A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels

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In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe–S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

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We extend our thanks to the affected individuals and families who participated in this study. We thank the reviewers for their time and insightful comments. We thank T. M. Dunn, D. Miller, S. J. Hayflick, A. J. Kastaniotis, M. S. Paul and H. Chung for helpful discussion, H. Pan and W.-W. Lin for injections to create transgenic flies and J. Kim for MS analyses. We also thank A. J. Kastaniotis at the University of Oulu for sharing human fibroblasts and N. Perrimon at Harvard Medical School for sharing S2 cells. We thank F. Missirlis at the Center for Research and Advanced Studies of the National Polytechnic Institute for the UAS-Fer1HCH, UAS-Fer2LCH stock. We also thank the Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Center and the Kyoto Drosophila Genetic Resource Center and the Developmental Studies Hybridoma Bank from the University of Iowa for providing fly stocks and reagents. We acknowledge support from the Shan and Lee-Jun Wong fellowship of Baylor College of Medicine to D.D. We thank the Baylor College of Medicine Intellectual and Developmental Disabilities Research Center confocal microscopy core, supported by the National Institute of Child Health & Human Development (U54 HD083092). H.J.B. is supported by the NIH Common Fund through the Office of Strategic Coordination/Office of the NIH Director and the NINDS (U54NS093793), NIH/ORIP (24OD022005 and R24OD031447), is a recipient of the Chair of the Neurological Research Institute of Texas Children’s Hospital and is supported by the Huffington foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Paul C. Marcogliese

Present address: Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada

A full list of Undiagnosed Diseases Network members and their affiliations appears in the Supplementary Information.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

Debdeep Dutta, Oguz Kanca, Paul C. Marcogliese, Zhongyuan Zuo, Rishi V. Shridharan, Jun Hyoung Park, Guang Lin, Ming Ge, Benny A. Kaipparettu & Hugo J. Bellen

Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX, USA

Debdeep Dutta, Oguz Kanca, Paul C. Marcogliese, Zhongyuan Zuo, Rishi V. Shridharan, Guang Lin, Ming Ge & Hugo J. Bellen

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

Seul Kee Byeon & Akhilesh Pandey

Pediatric Neurology Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel

Gali Heimer

The Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Gali Heimer

Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA

Jennefer N. Kohler & Matthew T. Wheeler

Manipal Academy of Higher Education, Manipal, India

Akhilesh Pandey

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Conceptualization: D.D. and H.J.B. Investigation: D.D., O.K., P.C.M., S.K.B., Z.Z., J.H.P, R.V.S., G.L., J.N.K., M.T.W., G.H. and M.G. Resources: H.J.B., A.P. and B.A.K. Writing, original draft: D.D. and H.J.B. Writing, review and editing: D.D., O.K., P.C.M., G.L., S.K.B., J.N.K., M.T.W., J.H.P, R.V.S., A.P., B.A.K., G.H. and H.J.B. Funding acquisition: H.J.B. Supervision: H.J.B.

Correspondence to Hugo J. Bellen.

The authors declare no competing interests.

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(a) Mitochondrial fatty acid synthesis pathway and its products. Briefly, an acetyl and malonyl moiety are condensed to make a four carbon long keto-acyl species, which remains attached to the mitochondrial Acyl Carrier Protein (mtACP), a protein that holds the growing acyl chain during the fatty acid synthesis within the mitochondria. Subsequently, this four-carbon long keto-acyl ACP undergoes a reduction-dehydration-reduction cycle to produce a butyryl-ACP (C4). C4 enters into the cycle, and two carbons from the malonyl moiety are attached to the butyryl species to make an acyl chain of six carbon length. This cycle continues until the carbon length of the growing acyl chain reaches up to 16–18 carbon. (b) Schema showing the mecr mutants used in this study. (c) Schema showing the Primers used for quantitative real-time PCR and the graphs showing the relative levels of mecr transcripts in mecrTG4 mutants. Each dot represents data from three technical replicates. (d) Western blot showing lipoic acid (LA) levels linked to Muc (ortholog of yeast Lat1 and a component of PDH complex in fruit fly) and CG5214 (ortholog of yeast Kgd2 and a component of OGDH complex in fruit fly) in mecr mutants. (e) Effects of a transgene containing mecr-GFP (Fosmid clone, CBGtg9060C0290D)36 or of ubiquitous expression of HA-tagged fly mecr on mecrTG4 homozygous mutants. One-way ANOVA followed by a Tukey’s post-hoc test was performed for the statistical analyses. Error bars represent SEM (****p < 0.0001).

