Genetic variation in ALDH4A1 is associated with muscle health over the lifespan and across species

Abstract

The influence of genetic variation on the aging process, including the incidence and severity of age-related diseases, is complex. Here, we define the evolutionarily conserved mitochondrial enzyme ALH-6/ALDH4A1 as a predictive biomarker for age-related changes in muscle health by combining Caenorhabditis elegans genetics and a gene-wide association scanning (GeneWAS) from older human participants of the US Health and Retirement Study (HRS). In a screen for mutations that activate oxidative stress responses, specifically in the muscle of C. elegans, we identified 96 independent genetic mutants harboring loss-of-function alleles of alh-6, exclusively. Each of these genetic mutations mapped to the ALH-6 polypeptide and led to the age-dependent loss of muscle health. Intriguingly, genetic variants in ALDH4A1 show associations with age-related muscle-related function in humans. Taken together, our work uncovers mitochondrial alh-6/ALDH4A1 as a critical component to impact normal muscle aging across species and a predictive biomarker for muscle health over the lifespan.

Keywords: C. elegans; HRS; aging; evolutionary biology; genetic variation; genetics; genomics; human; muscle; sarcopenia.

Figure 1.. Mutation of alh-6 uniquely activates age-dependent and activation of the gst-4p::gfp oxidative stress reporter in muscle.

(a) Schematic representation of genetic screen for mutants that phenocopy alh-6(lax105). (b) Schematic representation of the ALH-6 protein with the molecular identity of mutants isolated and sequenced annotated. Alleles that were selected for additional functional tests of muscle function (Figure 4) are highlighted in red and the location of the canonical alh-6(lax105) allele is highlighted in green. These alleles represent all the sequenced mutations in alh-6 that were isolated from the ethyl methanesulfonate (EMS) screen. (c) Quantification of stress reporter activation in the muscle in the new alh-6 mutant alleles, as measured by the intensity of GFP fluorescence from the oxidative stress reporter gst-4p::gfp (see Figure 1—figure supplement 1 for representative images). t-Test relative to gst-4p::gfp reporter animals (control); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 1—figure supplement 1.. Novel alleles of alh-6 induce muscle-specific activation of the gst-4p::gfp stress reporter reporter.

GFP fluorescence images of gst-4p::gfp animals harboring alh-6 mutations, as indicated. Scale bar = 100 μm. Quantification of fluorescence is shown in Figure 1c.

Figure 1—figure supplement 2.. Location of amino acid substitutions in alh-6 mutants.

Location of individual missense mutations of ALH-6 mutants on the predicted structure of the wild-type ALH-6 protein by Phyre2 (Kelley et al., 2015). Mutated residues are colored (purple) and circled.

Figure 2.. ALDH4A1 variants associate with human age-related phenotypes for change in muscle function.

Plot of association between variants in the ALDH4A1 gene and normative aging-related muscle decline in (a) gait speed and (b) grip strength in the US Health and Retirement Study (HRS). The x-axis shows the beta estimate for the effect of each SNP, represented by a dot, on the phenotype. The y-axis shows the log of the p value for the association between the SNP and the phenotype. SNPs that surpassed the empirical p value threshold, shown as a red line, for decline in gait speed (empirical p value = 0.006) and grip strength (empirical p value = 0.0019) are depicted as red dots. SNPs that surpassed a suggestive threshold (p value = 0.009 for gait speed) are depicted as purple dots.

Figure 3.. Effects of ALDH4A1 variation on phenotypes representing association with change in aging-related function in a normative, population-based sample of older adults.

(a) Change in gait speed over 10 years. Effect of SNP rs77608580 on aging-related changes in gait speed (b = 0.052, p = 0.0025). Over the span of one decade, on average, those with one or two effect alleles will have faster gait speeds with a difference of 0.52 and 1.04 m/s, respectively, compared to those without an effect allele. (b) Decline in grip strength over 10 years. Variation in ALDH4A1 (SNP rs28665699) is inversely associated with decline in aging-related grip strength (b = −0.045, p = 0.0009). Individuals with one or two effect alleles have slower progression of weakened grip strength over 10 years by 0.5 and 1.0 kg, respectively, compared to the same aged individuals without the effect allele.

Figure 3—figure supplement 1.. Association between rs77608580 and ALDH4A1 gene expression levels in whole blood.

Normalized gene expression levels for ALDH4A1 (y-axis) by SNP genotype (x-axis) are shown by violin plot. Also on the x-axis is the sample size by genotype in parenthesis. Violin plots show the density plot of the data (green cloud) with the median of the data shown by the white line of the black box plot within, the lower and upper border of the box plot corresponding to the first and third quartiles, respectively. The black dots represent sample points for the genotype in which there were too few samples to depict by box plot. A linear regression model was used to estimate the mean difference in expression levels, calculated as a normalized effect size (NES) to compare the alternative allele, G, to the minor allele, A, figure and data source: GTEx Analysis Release V8 (dbGaP Accession phs000424.v8.p2) (Battle et al., 2017).

Figure 4.. alh-6 mutations accelerate loss of muscle function.

WormLab software analysis of adjusted center point speed of individual animals of the given genotypes at the L4 stage (a) or day 3 of adulthood (b). Brown–Forsythe and Welch analysis of variance (ANOVA) test with Dunnett’s T3 multiple comparisons test, with individual variances computed for each comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4—figure supplement 1.. alh-6 mutations accelerate loss of muscle function.

Rate of thrashing for individual animals of the given genotypes at the L4 stage (a) or day 3 of adulthood (b). Brown–Forsythe and Welch test with Dunnett’s T3 multiple comparisons test, with individual variances computed for each comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.