Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditis elegans

Ruofeng Tang^{a, b, c},
Xin Wang^c,
Junxiang Zhou^{a, b, c},
Fengxia Zhang^d,
Shan Zhao^c,
Qiwen Gan^{a, b, c},
Liyuan Zhao^{a, b},
Fengyang Wang^{a, b},
Qian Zhang^{a, b},
Jie Zhang^c,
Guodong Wang^d,
Chonglin Yang^{a
, ,}

a.
State Key Laboratory of Natural Resource Conservation and Utilization in Yunnan, Center for Life Science, School of Life Sciences, Yunnan University, Kunming, 650021, China
b.
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
c.
Graduate University of Chinese Academy of Sciences, Beijing, 100049, China
d.
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

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Corresponding author: E-mail address: clyang@ynu.edu.cn (Chonglin Yang)

摘要

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Abstract: Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol, defects in which may lead to hyperargininemia, a devastating developmental disorder. It is largely unknown if defective arginine catabolism has any effects on mitochondria. Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditis elegans. Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate (ATP) production in C. elegans hypodermal cells. ARGN-1 localizes to mitochondria and its loss causes arginine accumulation, which disrupts mitochondrial dynamics. Heterologous expression of human ARG1 or ARG2 rescued the mitochondrial defects of argn-1 mutants. Importantly, genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations. These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.
- Arginine /
- Arginase /
- Hyperargininemia /
- Mitochondrial homeostasis

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Fig. 1. Loss of argn-1 causes abnormal enlargement of mitochondria in C. elegans. A: Representative images of Mito-GFP-labeled structures in the hypodermis of N2, argn-1(yq187), and argn-1(gk315316) animals carrying yqIs157 at the indicated developmental stages. Scale bars, 5 μm. B: Quantification of animals with abnormally enlarged mitochondria (area ≥12 μm²) as shown in (A). Fifty animals were scored for each genotype. Comparisons are between N2 and mutants. Error bars represent SEM. ^∗∗∗, P < 0.001.C: Representative fluorescence and DIC (differential interference contrast) images of mtLS::GFP-labeled structures in muscle and intestinal cells in the indicated animals carrying hqIs181. Scale bars, 5 μm. D: Quantification of animals with abnormally enlarged mitochondria (area ≥12 μm²) as shown in (C). Fifty animals were scored for each genotype. Error bars represent SEM. NS, no statistical significance. E: Representative images of mitochondria labeled with F54A3.5::GFP and TOMM-20::mCh in the hypodermis of animals with the indicated genotypes. Scale bars, 5 μm. F: Representative TEM images of mitochondria in the hypodermis in adult animals with the indicated genotypes. Scale bars, 1 μm. G: Schematic diagram of the argn-1 gene. Black boxes represent exons, and thin lines indicate introns. The point mutations of argn-1 are indicated with asterisks. H: Comparison of C. elegans ARGN-1 with human ARG1 and ARG2. The point mutations in ARGN-1 are indicated with asterisks. MTS, mitochondrion-targeting sequence. I: Graphic description of the arginase-mediated arginine degradation pathway.

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Fig. 2. Mutations in argn-1 lead to mitochondrial abnormalities. A: The expression pattern of Pgfp in embryos and adult worms. Scale bars, 5 μm. B–E: Images of the rescuing effects on argn-1(yq187) mitochondria of ectopically expressed ARGN-1::mCh (B), ARGN-1(G16E)::mCh (C), ARGN-1(D125A/H127A)::mCh (D), and ARGN-1(D229A/D231A)::mCh (E). Scale bars, 5 μm. F: Quantification of animals with abnormally enlarged mitochondria (area ≥12 μm²) as shown in (B–E). Fifty animals were scored for each genotype. Error bars represent SEM. ^∗∗∗, P < 0.001; NS, no statistical significance.

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Fig. 3. Loss of slc-25A29 suppresses the mitochondrial enlargement in argn-1(yq187) mutants. A: Schematic diagrams of arginine metabolism pathways in different tissues of mammals and C. elegans. The inner boxes represent mitochondria. Arg, arginine; Orn: ornithine; Cit, citrilline; Glu, glutamate; Gln, glutamine; Pro, proline; CP, carbamyl phosphate; P5C, pyrroline-5-carboxylate; ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; OTC, ornithine carbamoyltransferase; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; CPS1, carbamoyl phosphate synthetase I; ARG1/2, arginase1/2; ALH, aldehyde dehydrogenase.B–D: Fold change of arginine (B), lysine (C) and ornithine (D) levels in animals with the indicated genotypes. Data (mean ± SEM) were derived from three independent experiments and normalized to arginine, lysine or ornithine intensities in N2 animals. ^∗, P < 0.05;^∗∗, P < 0.01;^∗∗∗, P < 0.001; NS, no statistical significance.E: Representative images of mitochondria in the hypodermis of N2 and argn-1(yq187) animals with control RNAi or slc-25A29 RNAi treatment. Scale bars, 5 μm. F: Representative images of mitochondria in the hypodermis of N2, argn-1(yq187), oatr-1(tm5454) and oatr-1(tm5454);argn-1(yq187) animals with control RNAi or odc-1 RNAi treatment. Scale bars, 5 μm.

