Zebrafish (Danio rerio) as a model for the study of aging and exercise: Physical ability and trainability decrease with age

AbstractA rapidly aging global population has motivated the development and use of models for human aging. Studies on aging have shown parallels between zebrafish and humans at the internal organization level; however, few parallels have been studied at the whole-organism level. Furthermore, the effectiveness of exercise as a method to mitigate the effects of aging has not been studied in zebrafish. We investigated the effects of aging and intermittent exercise on swimming performance, kinematics and behavior. Young, middle-aged and old zebrafish (20–29, 36–48 and 60–71% of average lifespan, respectively) were exercised to exhaustion in endurance and sprint swimming tests once a week for four weeks. Both endurance and sprint performance decreased with increased age. Swimming performance improved with exercise training in young and middle-aged zebrafish, but not in old zebrafish. Tail-beat amplitude, which is akin to stride length in humans, increased for all age groups with training. Zebrafish turning frequency, which is an indicator of routine activity, decreased with age but showed no change with exercise. In sum, our results show that zebrafish exhibit a decline in whole-organism performance and trainability with age. These findings closely resemble the senescence-related declines in physical ability experienced by humans and mammalian aging models and therefore support the use of zebrafish as a model for human exercise and aging

Growth differentiation factor 6 as a putative risk factor in neuromuscular degeneration.

Mutation of Glass bottom boat, the Drosophila homologue of the bone morphogenetic protein or growth/differentiation factor (BMP/GDF) family of genes in vertebrates, has been shown to disrupt development of neuromuscular junctions (NMJ). Here we tested whether this same conclusion can be broadened to vertebrate BMP/GDF genes. This analysis was also extended to consider whether such genes are required for NMJ maintenance in post-larval stages, as this would argue that BMP genes are viable candidates for analysis in progressive neuromuscular disease. Zebrafish mutants harboring homozygous null mutations in the BMP-family gene gdf6a were raised to adulthood and assessed for neuromuscular deficits. Fish lacking gdf6a exhibited decreased endurance (∼ 50%, p = 0.005) compared to wild type, and this deficit progressively worsened with age. These fish also presented with significantly disrupted NMJ morphology (p = 0.009), and a lower abundance of spinal motor neurons (∼ 50%, p<0.001) compared to wild type. Noting the similarity of these symptoms to those of Amyotrophic Lateral Sclerosis (ALS) model mice and fish, we asked if mutations in gdf6a would enhance the phenotypes observed in the latter, i.e. in zebrafish over-expressing mutant Superoxide Dismutase 1 (SOD1). Amongst younger adult fish only bigenic fish harboring both the SOD1 transgene and gdf6a mutations, but not siblings with other combinations of these gene modifications, displayed significantly reduced endurance (75%, p<0.05) and strength/power (75%, p<0.05), as well as disrupted NMJ morphology (p<0.001) compared to wild type siblings. Bigenic fish also had lower survival rates compared to other genotypes. Thus conclusions regarding a role for BMP ligands in effecting NMJ can be extended to vertebrates, supporting conservation of mechanisms relevant to neuromuscular degenerative diseases. These conclusions synergize with past findings to argue for further analysis of GDF6 and other BMP genes as modifier loci, potentially affecting susceptibility to ALS and perhaps a broader suite of neurodegenerative diseases

Disruption of <i>gdf6</i> function exacerbates neuromuscular junction abnormalities in ALS model zebrafish.

<p>ALS model zebrafish possess disruptions to neuromuscular junctions (NMJ), and loss of <i>gdf6</i> function exacerbates this by 7 months of age. <b>A.</b> The presynaptic junctions (labeled with synaptophysin antibody) and postsynaptic junctions (labeled with fluorescently tagged αBTX) in ALS model zebrafish expressing the mutant SOD1^G93R show punctate morphology, deviations in presynaptic volume and less overall colocalization compared to WT sibling junctions. Some abnormalities are exacerbated in bigenic siblings expressing the mutant SOD1^G93R that are also <i>gdf6^−/−</i> (scale bar is 40 µm). <b>B.</b> Quantification of these NMJs suggests the presynaptic/postsynaptic volume ratios of SOD1^G93R and bigenic <i>gdf6^−/−</i>;SOD1^G93R zebrafish are larger than those of WT siblings at this age, though these differences do not rise to statistical significance (Kruskall-Wallis ANOVA, p = 0.134; n = 6,4,6 for WT, SOD1^G93R, and bigenic fish respectively). Colocalization coefficients, that measure overall colocalization of presynaptic and postsynaptic junctions, are altered in these fish. The values for SOD1^G93R zebrafish are significantly lower than wild type sibling values, indicating that presynapses and postsynapses overlap less, as characterized previously for this transgenic ALS model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089183#pone.0089183-Ramesh1" target="_blank">[28]</a>. Bigenic SOD1^G93R zebrafish that are also <i>gdf6−/−</i> have a dramatically lower coefficient than either sets of siblings, including being 30% lower than ALS model SOD1^G93R fish with normal <i>gdf6a</i> (*p<0.05; ***p<0.001. Kruskall-Wallis ANOVA with pairwise comparisons).</p

