microRNAs (miRNAs) are short, non-coding RNAs essential for gene regulation in many different processes, including neuronal development. However, the role of the miRNA pathway in maintaining neuronal health throughout aging is less understood. Here, we ask how the miRNA pathway in adulthood impacts neurobehaviors in C. elegans. Argonaute-like Gene 2 (ALG-2) is a protein required for the accumulation and function of certain miRNAs in C. elegans. Using the auxin-inducible degron (AID) system for temporal knockdown, we demonstrate that the miRNA Argonaute ALG-2 is required throughout adulthood to maintain two well-characterized neurobehaviors, basal slowing response and mechanosensation.
","acknowledgements":"This work was supported by grants from the National Institutes of Health [R35 GM127012 to A.E.P.]; and the Hevolution Foundation [HF-GRO-23-1199180]. E.C.S. was supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine [T32 GM007240]. S.D.P was supported by a Faculty Research Support Grant provided by Swarthmore College.
","authors":[{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","writing_originalDraft","writing_reviewEditing"],"email":"achon1@swarthmore.edu","firstName":"Ava","lastName":"Chon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0000-8049-6146"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","methodology","investigation","dataCuration","validation"],"email":"ruj016@ucsd.edu","firstName":"Runtian","lastName":"Jiang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0009-0069-777X"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation"],"email":"yphyu1@swarthmore.edu","firstName":"Yamin K.","lastName":"Phyu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-7961-3082"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["supervision","conceptualization","investigation"],"email":"eschiksn@ucsd.edu","firstName":"Erin C.","lastName":"Schiksnis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2756-8168"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","fundingAcquisition","writing_reviewEditing","supervision","methodology"],"email":"apasquinelli@ucsd.edu","firstName":"Amy E.","lastName":"Pasquinelli","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9511-0039"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","supervision","writing_originalDraft","writing_reviewEditing","visualization"],"email":"spalumb1@swarthmore.edu","firstName":"Sierra D.","lastName":"Palumbos","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3595-984X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"WBPerson39614
","image":{"url":"https://portal.micropublication.org/uploads/131f96e5348751094e69df4640ad491a.png"},"imageCaption":"A) Graphic depicting insertion of AID at the alg-2 gene locus. B) Immunoblot of ALG-2 protein levels from PQ668 worms following 30 minutes of auxin treatment at ranging auxin concentrations. Actin was probed as positive control. C) Soft touch response assay of day 3 adult PQ668 and N2 strains with or without auxin treatment. Defective movement defined as failure to complete two “S” turns following two soft touches behind pharynx. N>50 worms for each condition. Fisher's exact test used to determine significance, ** indicates p<0.01. D) BSR assay of day 1 adult PQ668 strain and E) day 4 adult PQ668 and N2 strains. For each condition, n=27. BSR was calculated by subtracting the number of body bends in the presence of food from the number of body bends when food was absent. One-way ANOVA with multiple comparisons used to determine significance; *** indicates p<0.001. F) Chemotaxis Index (CI) of day 2 adult PQ668 and N2 C.elegans with and without auxin treatment in response to isoamyl alcohol (IA). CI was calculated as the number of worms at IA minus the number of worms at H₂O, divided by the total number of worms. Error bars represent SD. n ≥10 per trial, 3 trials. One-way ANOVA with multiple comparisons used to determine significance.
","imageTitle":"ALG-2 function in adulthood required for neurobehaviors
","methods":"Strain Preparation: The N2 strain was obtained from the Caenorhabditis Genetics Center. The PQ668 (AID::mNeonGreen::3Xflag::alg2) strain was developed using CRISPR-Cas9 to insert AID::mNeonGreen::3Xflag before the 5' end of alg-2 before exon 1 of isoform A and exon 0 of isoform B (See Jiang, 2022). Briefly, four plasmids were injected into young adult N2 worms: 1) 50ng/ul of homologous repair template (aid::mNeonGreen::3xflag with ALG-2 homology arms), 2) 50ng/ul of pJB53 (Cas9 plasmid with ALG-2 specific sgRNA, modified from pJW1219, Addgene #61250), 3) 10ng/ul pGH8 (pRAB-3::mCherry::unc-54utr, Addgene plasmid #19359) and 4) 5ng/μL pCFJ104 (Pmyo-3::mCherry::unc-54, Addgene plasmid #19328). Recombinant worms were isolated as previously described (Dickinson et al. 2015) and then backcrossed 3X to N2 to generate PQ668.
Auxin Plates: Nematode growth media (NGM) plates were prepared as described previously (Stiernagle, 2006). Auxin-containing NGM plates were prepared by adding 0.89 mg/mL auxin after autoclaving (Sharma et al., 2024). Plates were seeded with OP50 for 48 hours.
Western Blot: Western blot was carried out as described previously and ALG-2 was detected based on 3X flag insertion using anti-FLAG antibody (Wynberghe et al., 2011).
Soft Touch Response: L4 C. elegans were plated on NGM plates with or without auxin and allowed to grow at 23℃ for 3 days. Day 3 adult worms were transferred to an unseeded NGM plate and allowed to acclimate. With an eyelash pick, worms were stroked behind the pharynx and behavior was scored (Chalfie et al., 2018). Defective movement was defined as a worm failing to perform 2 reverse “S” turns following 2 strokes.
Basal Slowing Response: L4 C. elegans were plated on NGM plates with or without auxin. Plates were kept at 23℃ and worms were allowed to grow for either 1 or 4 days before being transferred to an unseeded NGM plate for analysis. After a 3-minute acclimation period, body bends were counted over a 20-second period. Worms were transferred to a seeded plate and the same procedure was repeated (Petratou et al., 2024). BSR was calculated for individual worms by subtracting the number of body bends in the presence of food from the number of body bends when no food was present.
Chemotaxis Assay: L4 C. elegans were transferred to OP50-seeded NGM plates with or without auxin and maintained at 23℃ for 2 days. Chemotaxis assay plates were prepared by dividing each plate into two halves and 5μL of isoamyl alcohol (IA) on one side and 5μL of deionized water (H₂O) on the other. 5μL of 0.5 M sodium azide was spotted on IA and H₂O to paralyze the worms upon contact. Day-2 adult worms were washed with 1 mL of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1M MgSO₄) before being transferred to chemotaxis plates for 1-hour. A minimum of 10 worms per plate was assayed across 3 trials. Worms were manually counted under a dissecting microscope. The chemotaxis index was calculated as CI = (# of worms at IA − # of worms at H₂O) / total # of worms, and ranged from +1 (maximum attraction) to -1 (maximum repulsion) (Bargmann et al., 1993).
Statistics: All statistical analyses were performed using Prism 10 and specific tests are specified in figure legend.
","reagents":"","patternDescription":"miRNAs are non-coding RNAs that are approximately 22 nucleotides long which repress gene expression of target mRNAs (Shang et al., 2023). miRNAs associate with an Argonaute protein, forming the miRNA-induced silencing complex (miRISC), which recognizes specific mRNA targets and induces their translational repression and degradation (Shang et al., 2023, Sala et al., 2020). C. elegans express two Argonaute proteins, ALG-1 and ALG-2, which exhibit distinct roles (Tops et al., 2006, Aalto et al., 2018). While miRNAs are well characterized in development (Ivey and Srivastava, 2015), their role in aging and neurodegeneration is still an emerging field (Elder and Pasquinelli, 2022). In human patients with Parkinson's disease (PD) and Alzheimer's disease (AD), elevated levels of circulating miRNAs have been observed, but whether this is a feature of disease or a compensatory response is unclear (Alkhazaali-Ali et al., 2024). In a cultured neuronal model of PD, exosomes containing miRNAs were found to be neuroprotective (Palumbos et al., 2024). These observations led us to ask whether the miRNA pathway has a role in maintaining neuronal homeostasis by tracking common neurobehaviors that are impaired following neurodegeneration (Caldwell et al., 2020).
To do this, we used CRISPR/Cas9 to insert an Auxin-inducible-degron sequence at the N-terminus of the coding sequence of alg-2 gene in C. elegans, to generate a worm strain, PQ668 (AID::mNeonGreen::3Xflag::alg-2) enabailing temporal depletion of the ALG-2 protein in adult C. elegans via the exogenous addition of auxin (Fig. 1A) (Nishimura et al., 2009, Dickinson et al., 2015). To confirm successful knockdown of ALG-2, we tracked ALG-2 protein levels using immunoblotting from animals treated with auxin compared to a vehicle control (Fig. 1B). In as few as 30 minutes on auxin-plates, ALG-2 protein levels were reduced below detection. This was also confirmed by visualization of the integrated GFP fluorescent reporter. Thus, we developed a tool where ALG-2 levels could be selectively reduced during adulthood, allowing us to ask if loss of ALG-2 impacts neurodegenerative phenotypes.
C. elegans depend on soft touch sensation which is communicated via mechanoreceptors expressed by six touch receptor neurons and requires glutamatergic signaling (Chen and Chalfie, 2014). We asked if depletion of ALG-2 in adulthood impacts mechanosensation behavior. A subset of L4 PQ668 worms were transferred to auxin plates to induce ALG-2 knockdown (+auxin), while remaining L4 PQ668 worms were kept on standard NGM plates (-auxin) for 3 days. We tracked mechanosensation in response to an eyelash pick on day 3 adults. Interestingly, we observed a significant reduction in soft touch response in PQ668 +auxin worms (p=.0017) (Fig. 1C). In order to verify that auxin did not account for this difference in behavior, we repeated the soft touch assay with N2 worms and observed no difference between the +auxin and -auxin groups (p =.9999), confirming auxin did not serve as a confounding variable (Fig. 1C). Our findings suggest that the miRNA Argonaute ALG-2 is important for mechanosensation in adult animals.
Given our finding that ALG-2 expression is required for proper mechanosensation, we next asked whether other behaviors were impacted by ALG-2 depletion in adulthood. C. elegans reduce their speed in the presence of food, a phenomenon termed Basal Slowing Response (BSR) (Rivard et al., 2010), which is dependent on dopaminergic signaling (Sawin et al., 2000). We measured BSR in PQ668 +auxin and PQ668 -auxin worms to determine how impairment of the ALG-2-miRNA pathway impacts dopaminergic signaling in C. elegans. In day 1 adult worms, there was no statistically significant difference in BSR between PQ668 +auxin and PQ668 -auxin worms (p=.2244) (Fig. 1D). However, following 4 days of ALG-2 depletion, PQ668 worms showed significantly reduced BSR (p=.0008) (Fig. 1E). We found there was no difference between N2 +auxin vs. N2 -auxin worms under the same conditions (p=.9559) (Fig. 1E). Thus, we observed an age-dependent defect in BSR, suggesting a role for ALG-2 in a dopaminergic signaling response in older adults.
C. elegans possess a well-organized chemosensory system that allows them to navigate a variety of olfactory and gustatory cues associated with food, danger, and mates (Bargmann, 2006). We used the well characterized attractant, isoamyl alcohol, (Bargmann et al., 1993) to ask if ALG-2 function in adulthood is similarly required for chemotaxis. We tracked the chemotaxis index in PQ668 and N2 wild type worms (day 2 adults) with and without auxin. In contrast to mechanosensation and BSR, we found no significant change in chemosensory behavior across any conditions (Fig. 1F). Of note that both our auxin groups had higher variability in their response, suggesting that auxin itself could be impairing chemotaxis. Thus, loss of ALG-2 function did not impair chemotaxis, suggesting that distinct circuits are impacted by loss of ALG-2 regulated microRNAs.