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(a) Protein alignments of Mecr proteins. Red boxes indicate the amino acid that are variant in MEPAN patients. Schema shows the relative position of the patient variants in MECR protein. (b) Effects of ubiquitous expression of human MECR variants when driven by da-GAL4 in mecr mutants. (c) Percentage of 15-day-old flies that are able to climb 8 cm within 30 seconds. Each dot represents the percentage of flies from three independent experiments. (d) Average time taken by 15-day-old flies to climb 8 cm. Each dot represents the time taken by one fly in each of the three experiments. n = 93 (MECRRef), n = 86 (MECRArg258Trp), n = 84 (MECRGly232Glu) flies. One-way ANOVA followed by a Tukey’s post-hoc test was performed for the statistical analyses. Error bars represent SEM (****p < 0.0001).

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(a, b) Western blot (a) and quantification (b) showing relative levels of Mecr protein upon neuronal knockdown (elav-GAL4) with two different RNAi lines at 25 °C. Each dot represents the data from three independent experiments. For statistical analyses, one-way ANOVA followed by a Dunnett’s multiple comparison test is carried out. Error bars represent SEM (**p < 0.01). (c) Lifespan of flies with neuronal knockdown of mecr. (d) Percentage of 25-day-old flies that can climb 8 cm within 30 seconds. Each dot represents the percentage of flies from three independent experiments. (e) Average time taken by 25-day-old flies to climb 8 cm. n = 128 (luci-RNAi) and n = 55 (mecr-RNAi) flies. For statistical analyses, two-tailed Student’s t-test is carried out. Error bars represent SEM (*p<0.05; ****p < 0.0001). (f–h) Quantification of ERG traces from 3–5 day-old flies. Each dot represents the data from one fly. n = 8 (Control and mecrA; GR), n = 10 (mecrA) flies. For statistical analyses, one-way ANOVA followed by a Tukey’s post-hoc test is carried out. Error bars represent SEM.

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(a) Expression of mCherry driven by mecrKG4 (mecrKozak-GAL4, where we replaced the coding region of the gene with a Kozak sequence followed by a GAL4 gene) in larval brains. Elav-positive cells are neurons and Repo-positive cells are glia. (b) Expression of mCherry driven by mecrKozak-GAL4 in the adult brain. Note that mecr expression is sparse in the adult brains. However, a few cells including the prospective medial neurons, which typically produce insulin-like peptides express it abundantly. Scale bar 50 µm. (c) Colocalization of Mecr-GFP and ATP5α in 3rd instar larval fatbody tissue. Scale bar 10 µm. (d) Colocalization of human MECR and ATP5α in S2 cells. Scale bar 3.5 µm. Immunostaining was performed using an antibody against human MECR protein and an antibody against ATP5α. All experiments were carried out at least twice.

(a) Pedigree of the two patients with MEPAN syndrome identified through Undiagnosed Diseases Network. (b) RNA-seq from blood showing reduced levels of MECR transcripts in both patients. (c–g) Relative phospholipids levels in MEPAN patient-derived fibroblasts compared to the parent-derived control fibroblasts: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylglycerol (PG). The dots represent values of technical replicates from one set of biological replicates. For statistical analyses one-way ANOVA followed by a Tukey’s post-hoc test are carried out. Error bars represent SEM (*p < 0.05) (h–l) Relative levels of different phospholipids in the mecrTG4 larvae compared to control. The dots represent values of technical replicates from one set of biological replicates (n = 350 2nd instar larva). For statistical analyses, two-tailed Student’s t-test are carried out. Error bars represent SEM (*p < 0.05; ***p < 0.001).

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(a) Graph showing the ceramide species in the mecrTG4 larvae. Data are presented as mean values +/− SEM (b) Graph showing the relative levels of ceramidephosphoethanolamine (CPE) in the mecrTG4 larvae. For statistical analyses, two-tailed Student’s t-test are carried out. Error bars represent SEM (***p < 0.001). For a and b, the dots represent values of technical replicates from one set of biological replicates (n = 350 2nd instar larva). (c) Graph showing the ceramide species in both patient fibroblasts. The dots represent values of technical replicates from one set of biological replicates. Data are presented as mean values +/− SEM.