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Fig. 4. Arginase functions are conserved between C. elegans and humans. A–G: Images of the rescuing effects on argn-1(yq187) mitochondria of ectopically expressed hARG1::mCh (A), MTS::hARG1::mCh (B), MTS::hARG1(T134I)::mCh (C), hARG2::mCh (D), hARG2(41–354)::mCh (E), MTS::hARG2(41–354)::mCh (F), and hARG2(T153I)::mCh (G). Scale bars, 5 μm. H: Quantification of animals with abnormally enlarged mitochondria (area ≥12 μm²) as shown in (A–G). Fifty animals were scored for each genotype. Error bars represent SEM. ^∗∗, P < 0.01;^∗∗∗, P < 0.001; NS, no statistical significance.

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Fig. 5. Mitochondrial dynamics is disrupted in argn-1 mutants. A–C: Time-lapse imaging of Mito-GFP-labeled mitochondria in the hypodermis of N2 (A), argn-1(yq187) (6–12 h post L4) (B) and argn-1(yq187) (>12 h post L4) (C) animals. White and red arrows indicate sites of mitochondrial fusion and fission, respectively. Mitochondria undergoing fusion or fission are indicated with dotted purple lines. Scale bars, 5 μm. D: Quantification of fusion and fission events in N2 and argn-1(yq187) animals. Error bars represent SEM. ^∗, P < 0.05; NS, no statistical significance.E: Representative images of mitochondria in the hypodermis of N2, argn-1(yq187), fzo-1(tm1133) and fzo-1(tm1133);argn-1(yq187) animals. Scale bars, 5 μm. F: Representative images of mitochondria in the hypodermis of control RNAi- or eat-3 RNAi-treated N2 and argn-1(yq187) animals. Scale bars, 5 μm.

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Fig. 6. Inactivation of slc-25A29 and slc-25A18.1 suppresses mitochondrial defects in argn-1(yq187) mutants. A: Images of mitochondria in the hypodermis of N2, argn-1(yq187), slc-25A29(yq261);argn-1(yq187) and slc-25A18.1(yq267);argn-1(yq187) animals. Scale bars, 5 μm. B: Quantification of animals with abnormally enlarged mitochondria (area ≥12 μm²) as shown in (A). Fifty animals were scored for each genotype. Error bars represent SEM. ^∗∗∗, P < 0.001.C: Relative ATP levels in the hypodermis of animals with the indicated genotypes. Data (mean ± SEM) are from three independent experiments and are normalized to the ATP level in N2 animals. Error bars represent SEM. ^∗, P < 0.05.D: Relative mitochondrial membrane potentials in the hypodermis of animals with the indicated genotypes. Data (mean ± SEM) are normalized to the mitochondrial membrane potential in N2 animals. Fifteen animals were scored for each genotype. Error bars represent SEM. ^∗∗∗, P < 0.001.E: Schematic diagram of the slc-25A29 gene and SLC-25A29 protein. Black boxes represent exons, and thin lines indicate introns. The point mutation of slc-25A29(yq261) is indicated with an asterisk. F: Schematic diagram of the slc-25A18.1 gene and SLC-25A18.1 protein. Black boxes represent exons, and thin lines indicate introns. The point mutation of slc-25A18.1(yq267) is indicated with an asterisk. G: Representative images of mitochondria in the hypodermis of N2, argn-1(yq187), slc-25A29(yq276), slc-25A29(yq276);argn-1(yq187) and control RNAi- or slc-25A18.1 RNAi-treated N2 and argn-1(yq187) animals. Scale bars, 5 μm. H: Images of the rescuing effects on argn-1(yq187);slc-25A18.1(yq267) mitochondria of ectopically expressed SLC-25A18.1::mCh. Scale bars, 5 μm. I: Representative images of mitochondria in the hypodermis of N2, argn-1(yq187), gdh-1(yq275), idh-2(ok3184), gdh-1(yq275);idh-2(ok3184), gdh-1(yq275);argn-1(yq187), idh-2(ok3184);argn-1(yq187), and gdh-1(yq275);idh-2(ok3184);argn-1(yq187) animals. Scale bars, 5 μm.

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