Given our observations that adult expression of ALG-2 is selectively required for specific neurobehaviors, we asked whether alg-2 expression was selectively elevated in specific neurons in adulthood. We tracked alg-2 mRNA expression using the C. elegans Neuronal Gene Expression Map & Network (CeNGEN) and identified three neurons with a > 5-fold increase in expression level at adulthood vs. L4 (AWA, PVR and RME). We reasoned that up-regulation of alg-2 in specific adult neurons may contribute to the preservation of their functions. Of note, both RME and AWA could impact BSR, as RME is a motor neuron important for foraging (Cinar et al., 2005) and AWA is critical for sensing diacetyl, a bacterial metabolite important for food detection (Bargmann et al., 1993). In conclusion, we find that ALG-2 is required throughout adulthood to maintain glutamatergic and dopaminergic processing in C. elegans. These findings have important implications regarding the role of the miRNA pathway in aging and neurodegeneration.
","references":[{"reference":"Aalto AP, Nicastro IA, Broughton JP, Chipman LB, Schreiner WP, Chen JS, Pasquinelli AE. 2018. Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics. 14: e1007379.","pubmedId":"","doi":"10.1371/journal.pgen.1007379"},{"reference":"Alkhazaali Ali Z, Sahab Negah S, Boroumand AR, Tavakol Afshari J. 2024. MicroRNA (miRNA) as a biomarker for diagnosis, prognosis, and therapeutics molecules in neurodegenerative disease. Biomedicine & Pharmacotherapy. 177: 116899.","pubmedId":"","doi":"10.1016/j.biopha.2024.116899"},{"reference":"Bargmann CI, Hartwieg E, Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74: 515.","pubmedId":"","doi":"10.1016/0092-8674(93)80053-h"},{"reference":"Bargmann CI. 2006. Chemosensation in C. elegans.. Wormbook: 1.","pubmedId":"","doi":"10.1895/wormbook.1.123.1"},{"reference":"Caldwell KA, Willicott CW, Caldwell GA. 2020. Modeling neurodegeneration in Caenorhabditis elegans. Disease Models & Mechanisms. 13: dmm046110.","pubmedId":"","doi":"10.1242/dmm.046110"},{"reference":"Chalfie M, Hart AC, Rankin CH, Goodman MB. 2018. Assaying mechanosensation. WormBook: The Online Review of C. elegans Biology [Internet]","pubmedId":"","doi":""},{"reference":"Chen X, Chalfie M. 2014. Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels. Journal of Neuroscience. 34: 6522.","pubmedId":"","doi":"10.1523/JNEUROSCI.0022-14.2014"},{"reference":"Cinar H, Keles S, Jin Y. 2005. Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans. Current Biology. 15: 340.","pubmedId":"","doi":"10.1016/j.cub.2005.02.025"},{"reference":"Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. 2015. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics. 200: 1035.","pubmedId":"","doi":"10.1534/genetics.115.178335"},{"reference":"Elder CR, Pasquinelli AE. 2022. New Roles for MicroRNAs in Old Worms. Frontiers in Aging. 3: 871226.","pubmedId":"","doi":"10.3389/fragi.2022.871226"},{"reference":"Ivey KN, Srivastava D. 2015. microRNAs as Developmental Regulators. Cold Spring Harbor Perspectives in Biology. 7: a008144.","pubmedId":"","doi":"10.1101/cshperspect.a008144"},{"reference":"Jiang R. 2022. Conditional depletion of ALG-1 and ALG-2 using auxin inducible degron 2 (AID2).","pubmedId":"","doi":""},{"reference":"Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. 2009. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods. 6: 917.","pubmedId":"","doi":"10.1038/nmeth.1401"},{"reference":"O Brien J, Hayder H, Zayed Y, Peng C. 2018. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology. 9: 402.","pubmedId":"","doi":"10.3389/fendo.2018.00402"},{"reference":"Palumbos SD, Popolow J, Goldsmith J, Holzbaur ELF. 2024. Autophagic stress activates distinct compensatory secretory pathways in neurons. bioRxiv: 2024.11.07.621551.","pubmedId":"","doi":"10.1101/2024.11.07.621551"},{"reference":"Petratou D, Fragkiadaki P, Lionaki E, Tavernarakis N. 2024. Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans. STAR Protocols. 5: 102801.","pubmedId":"","doi":"10.1016/j.xpro.2023.102801"},{"reference":"Rivard L, Srinivasan J, Stone A, Ochoa S, Sternberg PW, Loer CM. 2010. A comparison of experience-dependent locomotory behaviors and biogenic amine neurons in nematode relatives of Caenorhabditis elegans. BMC Neuroscience. 11: 22.","pubmedId":"","doi":"10.1186/1471-2202-11-22"},{"reference":"Sala L, Chandrasekhar S, Vidigal JA. 2020. AGO unchained: Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in bioscience (Landmark edition). 25: 1.","pubmedId":"","doi":"10.2741/4793"},{"reference":"Sawin ER, Ranganathan R, Horvitz HR. 2000. C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway. Neuron. 26: 619.","pubmedId":"","doi":"10.1016/S0896-6273(00)81199-X"},{"reference":"Shang R, Lee S, Senavirathne G, Lai EC. 2023. microRNAs in action: biogenesis, function and regulation. Nature Reviews Genetics. 24: 816.","pubmedId":"","doi":"10.1038/s41576-023-00611-y"},{"reference":"Sharma N, Marques F, Kratsios P. 2024. Protocol for auxin-inducible protein degradation in C. elegans using different auxins and TIR1-expressing strains. STAR Protocols. 5: 103133.","pubmedId":"","doi":"10.1016/j.xpro.2024.103133"},{"reference":"Stiernagle T. 2006. Maintenance of C. elegans. WormBook: The Online Review of C. elegans Biology [Internet]","pubmedId":"","doi":""},{"reference":"Tops BBJ, Plasterk RHA, Ketting RF. 2006. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: Almost Identical yet Different. Cold Spring Harbor Symposia on Quantitative Biology. 71: 189.","pubmedId":"","doi":"10.1101/sqb.2006.71.035"}],"title":"The miRNA Argonaute protein, ALG-2, maintains neurobehaviors in adult C. elegans
","reviews":[{"reviewer":{"displayName":"Guy Caldwell"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"922f81e8-031b-4755-814a-476b225bdc92","decision":"edit","abstract":"microRNAs (miRNAs) are short, non-coding RNAs essential for gene regulation in many different processes, including neuronal development. However, the role of the miRNA pathway in maintaining neuronal health throughout aging is less understood. Here, we ask how the miRNA pathway in adulthood impacts neurobehaviors in C. elegans. Argonaute-like Gene 2 (ALG-2) is a protein required for the accumulation and function of certain miRNAs in C. elegans. Using the auxin-inducible degron (AID) system for temporal knockdown, we demonstrate that the miRNA Argonaute protein, ALG-2, is required throughout adulthood to maintain two well-characterized neurobehaviors, basal slowing response and mechanosensation.
","acknowledgements":"This work was supported by grants from the National Institutes of Health [R35 GM127012 to A.E.P.]; and the Hevolution Foundation [HF-GRO-23-1199180]. E.C.S. was supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine [T32 GM007240]. S.D.P was supported by a Faculty Research Support Grant provided by Swarthmore College.
We thank the C. elegans Genetic Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for providing strains used in this study.
","authors":[{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","writing_originalDraft","writing_reviewEditing"],"email":"achon1@swarthmore.edu","firstName":"Ava","lastName":"Chon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0000-8049-6146"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","methodology","investigation","dataCuration","validation"],"email":"ruj016@ucsd.edu","firstName":"Runtian","lastName":"Jiang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0009-0069-777X"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing","formalAnalysis"],"email":"yguo2@swarthmore.edu","firstName":"Yuxuan","lastName":"Guo","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5751-1255"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation"],"email":"yphyu1@swarthmore.edu","firstName":"Yamin K.","lastName":"Phyu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-7961-3082"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing"],"email":"lgoldbe1@swarthmore.edu","firstName":"Lilly M.","lastName":"Goldberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-8631-3723"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["supervision","conceptualization","investigation"],"email":"eschiksn@ucsd.edu","firstName":"Erin C.","lastName":"Schiksnis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2756-8168"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","fundingAcquisition","writing_reviewEditing","supervision","methodology"],"email":"apasquinelli@ucsd.edu","firstName":"Amy E.","lastName":"Pasquinelli","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9511-0039"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","supervision","writing_originalDraft","writing_reviewEditing","visualization"],"email":"spalumb1@swarthmore.edu","firstName":"Sierra D.","lastName":"Palumbos","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3595-984X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"WBPerson39614
","image":{"url":"https://portal.micropublication.org/uploads/e680aae95387f8728a08eed2a427f1f2.png"},"imageCaption":"A) Graphic depicting insertion of AID at the alg-2 gene locus. B) Immunoblot of ALG-2 protein levels from PQ668 worms following 30 minutes of auxin treatment at ranging auxin concentrations. Actin was probed as positive control. C) BSR assay of day 1 adult PQ668 strain and D) day 4 adult PQ668 and N2 strains. For each condition, n=27. BSR was calculated by subtracting the number of body bends in the presence of food from the number of body bends when food was absent. One-way ANOVA with multiple comparisons used to determine significance; *** indicates p<0.001. E) Soft touch response assay of day 2 adult PQ668 and N2 strains with or without auxin treatment. N=30 worms for each condition across 3 trials. One-way ANOVA test with multiple comparisons was used to determine significance, *** indicates p<0.001. F) Chemotaxis Index (CI) of day 2 adult PQ668 and N2 C.elegans with and without auxin treatment in response to isoamyl alcohol (IA). CI was calculated as the number of worms at IA minus the number of worms at H₂O, divided by the total number of worms. Error bars represent SD. n ≥10 per trial, 3 trials. One-way ANOVA with multiple comparisons used to determine significance. G) alg-2 expression in transcripts per million (TPM) at L1, L4 and Adult stages in touch receptor neurons. Data taken from single cell data reported by the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021).
","imageTitle":"ALG-2 function in adulthood required for neurobehaviors
","methods":"Strain Preparation: The N2 strain was obtained from the Caenorhabditis Genetics Center. The PQ668 (AID::mNeonGreen::3Xflag::alg-2) strain was developed using CRISPR-Cas9 to insert AID::mNeonGreen::3Xflag before the 5' end of alg-2 before exon 1 of isoform A and exon 0 of isoform B (See Jiang, 2022). Briefly, four plasmids were injected into young adult N2 worms: 1) 50ng/ul of homologous repair template (AID::mNeonGreen::3xflag with ALG-2 homology arms), 2) 50ng/ul of pJB53 (Cas9 plasmid with ALG-2 specific sgRNA, modified from pJW1219, Addgene #61250), 3) 10ng/ul pGH8 (Prab-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19359) and 4) 5ng/μL pCFJ104 (Pmyo-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19328). Recombinant worms were isolated as previously described (Dickinson et al. 2015) and then backcrossed 3X to N2 to generate PQ668.
Auxin Plates: Nematode growth media (NGM) plates were prepared as described previously (Stiernagle, 2006). Auxin-containing NGM plates were prepared by adding 0.89 mg/mL auxin after autoclaving (Sharma et al., 2024). Plates were seeded with OP50 for 48 hours.
Western Blot: Western blot was carried out as described previously and ALG-2 was detected based on 3X flag insertion using anti-FLAG antibody (Van Wynsberghe et al., 2011).
Soft Touch Response: L4 PQ668 C. elegans were transferred to NGM plates with or without auxin and maintained at 23°C for 2 days. Adult Day 2 worms were transferred to an unseeded NGM plate and allowed to acclimate before testing. Gentle mechanical stimuli were applied using an eyelash pick by alternately stroking the anterior of the worm (posterior to the pharynx) and the posterior of the worm (anterior of the anus) for a total of 10 touches per worm (Chalfie et al., 2018). Responses to anterior touch were scored as reversals, whereas responses to posterior touch were scored as forward movement. Failure to produce a movement response following soft touch stimulation was scored as defective.
