@gmail.com","firstName":"London","lastName":"Blackwell","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology",""],"credit":["dataCuration","formalAnalysis","investigation","writing_originalDraft"],"email":"josephsharp47@gmail.com","firstName":"Joseph","lastName":"Sharp","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas"],"departments":["Biology"],"credit":["investigation"],"email":"Gisselle726@gmail.com","firstName":"Gisselle","lastName":"Hernandez","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["University of Central Arkansas, Conway, AR, United States"],"departments":["Biology"],"credit":["conceptualization","project","supervision","writing_reviewEditing"],"email":"mfarris@uca.edu","firstName":"Mindy","lastName":"Farris","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-9559-5761"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":"https://portal.micropublication.org/uploads/c1b1bc3e86a98f5bce066e5f61f87f57.csv"},"extendedData":[],"funding":"Funding was provided by the University of Central Arkansas University Research Council, the UCA Graduate School, the UCA College of Science and Engineering, and the UCA Department of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/3ef2751a0307d8ceff63b64e17bc648f.png"},"imageCaption":"A) Wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan with glucose (p=0.0234), but Q40 worms showed a 16.7% decrease in post-heat stress lifespan after glucose treatment (p=0.0229). B-D) Neither vitamin D3 concentration had a significant effect on post-heat stress lifespan for wild-type (N2), Q24, or Q40 worms. There was a non-significant trend for 400µM to slightly lengthen Q40 post-heat stress lifespan (20.4%, p=0.0527).
*The Q40 800µM VD3 line ends before 0 survival as the last worms in this condition died censored deaths.
","imageTitle":"Kaplan-Meier survival curve for C. elegans heat-stressed at 35ºC for 7 hours, at day 1 of adulthood, after overnight treatment with A) 250mM glucose (sorbitol control), or B-D) 400µM or 800µM Vitamin D3 (ethanol control)
","methods":"C. elegans maintenance
C. elegans strains (see below) were purchased from the Caenorhabditis Genetics Center (CGC). Escherichia coli OP50 (OP50) was also obtained from the CGC, and was propagated into liquid cultures grown in Luria Broth. For growth of worm populations, 250μL live OP50 was spotted onto Nematode Growth Media (NGM) plates (Brenner, S., 1974) and allowed to grow at least one night before use. All strains were maintained at 20°C and on standard NGM plates spotted with OP50. Populations were synchronized by bleach preparation if stock strains were contaminated, otherwise, populations were synchronized by picking young adult worms onto fresh plates.
Heat stress assay
Heat stress response analyses were performed for N2, Q24, and Q40 strains on 6cm plates of regular NGM spotted with 250µL OP50 bacteria and treated with either 250mM glucose (250mM sorbitol control) or 400µM/800µM Vitamin D3 (ethanol control). Worms were placed on the respective treatment 1 day before heat stress, at day 0 of adulthood (L4), 25 worms per plate. Worms were exposed to 35ºC heat stress for 7 hours, at day 1 of adulthood. All worms were hermaphrodites. No FUdR was used. Worms were transferred away from progeny and scored for response to touch every other day, until all worms were dead. Worms that died of non-natural deaths, such as internal hatching of progeny, were censored when identified, but censored worms were included in the population up to this day. Results represent the survival rate from a minimum of two biological replicates.
Kaplan-Meier curves were generated using JMP software, and the nonparametric test, log-rank (Mantel-Cox) was utilized to identify differences in mean lifespans.
","reagents":"C. elegans strains
Strain | Genotype | Source |
wild-type | CGC | |
AM138 (Q24) | rmls130 [unc-54p::Q24::YFP], expresses in body-wall muscle cells | CGC |
AM101 (Q40) | rmls110 [F25B3.3p::Q40::YFP], pan-neuronal expression | CGC |
Huntington's disease (HD) and eight other polyglutamine diseases are characterized by a CAG expansion in the nine causative genes, leading to a polyglutamine (polyQ) expansion in the respective resulting proteins, with longer poly-Q stretches (greater than 35Q in HD) leading to disease phenotypes, and the length of expansion inversely correlated to age-of-onset (Kikis et al., 2010; Pradhan et al., 2022; Weydt and La Spada, 2006). While Caenorhabditis elegans lack the huntingtin gene, C. elegans models of HD have shown protein aggregation and shortened lifespans conferred by expression of a transgenic polyQ stretch alone (Gidalevitz et al., 2006). This transgenic polyQ model provides a convenient system to study the mechanisms of polyQ pathogenesis and to test potential interventions.
Glucose enrichment typically reduces C. elegans lifespan, but can be protective at early ages, particularly against stressors like prolonged heat (Schulz et al., 2007; Smith et al., 2024). Dietary restriction (DR) protects against proteotoxic stress at least in some cellular backgrounds, but tissue of expression appears to play a role (Steinkraus et al., 2008; Tauffenberger et al., 2012). Studies in C. elegans HD models also show high concentrations of glucose could protect against polyQ proteotoxic stress, with tissue of expression important for efficacy (Gatrell et al., 2020; Tauffenberger et al., 2012). A strong link has been shown between longevity and resistance to various types of stressors: severe stress shortens lifespan, and many interventions that extend lifespan appear to also confer resistance to severe stressors (Kenyon, 2010). While the link between stress resistance and longevity is complex, stressors like heat exposure can exacerbate shortened lifespan phenotypes and mimic effects of age with regard to induction of protein misfolding; proteotoxic stress in particular can be simulated by increased temperature (Calderwood et al., 2009; Gidalevitz et al., 2006). Measurements of response to prolonged heat can thus be a highly predictive measure of longevity, particularly in an expanded polyQ (Q40) background.