Mutations in <i>gdf6</i> sensitize SOD1∧G93R zebrafish to develop ALS-like symptoms.

<p>Six genotypes combining <i>gdf6^−/−</i> alleles and SOD1∧G93R alleles were examined in endurance and sprint tests, which primarily measure red and white muscle respectively. The SOD1∧G93R fish mimic many aspects of ALS progression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089183#pone.0089183-Ramesh1" target="_blank">[28]</a>. Swimming performance was measured in all six genotypes (determined by outcrosses and examining progeny) at 4.5 months of age. <b>A.</b> bigenic fish, i.e. <i>gdf6^−/−</i> mutants expressing SOD1∧G93R, had decreased survival compared to all other genotypes, i.e. their siblings ( = <i>gdf6^+/+</i> or <i>gdf6^+/−</i>) with or without SOD1∧G93R. <b>B.</b> bigenic <i>gdf6^−/−</i> mutants expressing SOD1∧G93R had significantly lower endurance compared to heterozygous siblings also expressing SOD1∧G93R, and to WT and heterozygous siblings without SOD1∧G93R. <b>C.</b> Sprint test demonstrates a significant deficit in white muscle function only when SOD1∧G93R and <i>gdf6^−/−</i> genotypes are combined ( = 25% deficit). (ANOVA and multiple linear regression, p<0.05; sample size indicated below graph).</p

Zebrafish harboring homozygous mutations in <i>gdf6</i> can be raised to adulthood and do not display overt skeletal defects.

<p><b>A... </b><i>gdf6^−/−</i> fish are viable into adulthood, exhibit variably penetrant microphthalmia and normal body morphology. <b>B... </b><i>gdf6^−/−</i> fish exhibit somewhat decreased survival compared to <i>gdf6^+/+</i> siblings (n = 11 <i>gdf6^−/−</i> fish; n = 4 <i>gdf6^+/+</i> siblings). <b>C, D... </b><i>gdf6^−/−</i> fish lack overt skeletal phenotypes, as revealed by (C) clearing and staining or by (D) microCT analysis. The latter is further represented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089183#pone.0089183.s003" target="_blank">Supplemental Movie S1</a>. Scale bars are 5 mm. A variety of fin morphologies were present in the fish examined, but these were neither different between experimental groups (genotypes) nor a significant covariant with swim performance (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089183#s2" target="_blank">Results</a>).</p

Zebrafish harboring homozygous mutations in <i>gdf6</i> exhibit decreased endurance.

<p><b>A. </b><i>gdf6^−/−</i> fish have a lower endurance compared to <i>gdf6^+/+</i> siblings as measured by increasing water velocity in a swim channel to determine the <i>U</i>_crit i.e. ‘critical swimming speed’ which is taken to be the highest speed that a fish can swim at for a period of several minutes before exhaustion. Each fish tested at 9 months is plotted. <b>B.</b> The same data in panel A (9 month) plotted along with 18 month old siblings. <i>gdf6^−/−</i> fish have approximately 50% lower endurance compared to <i>gdf6^+/+</i> siblings at each age (*p = 0.005, **p<0.01). Endurance trends towards being decreased in older fish of each genotype, but this difference is not significant. <b>C.</b> Open field tests of average swim velocity during 10 minutes of each hour through a circadian cycle, in tanks replicating lifetime husbandry conditions, show a lower mean movement at night (in either genotype, **p<0.01) but no difference between genotypes in any measure during day (p = 0.247) or night (p = 0.814).</p