Basal Slowing Response: L4 C. elegans were plated on NGM plates with or without auxin. Plates were kept at 23℃ and worms were allowed to grow for either 1 or 4 days before being transferred to an unseeded NGM plate for analysis. After a 3-minute acclimation period, body bends were counted over a 20-second period. Worms were transferred to a seeded plate and the same procedure was repeated (Petratou et al., 2024). BSR was calculated for individual worms by subtracting the number of body bends in the presence of food from the number of body bends when no food was present.
Chemotaxis Assay: L4 C. elegans were transferred to OP50-seeded NGM plates with or without auxin and maintained at 23℃ for 2 days. Chemotaxis assay plates were prepared by dividing each plate into two halves and 5μL of isoamyl alcohol (IA) on one side and 5μL of deionized water (H₂O) on the other. 5μL of 0.5 M sodium azide was spotted on IA and H₂O to paralyze the worms upon contact. Day-2 adult worms were washed with 1 mL of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1M MgSO₄) before being transferred to chemotaxis plates for 1-hour. A minimum of 10 worms per plate was assayed across 3 trials. Worms were manually counted under a dissecting microscope. The chemotaxis index was calculated as CI = (# of worms at IA − # of worms at H₂O) / total # of worms, and ranged from +1 (maximum attraction) to -1 (maximum repulsion) (Bargmann et al., 1993).
Statistics:
All statistical analyses were performed using GraphPad Prism (ver. 10) and specific tests are specified in figure legend.
","reagents":"","patternDescription":"miRNAs are non-coding RNAs that are approximately 22 nucleotides long which repress gene expression of target mRNAs (Shang et al., 2023). miRNAs associate with an Argonaute protein, forming the miRNA-induced silencing complex (miRISC), which recognizes specific mRNA targets and induces their translational repression and degradation (Shang et al., 2023, Sala et al., 2020). C. elegans express two argonaute proteins that mediate miRNA repression, ALG-1 and ALG-2, which exhibit distinct roles (Tops et al., 2006, Aalto et al., 2018). While miRNAs are well characterized in development (Ivey and Srivastava, 2015), their role in aging and neurodegeneration is still an emerging field (Elder and Pasquinelli, 2022). Here, we ask whether the miRNA pathway has a role in maintaining neuronal homeostasis in adult C. elegans by tracking common neurobehaviors (Caldwell et al., 2020).
To do this, we used CRISPR/Cas9 to insert an Auxin-inducible-degron sequence at the N-terminus of alg-2 coding sequence, to generate a worm strain, PQ668 (AID::mNeonGreen::3Xflag::alg-2) where we could temporally regulate alg-2 expression in adult C. elegans via the exogenous addition of auxin (Fig. 1A) (Nishimura et al., 2009, Dickinson et al., 2015). To confirm successful knockdown of ALG-2, we tracked ALG-2 protein levels using immunoblotting from worms treated with and without auxin (Fig. 1B). Following as short as 30 minutes on auxin-plates, ALG-2 protein levels were reduced below detection. This was also confirmed by tracking the inserted GFP fluorescent reporter. Thus, we developed a tool where ALG-2 levels could be selectively reduced during adulthood, allowing us to ask if loss of ALG-2 impacted neuronal homeostasis.
C. elegans reduce their speed in the presence of food, a phenomenon termed Basal Slowing Response (BSR) (Rivard et al., 2010), which is dependent on dopaminergic transmission (Sawin et al., 2000). We measured BSR in control (PQ668 – auxin) and ALG-2 depleted worms (PQ668 + auxin) to ask how impairment of the ALG-2-miRNA pathway impacts dopaminergic transmission in C. elegans. In day 1 adult worms, there was no statistically significant difference in BSR between PQ668 - auxin and PQ668 + auxin worms (p=0.2244) (Fig. 1C). However, following 4 days without ALG-2, PQ668 + auxin worms showed significantly reduced BSR compared to their matched controls (1.5185 vs. 5.4815, p=0.0008) (Fig. 1D). To confirm this observed effect was due to ALG-2 depletion and not the presence of auxin, we measured BSR in N2 worms in the presence or absence of auxin and observed no difference (p=0.9559) (Fig. 1E). Thus, we observed an age-dependent defect in BSR, suggesting a role for ALG-2 in maintaining dopaminergic transmission in adulthood.
We next asked whether other behaviors were impacted. C. elegans depend on soft touch sensation to navigate their world. Soft touch is communicated via mechanoreceptors expressed by six glutamatergic touch receptor neurons located in either the anterior or posterior of the worm (Chen and Chalfie, 2014). To determine whether depletion of ALG-2 in adulthood affects mechanosensation, we monitored soft touch response in Day 2 adult worms with control levels of ALG-1 (PQ668 - auxin) and those with ALG-2 depletion (PQ668 + auxin, L4-Day 2) following alternating anterior and posterior stimulation with an eyelash pick. While we observed a significant reduction in overall responses (64.33% vs. 83.00%, p=0.002), we noted that failure to respond was only observed following anterior stimuli. We therefore quantified anterior soft touch response and noted ALG-2 depleted worms (PQ668 + auxin) exhibited a defect in anterior mechanosensation compared to untreated controls (PQ668 - auxin) (28.67% vs. 66.00%, p=0.0002) (Fig. 1E). To determine whether auxin itself affected mechanosensation, we repeated the assay using N2 worms and found no significant difference between N2 worms grown on auxin vs. control plates (p=0.8283) (Fig. 1E). Our findings suggest that the miRNA Argonaute protein, ALG-2, is selectively required for anterior mechanosensation in adult animals, potentially through maintaining glutamatergic transmission in a subset of touch receptor neurons.
C. elegans possess a well-organized chemosensory system that allows them to navigate a variety of olfactory and gustatory cues associated with food, danger, and mates (Bargmann, 2006). We used the well characterized attractant, isoamyl alcohol, (Bargmann et al., 1993) to ask if ALG-2 function in adulthood is similarly required for chemotaxis. We tracked the chemotaxis index in PQ668 and N2 wild type worms (day 2 adults) with and without auxin. In contrast to mechanosensation and BSR, we found no significant change in chemosensory behavior across any conditions (Fig. 1F). It is of note that both our auxin groups had higher variability in their response, suggesting that auxin itself could be impairing chemotaxis. Thus, loss of ALG-2 function did not impair chemotaxis, suggesting that distinct circuits are impacted by loss of ALG-2 activity.
Given our observations that adult expression of ALG-2 is selectively required for specific neurobehaviors (BSR and anterior mechanosensation), but not others (chemosensation) we analyzed alg-2 expression dynamics in touch receptor and dopaminergic neurons across developmental stages using the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021). Touch receptor neurons (ALM, AVM, PLM, PVM) displayed a biphasic developmental regulatory pattern of alg-2 expression (high in L1, low in L4, high in adults) (Figure 1G). As this developmental expression pattern was true in both anterior and posterior touch neurons, local alg-2 transcription alone does not explain selective defect in anterior soft touch response. Similarly, alg-2 expression in dopaminergic neurons was inconsistent with a simple cell-autonomous mechanism. While adult CEP neurons showed a modest increase in alg-2, ADE and PDE expressed alg-2 at levels far below their developmental peaks. Given that miRNAs can be secreted and act extracellularly to impact protein expression in neighboring cells (Shang et al., 2023, Alkhazaali-Ali et al., 2024, Palumbos et al., 2025), we hypothesize that ALG-2 regulates neurotransmission, at least in part, in a non-cell-autonomous manner during aging. In summary, these results demonstrate that ALG-2 is required during adulthood to maintain glutamatergic and dopaminergic neurotransmission, suggesting miRNAs play an ongoing role in preserving neural function during aging.
","references":[{"reference":"Aalto AP, Nicastro IA, Broughton JP, Chipman LB, Schreiner WP, Chen JS, Pasquinelli AE. 2018. Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics. 14: e1007379.","pubmedId":"","doi":"10.1371/journal.pgen.1007379"},{"reference":"Alkhazaali Ali Z, Sahab Negah S, Boroumand AR, Tavakol Afshari J. 2024. MicroRNA (miRNA) as a biomarker for diagnosis, prognosis, and therapeutics molecules in neurodegenerative disease. Biomedicine & Pharmacotherapy. 177: 116899.","pubmedId":"","doi":"10.1016/j.biopha.2024.116899"},{"reference":"Bargmann CI, Hartwieg E, Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74: 515.","pubmedId":"","doi":"10.1016/0092-8674(93)80053-h"},{"reference":"Bargmann CI. 2006. Chemosensation in C. elegans.. Wormbook: 1.","pubmedId":"","doi":"10.1895/wormbook.1.123.1"},{"reference":"Caldwell KA, Willicott CW, Caldwell GA. 2020. Modeling neurodegeneration in Caenorhabditis elegans. Disease Models & Mechanisms. 13: dmm046110.","pubmedId":"","doi":"10.1242/dmm.046110"},{"reference":"Chalfie M, Hart AC, Rankin CH, Goodman MB. 2018. Assaying mechanosensation. WormBook: The Online Review of C. elegans Biology [Internet]","pubmedId":"","doi":""},{"reference":"Chen X, Chalfie M. 2014. Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels. Journal of Neuroscience. 34: 6522.","pubmedId":"","doi":"10.1523/JNEUROSCI.0022-14.2014"},{"reference":"Cinar H, Keles S, Jin Y. 2005. Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans. Current Biology. 15: 340.","pubmedId":"","doi":"10.1016/j.cub.2005.02.025"},{"reference":"Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. 2015. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics. 200: 1035.","pubmedId":"","doi":"10.1534/genetics.115.178335"},{"reference":"Elder CR, Pasquinelli AE. 2022. New Roles for MicroRNAs in Old Worms. Frontiers in Aging. 3: 871226.","pubmedId":"","doi":"10.3389/fragi.2022.871226"},{"reference":"Ivey KN, Srivastava D. 2015. microRNAs as Developmental Regulators. Cold Spring Harbor Perspectives in Biology. 7: a008144.","pubmedId":"","doi":"10.1101/cshperspect.a008144"},{"reference":"Jiang, R. 2022. Conditional depletion of ALG-1 and ALG-2 using auxin inducible degron 2 (AID2). UC San Diego. ProQuest ID: Jiang_ucsd_0033M_21343. Merritt ID: ark:/13030/m5hx8j4s. Retrieved from https://escholarship.org/uc/item/8646322z
","pubmedId":"","doi":""},{"reference":"Li W, Kang L, Piggott BJ, Feng Z, Xu XZS. 2011. The neural circuits and sensory channels mediating harsh touch sensation in Caenorhabditis elegans. Nature Communications 2: 10.1038/ncomms1308.
","pubmedId":"","doi":"10.1038/ncomms1308 "},{"reference":"Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. 2009. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods. 6: 917.","pubmedId":"","doi":"10.1038/nmeth.1401"},{"reference":"O Brien J, Hayder H, Zayed Y, Peng C. 2018. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology. 9: 402.","pubmedId":"","doi":"10.3389/fendo.2018.00402"},{"reference":"Palumbos SD, Popolow J, Goldsmith J, Holzbaur ELF. 2025. Autophagic stress activates distinct compensatory secretory pathways in neurons. Proceedings of the National Academy of Sciences 122: 10.1073/pnas.2421886122.
