Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration

Abstract

Regular physical exercise is the most efficient and accessible intervention known to promote healthy aging in humans. The molecular and cellular mechanisms that mediate system-wide exercise benefits, however, remain poorly understood, especially as applies to tissues that do not participate directly in training activity. The establishment of exercise protocols for short-lived genetic models will be critical for deciphering fundamental mechanisms of transtissue exercise benefits to healthy aging. Here we document optimization of a long-term swim exercise protocol for Caenorhabditis elegans and we demonstrate its benefits to diverse aging tissues, even if exercise occurs only during a restricted phase of adulthood. We found that multiple daily swim sessions are essential for exercise adaptation, leading to body wall muscle improvements in structural gene expression, locomotory performance, and mitochondrial morphology. Swim exercise training enhances whole-animal health parameters, such as mitochondrial respiration and midlife survival, increases functional healthspan of the pharynx and intestine, and enhances nervous system health by increasing learning ability and protecting against neurodegeneration in models of tauopathy, Alzheimer's disease, and Huntington's disease. Remarkably, swim training only during early adulthood induces long-lasting systemic benefits that in several cases are still detectable well into midlife. Our data reveal the broad impact of swim exercise in promoting extended healthspan of multiple C. elegans tissues, underscore the potency of early exercise experience to influence long-term health, and establish the foundation for exploiting the powerful advantages of this genetic model for the dissection of the exercise-dependent molecular circuitry that confers system-wide health benefits to aging adults.

Keywords: Caenorhabditis elegans; aging; exercise; muscle; neurodegeneration.

Fig. 1.

Multiple daily swim sessions are essential for C. elegans exercise adaptation. (A) Diagram of the 90-min swim session protocol performed for each exercise bout. We transferred control animals to an unseeded NGM plate for 90 min, whereas exercise animals were transferred to an unseeded NGM plate flooded with M9 buffer for the same 90 min to swim. (B) Diagram indicating the time of the day of each exercise session for the 5 long-term exercise regimens tested. (C) qPCR results for 10 muscle structural genes in Ad5 WT animals exposed to the 5 long-term exercise regimens indicated. n = 4 to 5 independent trials. (D) Percentage increase in crawling maximum velocity in Ad5 and Ad8 WT exercised animals exposed to the 5 tested long-term exercise regimens, relative to nonexercised control counterparts. n = 3 to 6 independent trials. *P ≤ 0.05, **P ≤ 0.01.

Fig. 2.

Long-term swim exercise improves burrowing performance and mitochondrial profiles of body wall muscle. (A) Diagram of burrowing assay. After the 3+3+2+2 regimen, we trapped Ad5 animals under a Pluronic F-127 gel and added attractant food E. coli OP50-1 to the center of the gel surface. Animals are attracted by the food and burrow to the surface of the gel at different rates. (B) Proportion of Ad5 WT animals exposed to the 3+3+2+2 regimen that reach the gel surface during the 3-h burrowing assay. n = 370 to 392 animals. (C) Representative confocal images of the 5 classes of body wall muscle mitochondrial network organization in P_myo-3mitoGFP animals. Fragmentation and disorganization of muscle mitochondria increases progressively from class 1 to class 5. (Scale bar, 10 µm.) (D) Distribution of mitochondrial classes in body wall muscle of Ad5, Ad8, and Ad11 P_myo-3mitoGFP animals exposed to the 3+3+2+2 regimen. n = 160 to 170 muscle images. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Fig. 3.

Long-term exercise improves mitochondrial respiration parameters and midlife survival. (A) Diagram of oxygen consumption rates measured in a Seahorse XFe24 Analyzer consequent to addition of mitochondrial inhibitors, allowing for the calculation of the 6 respiration parameters represented by different colors. (B–G) Basal respiration (B), maximal respiration (C), spare capacity (D), ATP-linked respiration (E), proton leak (F), and nonmitochondrial respiration (G) values of Ad5 and Ad8 WT animals exposed to the 3+3+2+2 regimen. n = 13 to 30 Seahorse XF24 Microplate wells. (H) Survival curve of WT animals exposed to the 3+3+2+2 regimen. n = 221 to 245 animals. *P ≤ 0.05.

Fig. 4.