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(a, b) Graphs showing the fold changes in different glucosylceramide species upon treatment with Desipramine and Deferiprone at 15- and 25-day timepoints. Each square indicates the average value of three technical replicates.

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(a) Relative activity of ETC complexes (CI-IV) in mecrTG4 mutants and controls. mecrTG4 mutant larvae display reduced activity of Complex-I, I+III, and IV and increased activity of Complex-II. Each dot represents data from three technical replicates (n = 150 2nd instar larva). For statistical analyses, two-tailed Student’s t test are carried out. Error bars represent SEM (**p < 0.01; ***p < 0.001****p < 0.0001). (b) Relative levels of ATP in fibroblasts from patients and parental control. Each dot represents the values from four experiments. For statistical analyses, one-way ANOVA followed by a Tukey’s post-hoc test are carried out. Error bars represent SEM (***p < 0.001). (c–e) Relative oxygen consumption rates in control and patient derived fibroblasts as measured by Seahorse analyses. (c) Basal respiration, (d) maximal respiration, and (e) spare respiratory capacity are reduced in the patient-derived fibroblasts compared to fibroblasts derived from parent control. Each dot represents the values of replicates in each well from one set of biological replicates. For statistical analyses one-way ANOVA followed by a Dunnett’s multiple comparisons test are carried out. Error bars represent SEM (*p < 0.05; **p < 0.01; ***p < 0.001). (f) Co-IP from one set of biological replicates shows the interaction between NFS1 and ISCU in the fibroblasts. After performing immunoprecipitation using Mouse Anti-ISCU antibody, the blot was probed for NFS1, stripped, and reblotted for ISCU.

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(a) Table showing MEPAN patients including mutations, symptoms, and ferritin levels in blood. Out of these six patients described in the table, one patient (Patient III) was described earlier by Heimer et al.10. The other five patients are newly identified individuals who have not been reported elsewhere. (b) Graph showing relative ATP levels in fibroblasts from patient III compared to the fibroblasts from unrelated controls. Each dot represents the values of replicates from three experiments. (c) Graph showing the relative iron levels in the fibroblasts from patient III. Each dot represents the values of replicates from three experiments. (d) Graph showing the relative aconitase activity in the fibroblasts from patient III. Each dot represents the values of replicates from three experiments. For statistical analyses, one-way ANOVA followed by a Tukey’s post-hoc test are carried out. Error bars represent SEM (**p < 0.01).

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(a, b) Average percentage (a) and climbing time (b) to reach 8 cm of 25-day-old flies with neuronal knockdown of mecr (elav-GAL4>mecr-RNAi) and expressing ferritins. n = 81 (luci-RNAi), n = 76 (mecr-RNAi), n = 74 (mecr-RNAi and Fer1HCH-Fer2LCH) flies. (c, d) Average percentage (c) and climbing time (d) of 25-day-old flies upon neuronal (by elav-GAL4) knockdown of mecr treated with and without low iron food as well as deferiprone. n = 62 (luci-RNAi), n = 155 (mecr-RNAi), n = 105 (mecr-RNAi with low iron food), n = 55 (mecr-RNAi with deferiprone) flies. For a and c, each dot represents the percentage of flies from at least three independent experiments. For b and d, each dot represents the time taken by one fly in at least three independent experiments. (e) Relative amount of iron in the untreated, desipramine and deferiprone-treated fly heads with neuronal knockdown of mecr. Each dot represents the values of replicates from three experiments each using 25 fly heads. (f) Co-IP shows the interaction between Nfs1 and Iscu in the fly heads with neuronal knockdown of mecr. One-way ANOVA followed by a Tukey’s post-hoc test is carried out for statistical analyses. Error bars represent SEM (***p < 0.001; ****p < 0.0001).

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Undiagnosed Disease Network Consortia member list and Supplementary Fig. 1 and Table 1.

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Dutta, D., Kanca, O., Byeon, S.K. et al. A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels. Nat Metab (2023). https://doi.org/10.1038/s42255-023-00873-0

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Received: 30 October 2022

Accepted: 21 July 2023

Published: 31 August 2023

DOI: https://doi.org/10.1038/s42255-023-00873-0

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