","pubmedId":"","doi":"10.1073/pnas.2421886122"},{"reference":"Petratou D, Fragkiadaki P, Lionaki E, Tavernarakis N. 2024. Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans. STAR Protocols. 5: 102801.","pubmedId":"","doi":"10.1016/j.xpro.2023.102801"},{"reference":"Rivard L, Srinivasan J, Stone A, Ochoa S, Sternberg PW, Loer CM. 2010. A comparison of experience-dependent locomotory behaviors and biogenic amine neurons in nematode relatives of Caenorhabditis elegans. BMC Neuroscience. 11: 22.","pubmedId":"","doi":"10.1186/1471-2202-11-22"},{"reference":"Sala L, Chandrasekhar S, Vidigal JA. 2020. AGO unchained: Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in bioscience (Landmark edition). 25: 1.","pubmedId":"","doi":"10.2741/4793"},{"reference":"Sawin ER, Ranganathan R, Horvitz HR. 2000. C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway. Neuron. 26: 619.","pubmedId":"","doi":"10.1016/S0896-6273(00)81199-X"},{"reference":"Shang R, Lee S, Senavirathne G, Lai EC. 2023. microRNAs in action: biogenesis, function and regulation. Nature Reviews Genetics. 24: 816.","pubmedId":"","doi":"10.1038/s41576-023-00611-y"},{"reference":"Sharma N, Marques F, Kratsios P. 2024. Protocol for auxin-inducible protein degradation in C. elegans using different auxins and TIR1-expressing strains. STAR Protocols. 5: 103133.","pubmedId":"","doi":"10.1016/j.xpro.2024.103133"},{"reference":"Stiernagle T. 2006. Maintenance of C. elegans. WormBook: The Online Review of C. elegans Biology [Internet]","pubmedId":"","doi":""},{"reference":"Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al., Miller. 2021. Molecular topography of an entire nervous system. Cell 184: 4329-4347.e23.
","pubmedId":"","doi":"10.1016/j.cell.2021.06.023"},{"reference":"Tops BBJ, Plasterk RHA, Ketting RF. 2006. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: Almost Identical yet Different. Cold Spring Harbor Symposia on Quantitative Biology. 71: 189.","pubmedId":"","doi":"10.1101/sqb.2006.71.035"},{"reference":"Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. 2011. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nature Structural & Molecular Biology 18: 302-308.
","pubmedId":"","doi":"doi.org/10.1038/nsmb.1986"},{"reference":"Vidigal JA. 2020. AGO unchained Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in Bioscience 25: 1-42.
","pubmedId":"","doi":"10.2741/4793"}],"title":"The miRNA Argonaute protein, ALG-2, maintains neurobehaviors in adult C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1786059499895"}]},{"id":"6657c03a-91da-4c33-b602-76aa13c80484","decision":"accept","abstract":"microRNAs (miRNAs) are short, non-coding RNAs essential for gene regulation in many different processes, including neuronal development. However, the role of the miRNA pathway in maintaining neuronal health throughout aging is less understood. Here, we ask how the miRNA pathway in adulthood impacts neurobehaviors in C. elegans. Argonaute-like Gene 2 (ALG-2) is a protein required for the accumulation and function of certain miRNAs in C. elegans. Using the auxin-inducible degron (AID) system for temporal knockdown, we demonstrate that the miRNA Argonaute protein, ALG-2, is required throughout adulthood to maintain two well-characterized neurobehaviors, basal slowing response and mechanosensation.
","acknowledgements":"This work was supported by grants from the National Institutes of Health [R35 GM127012 to A.E.P.]; and the Hevolution Foundation [HF-GRO-23-1199180]. E.C.S. was supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine [T32 GM007240]. S.D.P was supported by a Faculty Research Support Grant provided by Swarthmore College.
We thank the C. elegans Genetic Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for providing strains used in this study.
","authors":[{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","writing_originalDraft","writing_reviewEditing"],"email":"achon1@swarthmore.edu","firstName":"Ava","lastName":"Chon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0000-8049-6146"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","methodology","investigation","dataCuration","validation"],"email":"ruj016@ucsd.edu","firstName":"Runtian","lastName":"Jiang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0009-0069-777X"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing","formalAnalysis"],"email":"yguo2@swarthmore.edu","firstName":"Yuxuan","lastName":"Guo","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5751-1255"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation"],"email":"yphyu1@swarthmore.edu","firstName":"Yamin K.","lastName":"Phyu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-7961-3082"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing"],"email":"lgoldbe1@swarthmore.edu","firstName":"Lilly M.","lastName":"Goldberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-8631-3723"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["supervision","conceptualization","investigation"],"email":"eschiksn@ucsd.edu","firstName":"Erin C.","lastName":"Schiksnis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2756-8168"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","fundingAcquisition","writing_reviewEditing","supervision","methodology"],"email":"apasquinelli@ucsd.edu","firstName":"Amy E.","lastName":"Pasquinelli","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9511-0039"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","supervision","writing_originalDraft","writing_reviewEditing","visualization"],"email":"spalumb1@swarthmore.edu","firstName":"Sierra D.","lastName":"Palumbos","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3595-984X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"WBPerson39614
","image":{"url":"https://portal.micropublication.org/uploads/e680aae95387f8728a08eed2a427f1f2.png"},"imageCaption":"A) Graphic depicting insertion of AID at the alg-2 gene locus. B) Immunoblot of ALG-2 protein levels from PQ668 worms following 30 minutes of auxin treatment at ranging auxin concentrations. Actin was probed as positive control. C) BSR assay of day 1 adult PQ668 strain and D) day 4 adult PQ668 and N2 strains. For each condition, n=27. BSR was calculated by subtracting the number of body bends in the presence of food from the number of body bends when food was absent. One-way ANOVA with multiple comparisons used to determine significance; *** indicates p<0.001. E) Soft touch response assay of day 2 adult PQ668 and N2 strains with or without auxin treatment. N=30 worms for each condition across 3 trials. One-way ANOVA test with multiple comparisons was used to determine significance, *** indicates p<0.001. F) Chemotaxis Index (CI) of day 2 adult PQ668 and N2 C.elegans with and without auxin treatment in response to isoamyl alcohol (IA). CI was calculated as the number of worms at IA minus the number of worms at H₂O, divided by the total number of worms. Error bars represent SD. n ≥10 per trial, 3 trials. One-way ANOVA with multiple comparisons used to determine significance. G) alg-2 expression in transcripts per million (TPM) at L1, L4 and Adult stages in touch receptor neurons. Data taken from single cell data reported by the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021).
","imageTitle":"ALG-2 function in adulthood required for neurobehaviors
","methods":"Strain Preparation: The N2 strain was obtained from the Caenorhabditis Genetics Center. The PQ668 (AID::mNeonGreen::3Xflag::alg-2) strain was developed using CRISPR-Cas9 to insert AID::mNeonGreen::3Xflag before the 5' end of alg-2 before exon 1 of isoform A and exon 0 of isoform B (See Jiang, 2022). Briefly, four plasmids were injected into young adult N2 worms: 1) 50ng/ul of homologous repair template (AID::mNeonGreen::3xflag with ALG-2 homology arms), 2) 50ng/ul of pJB53 (Cas9 plasmid with ALG-2 specific sgRNA, modified from pJW1219, Addgene #61250), 3) 10ng/ul pGH8 (Prab-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19359) and 4) 5ng/μL pCFJ104 (Pmyo-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19328). Recombinant worms were isolated as previously described (Dickinson et al. 2015) and then backcrossed 3X to N2 to generate PQ668.
Auxin Plates: Nematode growth media (NGM) plates were prepared as described previously (Stiernagle, 2006). Auxin-containing NGM plates were prepared by adding 0.89 mg/mL auxin after autoclaving (Sharma et al., 2024). Plates were seeded with OP50 for 48 hours.
Western Blot: Western blot was carried out as described previously and ALG-2 was detected based on 3X flag insertion using anti-FLAG antibody (Van Wynsberghe et al., 2011).
Soft Touch Response: L4 PQ668 C. elegans were transferred to NGM plates with or without auxin and maintained at 23°C for 2 days. Adult Day 2 worms were transferred to an unseeded NGM plate and allowed to acclimate before testing. Gentle mechanical stimuli were applied using an eyelash pick by alternately stroking the anterior of the worm (posterior to the pharynx) and the posterior of the worm (anterior of the anus) for a total of 10 touches per worm (Chalfie et al., 2018). Responses to anterior touch were scored as reversals, whereas responses to posterior touch were scored as forward movement. Failure to produce a movement response following soft touch stimulation was scored as defective.
Basal Slowing Response: L4 C. elegans were plated on NGM plates with or without auxin. Plates were kept at 23℃ and worms were allowed to grow for either 1 or 4 days before being transferred to an unseeded NGM plate for analysis. After a 3-minute acclimation period, body bends were counted over a 20-second period. Worms were transferred to a seeded plate and the same procedure was repeated (Petratou et al., 2024). BSR was calculated for individual worms by subtracting the number of body bends in the presence of food from the number of body bends when no food was present.
Chemotaxis Assay: L4 C. elegans were transferred to OP50-seeded NGM plates with or without auxin and maintained at 23℃ for 2 days. Chemotaxis assay plates were prepared by dividing each plate into two halves and 5μL of isoamyl alcohol (IA) on one side and 5μL of deionized water (H₂O) on the other. 5μL of 0.5 M sodium azide was spotted on IA and H₂O to paralyze the worms upon contact. Day-2 adult worms were washed with 1 mL of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1M MgSO₄) before being transferred to chemotaxis plates for 1-hour. A minimum of 10 worms per plate was assayed across 3 trials. Worms were manually counted under a dissecting microscope. The chemotaxis index was calculated as CI = (# of worms at IA − # of worms at H₂O) / total # of worms, and ranged from +1 (maximum attraction) to -1 (maximum repulsion) (Bargmann et al., 1993).
Statistics:
All statistical analyses were performed using GraphPad Prism (ver. 10) and specific tests are specified in figure legend.
","reagents":"","patternDescription":"miRNAs are non-coding RNAs that are approximately 22 nucleotides long which repress gene expression of target mRNAs (Shang et al., 2023). miRNAs associate with an Argonaute protein, forming the miRNA-induced silencing complex (miRISC), which recognizes specific mRNA targets and induces their translational repression and degradation (Shang et al., 2023, Sala et al., 2020). C. elegans express two argonaute proteins that mediate miRNA repression, ALG-1 and ALG-2, which exhibit distinct roles (Tops et al., 2006, Aalto et al., 2018). While miRNAs are well characterized in development (Ivey and Srivastava, 2015), their role in aging and neurodegeneration is still an emerging field (Elder and Pasquinelli, 2022). Here, we ask whether the miRNA pathway has a role in maintaining neuronal homeostasis in adult C. elegans by tracking common neurobehaviors (Caldwell et al., 2020).
To do this, we used CRISPR/Cas9 to insert an Auxin-inducible-degron sequence at the N-terminus of alg-2 coding sequence, to generate a worm strain, PQ668 (AID::mNeonGreen::3Xflag::alg-2) where we could temporally regulate alg-2 expression in adult C. elegans via the exogenous addition of auxin (Fig. 1A) (Nishimura et al., 2009, Dickinson et al., 2015). To confirm successful knockdown of ALG-2, we tracked ALG-2 protein levels using immunoblotting from worms treated with and without auxin (Fig. 1B). Following as short as 30 minutes on auxin-plates, ALG-2 protein levels were reduced below detection. This was also confirmed by tracking the inserted GFP fluorescent reporter. Thus, we developed a tool where ALG-2 levels could be selectively reduced during adulthood, allowing us to ask if loss of ALG-2 impacted neuronal homeostasis.