Pharyngeal and intestinal healthspans are extended after long-term swim exercise. (A) Pharyngeal pumping rate of Ad5, Ad8, Ad11, and Ad15 WT animals exposed to the 3+3+2+2 regimen. Each point represents a single animal. n = 66 to 87 animals. (B) Representative image of non-Smurf (first and third animals from left to right) and Smurf (second and fourth animals from left to right, arrows indicate leaks) animals. (C and D) Higher-magnification images of Smurf animals showing intestinal leakage in the anterior (C) and posterior (D) regions. Arrows in B–D indicate areas of blue dye leakage into the body cavity. (Scale bars, 50 µm.) (E) Percentage of Smurf animals at Ad8, Ad11, and Ad15 in WT nematodes exposed to the 3+3+2+2 regimen. n = 4 to 8 independent trials. *P ≤ 0.05, ***P ≤ 0.001.

Fig. 5.

Learning ability is enhanced in exercised C. elegans. (A) Diagram of the associative learning assay. After the 3+3+2+2 regimen, we starved Ad5 animals for 1 h followed by food-butanone conditioning in a seeded NGM plate with 10% butanone solution on the inside of the lid. We performed chemotaxis assays of naïve and conditioned animals by testing attraction to butanone vs. isoamyl alcohol. (B) Learning index of Ad5 WT animals 0 h postconditioning after exposure to the 3+3+2+2 regimen. Chemotaxis index (CI) = (animal number at butanone − animal number at isoamyl alcohol)/(total animal number − immobile animal number at origin). We calculated learning index by subtraction of naïve CI from postconditioning CI. n = 8 independent trials. **P ≤ 0.01.

Fig. 6.

Long-term swim exercise improves neuronal health in multiple C. elegans neurodegeneration models. (A) Mean velocity of Ad5, Ad8, and Ad11 animals expressing aggregating Tau in all neurons (P_rab-3F3ΔK280; P_aex-3h4R1NTauV337M) exposed to the 3+3+2+2 regimen. Note that Tau-expressing animals did not exhibit a standard swimming motion due to impaired motility but were more active in liquid than on agar so enhanced activity during the assay was confirmed. Each point represents a single animal. n = 54 to 60 animals. (B) Representative confocal images of Ad3 animals expressing aggregating Tau in all neurons and GFP in GABAergic motor neurons (P_rab-3F3ΔK280; P_aex-3h4R1NTauV337M; P_unc-25GFP). Arrows indicate gaps in the ventral cords. (Scale bar, 30 µm.) (C) Average number of gaps detectable in ventral and dorsal cords of Ad3 animals expressing aggregating Tau in all neurons and GFP in GABAergic motor neurons exposed to the 3+2 regimen. n = 5 independent trials. Note that this strain did not exhibit a standard swimming motion due to the severe uncoordinated phenotype, which may explain the lack of up-regulation of muscle structural genes after the 3+2 regimen (SI Appendix, Fig. S9A). Nevertheless, animals were still more active in liquid than on agar so enhanced activity was confirmed. (D) CI toward benzaldehyde of Ad3 animals expressing neuronal Aβ_1–42 [smg-1(cc546^ts) P_snb-1Aβ_1–42::long 3′-UTR] exposed to the 3+3 regimen. We raised animals at 23 °C from the egg stage onward. CI = (animal number at benzaldehyde half − animal number at ethanol half)/(total animal number − immobile animal number at origin). n = 5 independent trials. Note that this strain swam slower than WT, which may explain the lack of significant up-regulation of muscle structural genes after the 3+3 regimen (SI Appendix, Fig. S9B). (E) Anterior touch sensitivity of Ad5, Ad8, and Ad11 animals expressing polyQ128 in the touch receptor neurons (P_mec-3htt57Q128) exposed to the 3+3+2+2 regimen. n = 119 to 248 animals. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 7.

Swim exercise of postreproductive C. elegans also improves locomotory vigor. (A) Diagram of the 2+2+2 exercise regimen performed with postreproductive animals (from Ad6 to Ad8) indicating the time of the day of each exercise session. We tested swim sessions with a duration of 90 and 60 min. (B and C) Crawling maximum velocity of Ad9 and Ad12 WT animals exposed to the 2+2+2 regimen with swim sessions of 90 min (B) or 60 min (C). Each point represents a single animal. n = 59 to 60 animals. *P ≤ 0.05, ***P ≤ 0.001.