Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

Abstract

Caenorhabditis elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear genes that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD(+)-dependent ketoacid oxidation occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations both among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease.

Figure 1. Tricarboxylic acid cycle (TCA) pathway gene upregulation in complex I mutants

Relative expression levels of all genes on the GeneChip C. elegans genome arrays obtained in two phenotypic groupings (i.e., mutant and wildtype) are given a ranking in GSEA used to calculate the likelihood genes comprising a given cluster (i.e., TCA cycle cluster) are overrepresented at a given end of the rank list. A) An enrichment plot indicates the rank of every gene in a pathway cluster (vertical black lines) as well as those genes which fall under the leading edge (between the left edge and the peak of the green curve). 21 of 26 TCA cycle transcripts annotated in the KEGG database are upregulated in the complex I mutant compared to wildtype. B) The table indicates the TCA gene transcripts in C. elegans, their individual rank among all genes on the array, and an indication of whether they contribute to the maximum enrichment score leading to a p value of 0.0066 (see Table 2). C) A visual representation of the overall upregulated TCA expression profile in complex I mutants can be obtained in pathway form (www.genMAPP.org). Red and green colors indicate genes overexpressed and underexpressed, respectively, in the mutant compared to wildtype, with shade intensity representing significance level.

Figure 1. Tricarboxylic acid cycle (TCA) pathway gene upregulation in complex I mutants

Relative expression levels of all genes on the GeneChip C. elegans genome arrays obtained in two phenotypic groupings (i.e., mutant and wildtype) are given a ranking in GSEA used to calculate the likelihood genes comprising a given cluster (i.e., TCA cycle cluster) are overrepresented at a given end of the rank list. A) An enrichment plot indicates the rank of every gene in a pathway cluster (vertical black lines) as well as those genes which fall under the leading edge (between the left edge and the peak of the green curve). 21 of 26 TCA cycle transcripts annotated in the KEGG database are upregulated in the complex I mutant compared to wildtype. B) The table indicates the TCA gene transcripts in C. elegans, their individual rank among all genes on the array, and an indication of whether they contribute to the maximum enrichment score leading to a p value of 0.0066 (see Table 2). C) A visual representation of the overall upregulated TCA expression profile in complex I mutants can be obtained in pathway form (www.genMAPP.org). Red and green colors indicate genes overexpressed and underexpressed, respectively, in the mutant compared to wildtype, with shade intensity representing significance level.

Figure 2. Quantitative profiling of free amino acids in respiratory chain mutants and wildtype worms

2a) Alanine is the most prevalent amino acid in C. elegans. Complex II (mev-1(kn1)) and complex III (isp-1(qm150)) mutants display largely similar patterns of alterations in amino acid profiles, each with 9 of 16 amino acid levels significantly different from wildtype at p < 0.05 (GLU, GLN, GLY, THR, VAL, PHE (in mev-1 only), IL, LEU, ORN, and LYS (in isp-1 only). Interestingly, the significant increase in alanine observed in the complex I mutant is not seen in the complex II or III mutants. 2b) Compared with wildtype worms, gas-1(fc21) complex I mutants show significant differences in 13 of 16 amino acid levels. Statistical significance is noted as ** p < 0.01 and *** p < 0.001. n represents # of synchronous adult worm aliquots of each strain studied. Values represent mean and standard error of whole worm free amino acid levels normalized to 1,000 adult worms.

Figure 3. Schematic overview of glutamate metabolism

Respiratory chain dysfunction presumably results in an increased NADH:NAD⁺ ratio (indicated by red arrows), which limits oxidation of ketoacids such as α-ketoisocaproate (KIC), the ketoacid of leucine. The result is greater accessibility of the ketoacid for conversion to its parent amino acid – in this illustration, leucine (Leu). A consequence would be accumulation of the parent amino acid and relative depletion of glutamate (Glu) (see Table 3). Some glutamate might be derived from reductive amination of −–ketoglutarate (αKG) in the glutamate dehydrogenase reaction, which is enhanced by an increased NADH:NAD⁺ ratio. However, this reaction is not sufficiently robust to prevent an overall diminution of the glutamate concentration and a marked increase in the ratio of leucine (and other amino acids) to glutamate (Table 3). This suggests that levels of amino acids such as leucine, which are formed from transamination reactions with glutamate, may be sensitive indicators of an altered redox state. Postulated direction of equilibrium reaction alterations occurring in primary respiratory chain dysfunction are indicated by blue arrows.