C. elegans reduce their speed in the presence of food, a phenomenon termed Basal Slowing Response (BSR) (Rivard et al., 2010), which is dependent on dopaminergic transmission (Sawin et al., 2000). We measured BSR in control (PQ668 – auxin) and ALG-2 depleted worms (PQ668 + auxin) to ask how impairment of the ALG-2-miRNA pathway impacts dopaminergic transmission in C. elegans. In day 1 adult worms, there was no statistically significant difference in BSR between PQ668 - auxin and PQ668 + auxin worms (p=0.2244) (Fig. 1C). However, following 4 days without ALG-2, PQ668 + auxin worms showed significantly reduced BSR compared to their matched controls (1.5185 vs. 5.4815, p=0.0008) (Fig. 1D). To confirm this observed effect was due to ALG-2 depletion and not the presence of auxin, we measured BSR in N2 worms in the presence or absence of auxin and observed no difference (p=0.9559) (Fig. 1E). Thus, we observed an age-dependent defect in BSR, suggesting a role for ALG-2 in maintaining dopaminergic transmission in adulthood.
We next asked whether other behaviors were impacted. C. elegans depend on soft touch sensation to navigate their world. Soft touch is communicated via mechanoreceptors expressed by six glutamatergic touch receptor neurons located in either the anterior or posterior of the worm (Chen and Chalfie, 2014). To determine whether depletion of ALG-2 in adulthood affects mechanosensation, we monitored soft touch response in Day 2 adult worms with control levels of ALG-1 (PQ668 - auxin) and those with ALG-2 depletion (PQ668 + auxin, L4-Day 2) following alternating anterior and posterior stimulation with an eyelash pick. While we observed a significant reduction in overall responses (64.33% vs. 83.00%, p=0.002), we noted that failure to respond was only observed following anterior stimuli. We therefore quantified anterior soft touch response and noted ALG-2 depleted worms (PQ668 + auxin) exhibited a defect in anterior mechanosensation compared to untreated controls (PQ668 - auxin) (28.67% vs. 66.00%, p=0.0002) (Fig. 1E). To determine whether auxin itself affected mechanosensation, we repeated the assay using N2 worms and found no significant difference between N2 worms grown on auxin vs. control plates (p=0.8283) (Fig. 1E). Our findings suggest that the miRNA Argonaute protein, ALG-2, is selectively required for anterior mechanosensation in adult animals, potentially through maintaining glutamatergic transmission in a subset of touch receptor neurons.
C. elegans possess a well-organized chemosensory system that allows them to navigate a variety of olfactory and gustatory cues associated with food, danger, and mates (Bargmann, 2006). We used the well characterized attractant, isoamyl alcohol, (Bargmann et al., 1993) to ask if ALG-2 function in adulthood is similarly required for chemotaxis. We tracked the chemotaxis index in PQ668 and N2 wild type worms (day 2 adults) with and without auxin. In contrast to mechanosensation and BSR, we found no significant change in chemosensory behavior across any conditions (Fig. 1F). It is of note that both our auxin groups had higher variability in their response, suggesting that auxin itself could be impairing chemotaxis. Thus, loss of ALG-2 function did not impair chemotaxis, suggesting that distinct circuits are impacted by loss of ALG-2 activity.
Given our observations that adult expression of ALG-2 is selectively required for specific neurobehaviors (BSR and anterior mechanosensation), but not others (chemosensation) we analyzed alg-2 expression dynamics in touch receptor and dopaminergic neurons across developmental stages using the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021). Touch receptor neurons (ALM, AVM, PLM, PVM) displayed a biphasic developmental regulatory pattern of alg-2 expression (high in L1, low in L4, high in adults) (Figure 1G). As this developmental expression pattern was true in both anterior and posterior touch neurons, local alg-2 transcription alone does not explain selective defect in anterior soft touch response. Similarly, alg-2 expression in dopaminergic neurons was inconsistent with a simple cell-autonomous mechanism. While adult CEP neurons showed a modest increase in alg-2, ADE and PDE expressed alg-2 at levels far below their developmental peaks. Given that miRNAs can be secreted and act extracellularly to impact protein expression in neighboring cells (Shang et al., 2023, Alkhazaali-Ali et al., 2024, Palumbos et al., 2025), we hypothesize that ALG-2 regulates neurotransmission, at least in part, in a non-cell-autonomous manner during aging. In summary, these results demonstrate that ALG-2 is required during adulthood to maintain glutamatergic and dopaminergic neurotransmission, suggesting miRNAs play an ongoing role in preserving neural function during aging.
","references":[{"reference":"Aalto AP, Nicastro IA, Broughton JP, Chipman LB, Schreiner WP, Chen JS, Pasquinelli AE. 2018. Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics. 14: e1007379.","pubmedId":"","doi":"10.1371/journal.pgen.1007379"},{"reference":"Alkhazaali Ali Z, Sahab Negah S, Boroumand AR, Tavakol Afshari J. 2024. MicroRNA (miRNA) as a biomarker for diagnosis, prognosis, and therapeutics molecules in neurodegenerative disease. Biomedicine & Pharmacotherapy. 177: 116899.","pubmedId":"","doi":"10.1016/j.biopha.2024.116899"},{"reference":"Bargmann CI, Hartwieg E, Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74: 515.","pubmedId":"","doi":"10.1016/0092-8674(93)80053-h"},{"reference":"Bargmann CI. 2006. Chemosensation in C. elegans. WormBook: 1-29.
","pubmedId":"18050433","doi":""},{"reference":"Caldwell KA, Willicott CW, Caldwell GA. 2020. Modeling neurodegeneration in Caenorhabditis elegans. Disease Models & Mechanisms. 13: dmm046110.","pubmedId":"","doi":"10.1242/dmm.046110"},{"reference":"Chalfie M, Hart AC, Rankin CH, Goodman MB. 2014. Assaying mechanosensation. WormBook: 10.1895/wormbook.1.172.1.
","pubmedId":"25093996","doi":""},{"reference":"Chen X, Chalfie M. 2014. Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels. Journal of Neuroscience. 34: 6522.","pubmedId":"","doi":"10.1523/JNEUROSCI.0022-14.2014"},{"reference":"Cinar H, Keles S, Jin Y. 2005. Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans. Current Biology. 15: 340.","pubmedId":"","doi":"10.1016/j.cub.2005.02.025"},{"reference":"Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. 2015. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics. 200: 1035.","pubmedId":"","doi":"10.1534/genetics.115.178335"},{"reference":"Elder CR, Pasquinelli AE. 2022. New Roles for MicroRNAs in Old Worms. Frontiers in Aging. 3: 871226.","pubmedId":"","doi":"10.3389/fragi.2022.871226"},{"reference":"Ivey KN, Srivastava D. 2015. microRNAs as Developmental Regulators. Cold Spring Harbor Perspectives in Biology. 7: a008144.","pubmedId":"","doi":"10.1101/cshperspect.a008144"},{"reference":"Jiang, R. 2022. Conditional depletion of ALG-1 and ALG-2 using auxin inducible degron 2 (AID2). UC San Diego. ProQuest ID: Jiang_ucsd_0033M_21343. Merritt ID: ark:/13030/m5hx8j4s. Retrieved from https://escholarship.org/uc/item/8646322z
","pubmedId":"","doi":""},{"reference":"Li W, Kang L, Piggott BJ, Feng Z, Xu XZS. 2011. The neural circuits and sensory channels mediating harsh touch sensation in Caenorhabditis elegans. Nature Communications 2: 10.1038/ncomms1308.
","pubmedId":"","doi":"10.1038/ncomms1308 "},{"reference":"Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. 2009. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods. 6: 917.","pubmedId":"","doi":"10.1038/nmeth.1401"},{"reference":"O Brien J, Hayder H, Zayed Y, Peng C. 2018. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology. 9: 402.","pubmedId":"","doi":"10.3389/fendo.2018.00402"},{"reference":"Palumbos SD, Popolow J, Goldsmith J, Holzbaur ELF. 2025. Autophagic stress activates distinct compensatory secretory pathways in neurons. Proceedings of the National Academy of Sciences 122: 10.1073/pnas.2421886122.
","pubmedId":"","doi":"10.1073/pnas.2421886122"},{"reference":"Petratou D, Fragkiadaki P, Lionaki E, Tavernarakis N. 2024. Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans. STAR Protocols. 5: 102801.","pubmedId":"","doi":"10.1016/j.xpro.2023.102801"},{"reference":"Rivard L, Srinivasan J, Stone A, Ochoa S, Sternberg PW, Loer CM. 2010. A comparison of experience-dependent locomotory behaviors and biogenic amine neurons in nematode relatives of Caenorhabditis elegans. BMC Neuroscience. 11: 22.","pubmedId":"","doi":"10.1186/1471-2202-11-22"},{"reference":"Sala L, Chandrasekhar S, Vidigal JA. 2020. AGO unchained: Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in bioscience (Landmark edition). 25: 1.","pubmedId":"","doi":"10.2741/4793"},{"reference":"Sawin ER, Ranganathan R, Horvitz HR. 2000. C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway. Neuron. 26: 619.","pubmedId":"","doi":"10.1016/S0896-6273(00)81199-X"},{"reference":"Shang R, Lee S, Senavirathne G, Lai EC. 2023. microRNAs in action: biogenesis, function and regulation. Nature Reviews Genetics. 24: 816.","pubmedId":"","doi":"10.1038/s41576-023-00611-y"},{"reference":"Sharma N, Marques F, Kratsios P. 2024. Protocol for auxin-inducible protein degradation in C. elegans using different auxins and TIR1-expressing strains. STAR Protocols. 5: 103133.","pubmedId":"","doi":"10.1016/j.xpro.2024.103133"},{"reference":"Stiernagle T. 2006. Maintenance of C. elegans. WormBook: 1-11.
","pubmedId":"18050451","doi":""},{"reference":"Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al., Miller. 2021. Molecular topography of an entire nervous system. Cell 184: 4329-4347.e23.
","pubmedId":"","doi":"10.1016/j.cell.2021.06.023"},{"reference":"Tops BBJ, Plasterk RHA, Ketting RF. 2006. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: Almost Identical yet Different. Cold Spring Harbor Symposia on Quantitative Biology. 71: 189.","pubmedId":"","doi":"10.1101/sqb.2006.71.035"},{"reference":"Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. 2011. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nature Structural & Molecular Biology 18: 302-308.
","pubmedId":"","doi":"doi.org/10.1038/nsmb.1986"},{"reference":"Vidigal JA. 2020. AGO unchained Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in Bioscience 25: 1-42.
","pubmedId":"","doi":"10.2741/4793"}],"title":"The miRNA Argonaute protein, ALG-2, maintains neurobehaviors in adult C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"fe548808-7956-4de6-819c-eccdcf6cd2a0","decision":"edit","abstract":"microRNAs (miRNAs) are short, non-coding RNAs essential for gene regulation in many different processes, including neuronal development. However, the role of the miRNA pathway in maintaining neuronal health throughout aging is less understood. Here, we ask how the miRNA pathway in adulthood impacts neurobehaviors in C. elegans. Argonaute-like Gene 2 (ALG-2) is a protein required for the accumulation and function of certain miRNAs in C. elegans. Using the auxin-inducible degron 2 (AID2) system for temporal knockdown, we demonstrate that the miRNA Argonaute protein, ALG-2, is required throughout adulthood to maintain two well-characterized neurobehaviors, basal slowing response and mechanosensation.
","acknowledgements":"This work was supported by grants from the National Institutes of Health [R35 GM127012 to A.E.P.]; and the Hevolution Foundation [HF-GRO-23-1199180]. E.C.S. was supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine [T32 GM007240]. S.D.P was supported by a Faculty Research Support Grant provided by Swarthmore College.
We thank the C. elegans Genetic Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for providing strains used in this study.