Another pathway showing lifespan extension in wild-type (N2) C. elegans and in C. elegans models of Alzheimer's disease (AD) is vitamin D3 (VD₃) supplementation, potentially through decreased nutrient signaling as a primary mechanism (Huggins and Farris, 2023; Leiteritz et al., 2021; Mark et al., 2016; Messing et al., 2013). We tested effects of glucose enrichment and of VD₃ supplementation on early-life resistance to a heat stressor in a C. elegans model of polyQ disease (Q40).
Glucose effects on HD early-life stress resistance
To investigate the effects of exposure to glucose on early-life stress resistance in a C. elegans model of HD, wild-type (N2) worms and worms expressing a 40-glutamine stretch (Q40) under the control of a pan-neuronal promoter were utilized. Worms were transferred to 250mM glucose or 250mM sorbitol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
The wild-type (N2) worms showed a 20.6% increase in post-heat stress lifespan when exposed to glucose (p=0.0234). The Q40 worms, however, showed a 16.7% decrease in post-heat stress lifespan on glucose compared to the sorbitol osmotic control (p=0.0229) (Figure A, Table A).
Vitamin D3 effects on HD early-life stress resistance
We investigated the effects of exposure to vitamin D3 on early-life heat stress resistance for wild-type (N2) worms and worms with pan-neuronal Q40 expression. We also examined VD3 effects on worms expressing a 24-glutamine stretch (Q24) under the control of a body-wall muscle promoter. As the Q24 strain is under the polyQ threshold for symptoms in 8 of the 9 polyQ diseases, at least in humans, the Q24 strain primarily served as another control strain. Polyglutamine stretches lead to length-dependent aggregation and cellular dysfunction in both neurons and body-wall muscle cells (Teschendorf and Link, 2009), although the different tissue of expression could certainly affect pathogenesis as well. Worms were transferred to 400µM or 800 µM VD₃ or ethanol control plates on day 0 of adulthood (L4 stage), followed by a 7-hour heat stress at 35°C on day 1 of adulthood. Worms were scored for post-heat-stress lifespan.
Neither VD3 concentration had a significant effect on post-heat stress lifespan in any of the worm strains (Table B). The sample sizes for the VD3 trials were low, so it is difficult to solidify conclusions; there was a non-significant trend, however, for 400µM VD3 to lengthen post-heat stress lifespans, particularly that of the Q40 strain (20.4%, p=0.0527) (Figure B-D, Table B).
C. elegans have several mechanisms to respond to high temperature environments, including the heat-shock transcription factor-1 (HSF-1) and DAF-16 transcription factor, which upregulate systems responsible for maintaining cellular and protein homeostasis (Park et al., 2017). These functions require energy sources, which leads to the reasoning that additional glucose could allow them to function at a higher capacity to protect the organism from damage. Indeed, neurons have a high glucose requirement, particularly in an HD background, seen in the wasting phenotype of HD patients and transgenic models (Aditi et al., 2016; Tramutola et al., 2023). While we did find that high glucose conferred a benefit to young adult wild-type worms enduring heat stress exposure, as has been documented previously (Smith et al., 2024), we did not see a benefit to polyQ (Q40) worms from glucose enrichment at this level (250mM) or at this early age of adulthood.
Vitamin D3, however, at the lower dose (400µM) did show a partial rescue of the Q40 worm post-heat stress lifespan, and improved post-heat stress lifespan for all strains, although not statistically significant. The higher dose of VD3 (800µM) likely crossed into toxicity.
These results show effects of glucose and VD3 interventions specifically early in the animal's lifespan. We have seen later-life interventions to have distinct effects (Smith et al., 2024), and are eager to test these during mid-life and late-life timepoints. Learning when these pathways are activated could uncover parts of the polyglutamine pathogenesis mechanism, as well as suggest treatment options.
","references":[{"reference":"Aditi K, Shakarad MN, Agrawal N. 2016. Altered lipid metabolism in Drosophila model of Huntington’s disease. Scientific Reports. 6: 31411. 194.","pubmedId":"","doi":"10.1038/srep31411"},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
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","pubmedId":"","doi":"10.1186/1750-1326-4-38"},{"reference":"Tramutola A, Bakels HS, Perrone F, Di Nottia M, Mazza T, Abruzzese MP, et al., Squitieri F. 2023. GLUT-1 changes in paediatric Huntington disease brain cortex and fibro. eBioMedicine. 97: 104849. 196.","pubmedId":"","doi":"10.1016/j.ebiom.2023.104849"},{"reference":"Weydt P, La Spada AR. 2006. Targeting protein aggregation in neurodegeneration – lessons from poly. Expert Opinion on Therapeutic Targets. 10: 505-513. 185.","pubmedId":"","doi":"10.1517/14728222.10.4.505"}],"title":"Effects of Glucose and Vitamin D3 on Heat Stress Resistance in a Caenorhabditis elegans Model of Polyglutamine Aggregation Disease
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