Developmental Defects of Caenorhabditis elegans Lacking Branched-chain α-Ketoacid Dehydrogenase Are Mainly Caused by Monomethyl Branched-chain Fatty Acid Deficiency

Abstract

Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD.

Keywords: Caenorhabditis elegans (C. elegans); amino acid; branched-chain amino acid (BCAA); branched-chain fatty acid; dbt-1; dihydrolipoamide branched-chain transacylase (DBT); fatty acid metabolism; inborn error of metabolism; maple syrup urine disease (MSUD); neurological disease.

FIGURE 1.

dbt-1(lf) worms have increased BCAAs and decreased mmBCFAs. A, a simple diagram to illustrate BCAA catabolism and mmBCFA metabolism in C. elegans and mammals. The multi-subunit BCKDH complex catalyzes the rate-limiting step in BCAA degradation, the products of which are degraded or serve as primers for mmBCFA synthesis. Specifically, C5ISO, produced from leucine, is the primer of odd-numbered iso-mmBCFAs, among which C15ISO and C17ISO are the major mmBCFA species in C. elegans; C5anteISO, produced from isoleucine, is the primer of odd numbered anteiso-mmBCFAs; C4ISO, produced from valine, is the primer of even-numbered iso-mmBCFAs. B and C, bar graphs showing changes in BCAA and mmBCFA levels in dbt-1(lf) L1 larvae. n = 3; error bars indicate standard deviation.

FIGURE 2.

RNAi knockdown of BCKDH subunit homologues reduced mmBCFA levels and caused larval arrest. F27D4.5 is a homologue of the E1β subunit, and dbt-1 is a homologue of the E2 subunit of human BCKDH, respectively. A, RNAi of F27D4.5 or dbt-1 reduced the levels of both C15ISO and C17ISO. n = 2; error bars indicate standard deviation. B–D, RNAi of F27D4.5 or dbt-1 caused larval arrest in the next generation. 5 days after transferring to new plates, worms on control RNAi all grew to adults (n = 100)(B), but worms on F27D4.5 (n = 200)(C) or dbt-1 (n = 200)(D) RNAi plates remained as larvae. Experiments were done using rrf-3 worms, which are hypersensitive to RNAi. Synchronized L1s were put on RNAi plates, and the subsequent young adults of the same generation were analyzed by GC. RNAi of Y39E4A.3, which is a homologue of the E1α subunit of human BCKDH, has been shown previously to also cause a reduction in mmBCFA levels (22) and was therefore not tested here.

FIGURE 3.

dbt-1(lf) causes larval arrest and embryonic lethal phenotypes that are both suppressed by mmBCFA supplementation. A, dose-response curves of C13ISO, C15ISO, C17ISO, and d17SPA in rescuing the larval arrest phenotype of dbt-1(lf). More than 100 worms were tested for each measurement. n = 3; error bars indicate standard deviation. B, hatching rate of eggs laid by dbt-1(lf) worms treated with different concentrations (Conc.) of C13ISO, C15ISO, C17ISO, or d17SPA. dbt-1^{C15ISO (low)} and dbt-1^{C15ISO (high)} (see “Results”) are highlighted in bold. The open arrow points to dbt-1^{C15ISO (low)}, and the filled arrow points to dbt-1^{C15ISO (high)}. n = 3; error bars indicate standard deviation.

FIGURE 4.

C15ISO supplementation restores mmBCFA levels without affecting BCAAs. A–D, the levels of mmBCFAs (A and C) and BCAAs (B and D) in samples of the indicated stages of WT, dbt-1^{C15ISO (low)}, or dbt-1^{C15ISO (high)} worms are shown. The difference between dbt-1^{C15ISO (low)} and dbt-1^{C15ISO (high)} worms indicates a positive correlation between the level of mmBCFA supplementation and BCAA levels. Due to lethality, dbt-1(lf) worms without supplementation at the similar stages could not be tested. However, the difference between BCAA levels in dbt-1(lf) worms and that in WT is expected to be similar to that shown in Fig. 1B for L1 larvae. Therefore, BCAA levels of mmBCFA supplemented dbt-1(lf) mutants are not likely to be significantly lower than that in dbt-1(lf) alone. n = 3; error bars indicate standard deviation.

FIGURE 5.

dbt-1 is ubiquitously expressed, and the neuronal expression of dbt-1 significantly represses the developmental defects. A, microscopic image showing the broad expression pattern of a dbt-1 promoter-driven GFP fusion gene. B–K, microscopic images showing the expression of a functional DBT-1-GFP fusion protein in a whole adult dbt-1(lf) worm carrying the translational dbt-1::gfp fusion gene. C and D, expression in the hypodermis and muscle; E and F, expression in the hypodermis and intestine; G–K, expression in the head sensory neurons. C, E, G, and J, bright field; D, F, H, I, and K, GFP channel. m, muscle; h, hypodermis; i, intestine. The dashed circle in G marks the pharyngeal bulb located beside a collection of head amphid neurons. The arrow in G–I points to a single head neuron with cytosolic DBT-1::GFP expression. I shows the neuron under a higher magnification where DBT-1 is expressed in puncta in the cytosol and in the neuronal projections that extend from opposing sides of the cell. J and K, DBT-1-GFP is also detected in patches in the dendrites of ciliated neurons. The dashed line in J marks the path of the dendrites. The arrowheads in K mark patches of DBT-1-GFP along the dendrites with strong expression in the cilia. Scale bars represent 50 μm in A and B and 10 μm in C–K. L and M, bar graphs showing the extent of suppression of the larval arrest (L) or embryonic lethal (M) phenotype of dbt-1(lf) by tissue-specific expression of the DBT-1-GFP fusion protein. myo-3Prom, rgef-1Prom, col-10Prom, and ges-1Prom are muscle, neuron, hypodermis, and intestine-specific promoters, respectively. n = 3; error bars indicate standard deviation.