","authors":[{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","writing_originalDraft","writing_reviewEditing"],"email":"achon1@swarthmore.edu","firstName":"Ava","lastName":"Chon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0000-8049-6146"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","methodology","investigation","dataCuration","validation"],"email":"ruj016@ucsd.edu","firstName":"Runtian","lastName":"Jiang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0009-0069-777X"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing","formalAnalysis"],"email":"yguo2@swarthmore.edu","firstName":"Yuxuan","lastName":"Guo","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5751-1255"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation"],"email":"yphyu1@swarthmore.edu","firstName":"Yamin K.","lastName":"Phyu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-7961-3082"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing"],"email":"lgoldbe1@swarthmore.edu","firstName":"Lilly M.","lastName":"Goldberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-8631-3723"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["supervision","conceptualization","investigation"],"email":"eschiksn@ucsd.edu","firstName":"Erin C.","lastName":"Schiksnis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2756-8168"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","fundingAcquisition","writing_reviewEditing","supervision","methodology"],"email":"apasquinelli@ucsd.edu","firstName":"Amy E.","lastName":"Pasquinelli","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9511-0039"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","supervision","writing_originalDraft","writing_reviewEditing","visualization"],"email":"spalumb1@swarthmore.edu","firstName":"Sierra D.","lastName":"Palumbos","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3595-984X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"WBPerson39614
","image":{"url":"https://portal.micropublication.org/uploads/e680aae95387f8728a08eed2a427f1f2.png"},"imageCaption":"A) Graphic depicting insertion of AID2 at the alg-2 gene locus. B) Immunoblot of ALG-2 protein levels from PQ668 worms following 30 minutes of auxin treatment at ranging auxin concentrations. Actin was probed as positive control. C) BSR assay of day 1 adult PQ668 strain and D) day 4 adult PQ668 and N2 strains. For each condition, n=27. BSR was calculated by subtracting the number of body bends in the presence of food from the number of body bends when food was absent. One-way ANOVA with multiple comparisons used to determine significance; *** indicates p<0.001. E) Soft touch response assay of day 2 adult PQ668 and N2 strains with or without auxin treatment. N=30 worms for each condition across 3 trials. One-way ANOVA test with multiple comparisons was used to determine significance, *** indicates p<0.001. F) Chemotaxis Index (CI) of day 2 adult PQ668 and N2 C.elegans with and without auxin treatment in response to isoamyl alcohol (IA). CI was calculated as the number of worms at IA minus the number of worms at H₂O, divided by the total number of worms. Error bars represent SD. n ≥10 per trial, 3 trials. One-way ANOVA with multiple comparisons used to determine significance. G) alg-2 expression in transcripts per million (TPM) at L1, L4 and Adult stages in touch receptor neurons. Data taken from single cell data reported by the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021).
","imageTitle":"ALG-2 function in adulthood required for neurobehaviors
","methods":"Strain Preparation: The N2 strain was obtained from the Caenorhabditis Genetics Center. The PQ668 (AID2::mNeonGreen::3Xflag::alg-2) strain was developed using CRISPR-Cas9 to insert AID2::mNeonGreen::3Xflag before the 5' end of alg-2 before exon 1 of isoform A and exon 0 of isoform B (See Jiang, 2022). Briefly, four plasmids were injected into young adult N2 worms: 1) 50ng/ul of homologous repair template (AID2::mNeonGreen::3xflag with ALG-2 homology arms), 2) 50ng/ul of pJB53 (Cas9 plasmid with ALG-2 specific sgRNA, modified from pJW1219, Addgene #61250), 3) 10ng/ul pGH8 (Prab-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19359) and 4) 5ng/μL pCFJ104 (Pmyo-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19328). Recombinant worms were isolated as previously described (Dickinson et al. 2015) and then backcrossed 3X to N2 to generate PQ668.
Auxin Plates: Nematode growth media (NGM) plates were prepared as described previously (Stiernagle, 2006). Auxin-containing NGM plates were prepared by adding 0.89 mg/mL auxin after autoclaving (Sharma et al., 2024). Plates were seeded with OP50 for 48 hours.
Western Blot: Western blot was carried out as described previously and ALG-2 was detected based on 3X flag insertion using anti-FLAG antibody (Van Wynsberghe et al., 2011).
Soft Touch Response: L4 PQ668 C. elegans were transferred to NGM plates with or without auxin and maintained at 23°C for 2 days. Adult Day 2 worms were transferred to an unseeded NGM plate and allowed to acclimate before testing. Gentle mechanical stimuli were applied using an eyelash pick by alternately stroking the anterior of the worm (posterior to the pharynx) and the posterior of the worm (anterior of the anus) for a total of 10 touches per worm (Chalfie et al., 2018). Responses to anterior touch were scored as reversals, whereas responses to posterior touch were scored as forward movement. Failure to produce a movement response following soft touch stimulation was scored as defective.
Basal Slowing Response: L4 C. elegans were plated on NGM plates with or without auxin. Plates were kept at 23℃ and worms were allowed to grow for either 1 or 4 days before being transferred to an unseeded NGM plate for analysis. After a 3-minute acclimation period, body bends were counted over a 20-second period. Worms were transferred to a seeded plate and the same procedure was repeated (Petratou et al., 2024). BSR was calculated for individual worms by subtracting the number of body bends in the presence of food from the number of body bends when no food was present.
Chemotaxis Assay: L4 C. elegans were transferred to OP50-seeded NGM plates with or without auxin and maintained at 23℃ for 2 days. Chemotaxis assay plates were prepared by dividing each plate into two halves and 5μL of isoamyl alcohol (IA) on one side and 5μL of deionized water (H₂O) on the other. 5μL of 0.5 M sodium azide was spotted on IA and H₂O to paralyze the worms upon contact. Day-2 adult worms were washed with 1 mL of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1M MgSO₄) before being transferred to chemotaxis plates for 1-hour. A minimum of 10 worms per plate was assayed across 3 trials. Worms were manually counted under a dissecting microscope. The chemotaxis index was calculated as CI = (# of worms at IA − # of worms at H₂O) / total # of worms, and ranged from +1 (maximum attraction) to -1 (maximum repulsion) (Bargmann et al., 1993).
Statistics:
All statistical analyses were performed using GraphPad Prism (ver. 10) and specific tests are specified in figure legend.
","reagents":"","patternDescription":"miRNAs are non-coding RNAs that are approximately 22 nucleotides long which repress gene expression of target mRNAs (Shang et al., 2023). miRNAs associate with an Argonaute protein, forming the miRNA-induced silencing complex (miRISC), which recognizes specific mRNA targets and induces their translational repression and degradation (Shang et al., 2023, Sala et al., 2020). C. elegans express two argonaute proteins that mediate miRNA repression, ALG-1 and ALG-2, which exhibit distinct roles (Tops et al., 2006, Aalto et al., 2018). While miRNAs are well characterized in development (Ivey and Srivastava, 2015), their role in aging and neurodegeneration is still an emerging field (Elder and Pasquinelli, 2022). Here, we ask whether the miRNA pathway has a role in maintaining neuronal homeostasis in adult C. elegans by tracking common neurobehaviors (Caldwell et al., 2020).
To do this, we used CRISPR/Cas9 to insert an Auxin-inducible degron 2 sequence at the N-terminus of alg-2 coding sequence, to generate a worm strain, PQ668 (AID2::mNeonGreen::3Xflag::alg-2) where we could temporally regulate alg-2 expression in adult C. elegans via the exogenous addition of auxin (Fig. 1A) (Nishimura et al., 2009, Dickinson et al., 2015, Yesbolatova et al., 2020). To confirm successful knockdown of ALG-2, we tracked ALG-2 protein levels using immunoblotting from worms treated with and without auxin (Fig. 1B). Following as short as 30 minutes on auxin-plates, ALG-2 protein levels were reduced below detection. This was also confirmed by tracking the inserted GFP fluorescent reporter. Thus, we developed a tool where ALG-2 levels could be selectively reduced during adulthood, allowing us to ask if loss of ALG-2 impacted neuronal homeostasis.
C. elegans reduce their speed in the presence of food, a phenomenon termed Basal Slowing Response (BSR) (Rivard et al., 2010), which is dependent on dopaminergic transmission (Sawin et al., 2000). We measured BSR in control (PQ668 – auxin) and ALG-2 depleted worms (PQ668 + auxin) to ask how impairment of the ALG-2-miRNA pathway impacts dopaminergic transmission in C. elegans. In day 1 adult worms, there was no statistically significant difference in BSR between PQ668 - auxin and PQ668 + auxin worms (p=0.2244) (Fig. 1C). However, following 4 days without ALG-2, PQ668 + auxin worms showed significantly reduced BSR compared to their matched controls (1.5185 vs. 5.4815, p=0.0008) (Fig. 1D). To confirm this observed effect was due to ALG-2 depletion and not the presence of auxin, we measured BSR in N2 worms in the presence or absence of auxin and observed no difference (p=0.9559) (Fig. 1E). Thus, we observed an age-dependent defect in BSR, suggesting a role for ALG-2 in maintaining dopaminergic transmission in adulthood.
We next asked whether other behaviors were impacted. C. elegans depend on soft touch sensation to navigate their world. Soft touch is communicated via mechanoreceptors expressed by six glutamatergic touch receptor neurons located in either the anterior or posterior of the worm (Chen and Chalfie, 2014). To determine whether depletion of ALG-2 in adulthood affects mechanosensation, we monitored soft touch response in Day 2 adult worms with control levels of ALG-1 (PQ668 - auxin) and those with ALG-2 depletion (PQ668 + auxin, L4-Day 2) following alternating anterior and posterior stimulation with an eyelash pick. While we observed a significant reduction in overall responses (64.33% vs. 83.00%, p=0.002), we noted that failure to respond was only observed following anterior stimuli. We therefore quantified anterior soft touch response and noted ALG-2 depleted worms (PQ668 + auxin) exhibited a defect in anterior mechanosensation compared to untreated controls (PQ668 - auxin) (28.67% vs. 66.00%, p=0.0002) (Fig. 1E). To determine whether auxin itself affected mechanosensation, we repeated the assay using N2 worms and found no significant difference between N2 worms grown on auxin vs. control plates (p=0.8283) (Fig. 1E). Our findings suggest that the miRNA Argonaute protein, ALG-2, is selectively required for anterior mechanosensation in adult animals, potentially through maintaining glutamatergic transmission in a subset of touch receptor neurons.
C. elegans possess a well-organized chemosensory system that allows them to navigate a variety of olfactory and gustatory cues associated with food, danger, and mates (Bargmann, 2006). We used the well characterized attractant, isoamyl alcohol, (Bargmann et al., 1993) to ask if ALG-2 function in adulthood is similarly required for chemotaxis. We tracked the chemotaxis index in PQ668 and N2 wild type worms (day 2 adults) with and without auxin. In contrast to mechanosensation and BSR, we found no significant change in chemosensory behavior across any conditions (Fig. 1F). It is of note that both our auxin groups had higher variability in their response, suggesting that auxin itself could be impairing chemotaxis. Thus, loss of ALG-2 function did not impair chemotaxis, suggesting that distinct circuits are impacted by loss of ALG-2 activity.
Given our observations that adult expression of ALG-2 is selectively required for specific neurobehaviors (BSR and anterior mechanosensation), but not others (chemosensation) we analyzed alg-2 expression dynamics in touch receptor and dopaminergic neurons across developmental stages using the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021). Touch receptor neurons (ALM, AVM, PLM, PVM) displayed a biphasic developmental regulatory pattern of alg-2 expression (high in L1, low in L4, high in adults) (Figure 1G). As this developmental expression pattern was true in both anterior and posterior touch neurons, local alg-2 transcription alone does not explain selective defect in anterior soft touch response. Similarly, alg-2 expression in dopaminergic neurons was inconsistent with a simple cell-autonomous mechanism. While adult CEP neurons showed a modest increase in alg-2, ADE and PDE expressed alg-2 at levels far below their developmental peaks. Given that miRNAs can be secreted and act extracellularly to impact protein expression in neighboring cells (Shang et al., 2023, Alkhazaali-Ali et al., 2024, Palumbos et al., 2025), we hypothesize that ALG-2 regulates neurotransmission, at least in part, in a non-cell-autonomous manner during aging. In summary, these results demonstrate that ALG-2 is required during adulthood to maintain glutamatergic and dopaminergic neurotransmission, suggesting miRNAs play an ongoing role in preserving neural function during aging.
","references":[{"reference":"Aalto AP, Nicastro IA, Broughton JP, Chipman LB, Schreiner WP, Chen JS, Pasquinelli AE. 2018. Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics. 14: e1007379.","pubmedId":"","doi":"10.1371/journal.pgen.1007379"},{"reference":"Alkhazaali Ali Z, Sahab Negah S, Boroumand AR, Tavakol Afshari J. 2024. MicroRNA (miRNA) as a biomarker for diagnosis, prognosis, and therapeutics molecules in neurodegenerative disease. Biomedicine & Pharmacotherapy. 177: 116899.","pubmedId":"","doi":"10.1016/j.biopha.2024.116899"},{"reference":"Bargmann CI, Hartwieg E, Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74: 515.","pubmedId":"","doi":"10.1016/0092-8674(93)80053-h"},{"reference":"Bargmann CI. 2006. Chemosensation in C. elegans. WormBook: 1-29.
","pubmedId":"18050433","doi":""},{"reference":"Caldwell KA, Willicott CW, Caldwell GA. 2020. Modeling neurodegeneration in Caenorhabditis elegans. Disease Models & Mechanisms. 13: dmm046110.","pubmedId":"","doi":"10.1242/dmm.046110"},{"reference":"Chalfie M, Hart AC, Rankin CH, Goodman MB. 2014. Assaying mechanosensation. WormBook: 10.1895/wormbook.1.172.1.
","pubmedId":"25093996","doi":""},{"reference":"Chen X, Chalfie M. 2014. Modulation of C. elegans Touch Sensitivity Is Integrated at Multiple Levels. Journal of Neuroscience. 34: 6522.","pubmedId":"","doi":"10.1523/JNEUROSCI.0022-14.2014"},{"reference":"Cinar H, Keles S, Jin Y. 2005. Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans. Current Biology. 15: 340.","pubmedId":"","doi":"10.1016/j.cub.2005.02.025"},{"reference":"Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. 2015. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette. Genetics. 200: 1035.","pubmedId":"","doi":"10.1534/genetics.115.178335"},{"reference":"Elder CR, Pasquinelli AE. 2022. New Roles for MicroRNAs in Old Worms. Frontiers in Aging. 3: 871226.","pubmedId":"","doi":"10.3389/fragi.2022.871226"},{"reference":"Ivey KN, Srivastava D. 2015. microRNAs as Developmental Regulators. Cold Spring Harbor Perspectives in Biology. 7: a008144.","pubmedId":"","doi":"10.1101/cshperspect.a008144"},{"reference":"Jiang, R. 2022. Conditional depletion of ALG-1 and ALG-2 using auxin inducible degron 2 (AID2). UC San Diego. ProQuest ID: Jiang_ucsd_0033M_21343. Merritt ID: ark:/13030/m5hx8j4s. Retrieved from https://escholarship.org/uc/item/8646322z
","pubmedId":"","doi":""},{"reference":"Li W, Kang L, Piggott BJ, Feng Z, Xu XZS. 2011. The neural circuits and sensory channels mediating harsh touch sensation in Caenorhabditis elegans. Nature Communications 2: 10.1038/ncomms1308.
","pubmedId":"","doi":"10.1038/ncomms1308 "},{"reference":"Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M. 2009. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature Methods. 6: 917.","pubmedId":"","doi":"10.1038/nmeth.1401"},{"reference":"O Brien J, Hayder H, Zayed Y, Peng C. 2018. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in Endocrinology. 9: 402.","pubmedId":"","doi":"10.3389/fendo.2018.00402"},{"reference":"Palumbos SD, Popolow J, Goldsmith J, Holzbaur ELF. 2025. Autophagic stress activates distinct compensatory secretory pathways in neurons. Proceedings of the National Academy of Sciences 122: 10.1073/pnas.2421886122.
","pubmedId":"","doi":"10.1073/pnas.2421886122"},{"reference":"Petratou D, Fragkiadaki P, Lionaki E, Tavernarakis N. 2024. Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans. STAR Protocols. 5: 102801.","pubmedId":"","doi":"10.1016/j.xpro.2023.102801"},{"reference":"Rivard L, Srinivasan J, Stone A, Ochoa S, Sternberg PW, Loer CM. 2010. A comparison of experience-dependent locomotory behaviors and biogenic amine neurons in nematode relatives of Caenorhabditis elegans. BMC Neuroscience. 11: 22.","pubmedId":"","doi":"10.1186/1471-2202-11-22"},{"reference":"Sala L, Chandrasekhar S, Vidigal JA. 2020. AGO unchained: Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in bioscience (Landmark edition). 25: 1.","pubmedId":"","doi":"10.2741/4793"},{"reference":"Sawin ER, Ranganathan R, Horvitz HR. 2000. C. elegans Locomotory Rate Is Modulated by the Environment through a Dopaminergic Pathway and by Experience through a Serotonergic Pathway. Neuron. 26: 619.","pubmedId":"","doi":"10.1016/S0896-6273(00)81199-X"},{"reference":"Shang R, Lee S, Senavirathne G, Lai EC. 2023. microRNAs in action: biogenesis, function and regulation. Nature Reviews Genetics. 24: 816.","pubmedId":"","doi":"10.1038/s41576-023-00611-y"},{"reference":"Sharma N, Marques F, Kratsios P. 2024. Protocol for auxin-inducible protein degradation in C. elegans using different auxins and TIR1-expressing strains. STAR Protocols. 5: 103133.","pubmedId":"","doi":"10.1016/j.xpro.2024.103133"},{"reference":"Stiernagle T. 2006. Maintenance of C. elegans. WormBook: 1-11.
","pubmedId":"18050451","doi":""},{"reference":"Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, et al., Miller. 2021. Molecular topography of an entire nervous system. Cell 184: 4329-4347.e23.
","pubmedId":"","doi":"10.1016/j.cell.2021.06.023"},{"reference":"Tops BBJ, Plasterk RHA, Ketting RF. 2006. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: Almost Identical yet Different. Cold Spring Harbor Symposia on Quantitative Biology. 71: 189.","pubmedId":"","doi":"10.1101/sqb.2006.71.035"},{"reference":"Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. 2011. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nature Structural & Molecular Biology 18: 302-308.
","pubmedId":"","doi":"doi.org/10.1038/nsmb.1986"},{"reference":"Vidigal JA. 2020. AGO unchained Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in Bioscience 25: 1-42.
","pubmedId":"","doi":"10.2741/4793"},{"reference":"Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, et al., Kanemaki MT. 2020. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun 11(1): 5701.
","pubmedId":"33177522","doi":""}],"title":"The miRNA Argonaute protein, ALG-2, maintains neurobehaviors in adult C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"beaa520b-3186-4be5-889f-538660fc423f","decision":"publish","abstract":"microRNAs (miRNAs) are short, non-coding RNAs essential for gene regulation in many different processes, including neuronal development. However, the role of the miRNA pathway in maintaining neuronal health throughout aging is less understood. Here, we ask how the miRNA pathway in adulthood impacts neurobehaviors in C. elegans. Argonaute-like Gene 2 (ALG-2) is a protein required for the accumulation and function of certain miRNAs in C. elegans. Using the auxin-inducible degron 2 (AID2) system for temporal knockdown, we demonstrate that the miRNA Argonaute protein, ALG-2, is required throughout adulthood to maintain two well-characterized neurobehaviors, basal slowing response and mechanosensation.
","acknowledgements":"This work was supported by grants from the National Institutes of Health [R35 GM127012 to A.E.P.]; and the Hevolution Foundation [HF-GRO-23-1199180]. E.C.S. was supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine [T32 GM007240]. S.D.P was supported by a Faculty Research Support Grant provided by Swarthmore College.
We thank the C. elegans Genetic Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for providing strains used in this study.
","authors":[{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","formalAnalysis","writing_originalDraft","writing_reviewEditing"],"email":"achon1@swarthmore.edu","firstName":"Ava","lastName":"Chon","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0000-8049-6146"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","methodology","investigation","dataCuration","validation"],"email":"ruj016@ucsd.edu","firstName":"Runtian","lastName":"Jiang","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0009-0069-777X"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing","formalAnalysis"],"email":"yguo2@swarthmore.edu","firstName":"Yuxuan","lastName":"Guo","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5751-1255"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["formalAnalysis","investigation"],"email":"yphyu1@swarthmore.edu","firstName":"Yamin K.","lastName":"Phyu","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-7961-3082"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["dataCuration","writing_reviewEditing"],"email":"lgoldbe1@swarthmore.edu","firstName":"Lilly M.","lastName":"Goldberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0001-8631-3723"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["supervision","conceptualization","investigation"],"email":"eschiksn@ucsd.edu","firstName":"Erin C.","lastName":"Schiksnis","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0003-2756-8168"},{"affiliations":["UC San Diego, San Diego, CA, United States"],"departments":["Molecular Biology"],"credit":["conceptualization","fundingAcquisition","writing_reviewEditing","supervision","methodology"],"email":"apasquinelli@ucsd.edu","firstName":"Amy E.","lastName":"Pasquinelli","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-9511-0039"},{"affiliations":["Swarthmore College, Swarthmore, PA, United States"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","supervision","writing_originalDraft","writing_reviewEditing","visualization"],"email":"spalumb1@swarthmore.edu","firstName":"Sierra D.","lastName":"Palumbos","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-3595-984X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"WBPerson39614
","image":{"url":"https://portal.micropublication.org/uploads/e680aae95387f8728a08eed2a427f1f2.png"},"imageCaption":"A) Graphic depicting insertion of AID2 at the alg-2 gene locus. B) Immunoblot of ALG-2 protein levels from PQ668 worms following 30 minutes of auxin treatment at ranging auxin concentrations. Actin was probed as positive control. C) BSR assay of day 1 adult PQ668 strain and D) day 4 adult PQ668 and N2 strains. For each condition, n=27. BSR was calculated by subtracting the number of body bends in the presence of food from the number of body bends when food was absent. One-way ANOVA with multiple comparisons used to determine significance; *** indicates p<0.001. E) Soft touch response assay of day 2 adult PQ668 and N2 strains with or without auxin treatment. N=30 worms for each condition across 3 trials. One-way ANOVA test with multiple comparisons was used to determine significance, *** indicates p<0.001. F) Chemotaxis Index (CI) of day 2 adult PQ668 and N2 C.elegans with and without auxin treatment in response to isoamyl alcohol (IA). CI was calculated as the number of worms at IA minus the number of worms at H₂O, divided by the total number of worms. Error bars represent SD. n ≥10 per trial, 3 trials. One-way ANOVA with multiple comparisons used to determine significance. G) alg-2 expression in transcripts per million (TPM) at L1, L4 and Adult stages in touch receptor neurons. Data taken from single cell data reported by the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021).
","imageTitle":"ALG-2 function in adulthood required for neurobehaviors
","methods":"Strain Preparation: The N2 strain was obtained from the Caenorhabditis Genetics Center. The PQ668 (AID2::mNeonGreen::3Xflag::alg-2) strain was developed using CRISPR-Cas9 to insert AID2::mNeonGreen::3Xflag before the 5' end of alg-2 before exon 1 of isoform A and exon 0 of isoform B (See Jiang, 2022). Briefly, four plasmids were injected into young adult N2 worms: 1) 50ng/ul of homologous repair template (AID2::mNeonGreen::3xflag with ALG-2 homology arms), 2) 50ng/ul of pJB53 (Cas9 plasmid with ALG-2 specific sgRNA, modified from pJW1219, Addgene #61250), 3) 10ng/ul pGH8 (Prab-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19359) and 4) 5ng/μL pCFJ104 (Pmyo-3::mCherry::unc-54 3'-UTR, Addgene plasmid #19328). Recombinant worms were isolated as previously described (Dickinson et al. 2015) and then backcrossed 3X to N2 to generate PQ668.
Auxin Plates: Nematode growth media (NGM) plates were prepared as described previously (Stiernagle, 2006). Auxin-containing NGM plates were prepared by adding 0.89 mg/mL auxin after autoclaving (Sharma et al., 2024). Plates were seeded with OP50 for 48 hours.
Western Blot: Western blot was carried out as described previously and ALG-2 was detected based on 3X flag insertion using anti-FLAG antibody (Van Wynsberghe et al., 2011).
Soft Touch Response: L4 PQ668 C. elegans were transferred to NGM plates with or without auxin and maintained at 23°C for 2 days. Adult Day 2 worms were transferred to an unseeded NGM plate and allowed to acclimate before testing. Gentle mechanical stimuli were applied using an eyelash pick by alternately stroking the anterior of the worm (posterior to the pharynx) and the posterior of the worm (anterior of the anus) for a total of 10 touches per worm (Chalfie et al., 2018). Responses to anterior touch were scored as reversals, whereas responses to posterior touch were scored as forward movement. Failure to produce a movement response following soft touch stimulation was scored as defective.
Basal Slowing Response: L4 C. elegans were plated on NGM plates with or without auxin. Plates were kept at 23℃ and worms were allowed to grow for either 1 or 4 days before being transferred to an unseeded NGM plate for analysis. After a 3-minute acclimation period, body bends were counted over a 20-second period. Worms were transferred to a seeded plate and the same procedure was repeated (Petratou et al., 2024). BSR was calculated for individual worms by subtracting the number of body bends in the presence of food from the number of body bends when no food was present.
Chemotaxis Assay: L4 C. elegans were transferred to OP50-seeded NGM plates with or without auxin and maintained at 23℃ for 2 days. Chemotaxis assay plates were prepared by dividing each plate into two halves and 5μL of isoamyl alcohol (IA) on one side and 5μL of deionized water (H₂O) on the other. 5μL of 0.5 M sodium azide was spotted on IA and H₂O to paralyze the worms upon contact. Day-2 adult worms were washed with 1 mL of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 mL 1M MgSO₄) before being transferred to chemotaxis plates for 1-hour. A minimum of 10 worms per plate was assayed across 3 trials. Worms were manually counted under a dissecting microscope. The chemotaxis index was calculated as CI = (# of worms at IA − # of worms at H₂O) / total # of worms, and ranged from +1 (maximum attraction) to -1 (maximum repulsion) (Bargmann et al., 1993).
Statistics:
All statistical analyses were performed using GraphPad Prism (ver. 10) and specific tests are specified in figure legend.
","reagents":"","patternDescription":"miRNAs are non-coding RNAs that are approximately 22 nucleotides long which repress gene expression of target mRNAs (Shang et al., 2023). miRNAs associate with an Argonaute protein, forming the miRNA-induced silencing complex (miRISC), which recognizes specific mRNA targets and induces their translational repression and degradation (Shang et al., 2023, Sala et al., 2020). C. elegans express two argonaute proteins that mediate miRNA repression, ALG-1 and ALG-2, which exhibit distinct roles (Tops et al., 2006, Aalto et al., 2018). While miRNAs are well characterized in development (Ivey and Srivastava, 2015), their role in aging and neurodegeneration is still an emerging field (Elder and Pasquinelli, 2022). Here, we ask whether the miRNA pathway has a role in maintaining neuronal homeostasis in adult C. elegans by tracking common neurobehaviors (Caldwell et al., 2020).
To do this, we used CRISPR/Cas9 to insert an Auxin-inducible degron 2 sequence at the N-terminus of alg-2 coding sequence, to generate a worm strain, PQ668 (AID2::mNeonGreen::3Xflag::alg-2) where we could temporally regulate alg-2 expression in adult C. elegans via the exogenous addition of auxin (Fig. 1A) (Nishimura et al., 2009, Dickinson et al., 2015, Yesbolatova et al., 2020). To confirm successful knockdown of ALG-2, we tracked ALG-2 protein levels using immunoblotting from worms treated with and without auxin (Fig. 1B). Following as short as 30 minutes on auxin-plates, ALG-2 protein levels were reduced below detection. This was also confirmed by tracking the inserted GFP fluorescent reporter. Thus, we developed a tool where ALG-2 levels could be selectively reduced during adulthood, allowing us to ask if loss of ALG-2 impacted neuronal homeostasis.
C. elegans reduce their speed in the presence of food, a phenomenon termed Basal Slowing Response (BSR) (Rivard et al., 2010), which is dependent on dopaminergic transmission (Sawin et al., 2000). We measured BSR in control (PQ668 – auxin) and ALG-2 depleted worms (PQ668 + auxin) to ask how impairment of the ALG-2-miRNA pathway impacts dopaminergic transmission in C. elegans. In day 1 adult worms, there was no statistically significant difference in BSR between PQ668 - auxin and PQ668 + auxin worms (p=0.2244) (Fig. 1C). However, following 4 days without ALG-2, PQ668 + auxin worms showed significantly reduced BSR compared to their matched controls (1.5185 vs. 5.4815, p=0.0008) (Fig. 1D). To confirm this observed effect was due to ALG-2 depletion and not the presence of auxin, we measured BSR in N2 worms in the presence or absence of auxin and observed no difference (p=0.9559) (Fig. 1E). Thus, we observed an age-dependent defect in BSR, suggesting a role for ALG-2 in maintaining dopaminergic transmission in adulthood.
We next asked whether other behaviors were impacted. C. elegans depend on soft touch sensation to navigate their world. Soft touch is communicated via mechanoreceptors expressed by six glutamatergic touch receptor neurons located in either the anterior or posterior of the worm (Chen and Chalfie, 2014). To determine whether depletion of ALG-2 in adulthood affects mechanosensation, we monitored soft touch response in Day 2 adult worms with control levels of ALG-1 (PQ668 - auxin) and those with ALG-2 depletion (PQ668 + auxin, L4-Day 2) following alternating anterior and posterior stimulation with an eyelash pick. While we observed a significant reduction in overall responses (64.33% vs. 83.00%, p=0.002), we noted that failure to respond was only observed following anterior stimuli. We therefore quantified anterior soft touch response and noted ALG-2 depleted worms (PQ668 + auxin) exhibited a defect in anterior mechanosensation compared to untreated controls (PQ668 - auxin) (28.67% vs. 66.00%, p=0.0002) (Fig. 1E). To determine whether auxin itself affected mechanosensation, we repeated the assay using N2 worms and found no significant difference between N2 worms grown on auxin vs. control plates (p=0.8283) (Fig. 1E). Our findings suggest that the miRNA Argonaute protein, ALG-2, is selectively required for anterior mechanosensation in adult animals, potentially through maintaining glutamatergic transmission in a subset of touch receptor neurons.
C. elegans possess a well-organized chemosensory system that allows them to navigate a variety of olfactory and gustatory cues associated with food, danger, and mates (Bargmann, 2006). We used the well characterized attractant, isoamyl alcohol, (Bargmann et al., 1993) to ask if ALG-2 function in adulthood is similarly required for chemotaxis. We tracked the chemotaxis index in PQ668 and N2 wild type worms (day 2 adults) with and without auxin. In contrast to mechanosensation and BSR, we found no significant change in chemosensory behavior across any conditions (Fig. 1F). It is of note that both our auxin groups had higher variability in their response, suggesting that auxin itself could be impairing chemotaxis. Thus, loss of ALG-2 function did not impair chemotaxis, suggesting that distinct circuits are impacted by loss of ALG-2 activity.
Given our observations that adult expression of ALG-2 is selectively required for specific neurobehaviors (BSR and anterior mechanosensation), but not others (chemosensation) we analyzed alg-2 expression dynamics in touch receptor and dopaminergic neurons across developmental stages using the C. elegans Neuronal Gene Expression Map & Network (Taylor et al., 2021). Touch receptor neurons (ALM, AVM, PLM, PVM) displayed a biphasic developmental regulatory pattern of alg-2 expression (high in L1, low in L4, high in adults) (Figure 1G). As this developmental expression pattern was true in both anterior and posterior touch neurons, local alg-2 transcription alone does not explain selective defect in anterior soft touch response. Similarly, alg-2 expression in dopaminergic neurons was inconsistent with a simple cell-autonomous mechanism. While adult CEP neurons showed a modest increase in alg-2, ADE and PDE expressed alg-2 at levels far below their developmental peaks. Given that miRNAs can be secreted and act extracellularly to impact protein expression in neighboring cells (Shang et al., 2023, Alkhazaali-Ali et al., 2024, Palumbos et al., 2025), we hypothesize that ALG-2 regulates neurotransmission, at least in part, in a non-cell-autonomous manner during aging. In summary, these results demonstrate that ALG-2 is required during adulthood to maintain glutamatergic and dopaminergic neurotransmission, suggesting miRNAs play an ongoing role in preserving neural function during aging.
","references":[{"reference":"Aalto AP, Nicastro IA, Broughton JP, Chipman LB, Schreiner WP, Chen JS, Pasquinelli AE. 2018. Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging. PLoS Genetics. 14: e1007379.","pubmedId":"","doi":"10.1371/journal.pgen.1007379"},{"reference":"Alkhazaali Ali Z, Sahab Negah S, Boroumand AR, Tavakol Afshari J. 2024. MicroRNA (miRNA) as a biomarker for diagnosis, prognosis, and therapeutics molecules in neurodegenerative disease. Biomedicine & Pharmacotherapy. 177: 116899.","pubmedId":"","doi":"10.1016/j.biopha.2024.116899"},{"reference":"Bargmann CI, Hartwieg E, Horvitz HR. 1993. Odorant-selective genes and neurons mediate olfaction in C. elegans. Cell. 74: 515.","pubmedId":"","doi":"10.1016/0092-8674(93)80053-h"},{"reference":"Bargmann CI. 2006. Chemosensation in C. elegans. WormBook: 1-29.
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","pubmedId":"","doi":"10.1016/j.cell.2021.06.023"},{"reference":"Tops BBJ, Plasterk RHA, Ketting RF. 2006. The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: Almost Identical yet Different. Cold Spring Harbor Symposia on Quantitative Biology. 71: 189.","pubmedId":"","doi":"10.1101/sqb.2006.71.035"},{"reference":"Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE. 2011. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nature Structural & Molecular Biology 18: 302-308.
","pubmedId":"","doi":"doi.org/10.1038/nsmb.1986"},{"reference":"Vidigal JA. 2020. AGO unchained Canonical and non-canonical roles of Argonaute proteins in mammals. Frontiers in Bioscience 25: 1-42.
","pubmedId":"","doi":"10.2741/4793"},{"reference":"Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, et al., Kanemaki MT. 2020. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun 11(1): 5701.
","pubmedId":"33177522","doi":""}],"title":"The miRNA Argonaute protein, ALG-2, maintains neurobehaviors in adult C. elegans
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes 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