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Mitochondrial respiratory complex IV deficiency recapitulates amyotrophic lateral sclerosis

Nature Neuroscience volume 28, pages 748–756 (2025)Cite this article

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An Author Correction to this article was published on 21 March 2025

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Abstract

Amyotrophic lateral sclerosis (ALS) is categorized into ~10% familial and ~90% sporadic cases. While familial ALS is caused by mutations in many genes of diverse functions, the underlying pathogenic mechanisms of ALS, especially in sporadic ALS (sALS), are largely unknown. Notably, about half of the cases with sALS showed defects in mitochondrial respiratory complex IV (CIV). To determine the causal role of this defect in ALS, we used transcription activator-like effector-based mitochondrial genome editing to introduce mutations in CIV subunits in rat neurons. Our results demonstrate that neuronal CIV deficiency is sufficient to cause a number of ALS-like phenotypes, including cytosolic TAR DNA-binding protein 43 redistribution, selective motor neuron loss and paralysis. These results highlight CIV deficiency as a potential cause of sALS and shed light on the specific vulnerability of motor neurons, marking an important advance in understanding and therapeutic development of sALS.

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Fig. 1: Defective CIV in patients with ALS.
Fig. 2: Neuronal targeting of respiratory chain CIV leads to ALS in rats.
Fig. 3: Motor-related neuronal deficits in neuronal CIV-deficient rats.
Fig. 4: Denervation and skeletal muscle atrophy in neuronal CIV-deficient rats.
Fig. 5: COXIII deficiency leads to increased necrosis, neuroinflammation, TDP-43 cytosolic distribution and impaired mitochondrial functions.

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Data availability

All raw RNA-seq and snRNA-seq data can be accessed via the Gene Expression Omnibus (accession nos. GSE252875 and GSE275999). Processed gene expression count matrices have been deposited with Zenodo62. Proteomics data have been uploaded to the PRIDE database under accession no. PXD058902. Source data are provided with this paper.

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Acknowledgements

We thank J. Lin, P. Guo, Y. Yu and J. Qiu for critical comments. We acknowledge the financial support provided by the Ministry of Science and Technology of China (grant nos. 2019YFA0508701 and 2022YFA1303300 to X.R.Z.), the National Natural Science Foundation of China (no. 82450106 to X.R.Z), the Haihe Laboratory of Cell Ecosystem Innovation Fund (no. HH23KYZX0003 to X.R.Z. and no. HH24KYZX0007 to Y.W.M.), the National Key R&D Program of China (no. 2022YEF0203200 to D.L.), the CAMS Innovation Fund for Medical Sciences (grant no. 2021-I2M-1-024 to Y.W.M. and grant no. 2022-I2M-1-020 to D.L.) and the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (no. 2023-PT180-01 to Y.W.M.).

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Author notes

  1. These authors contributed equally: Man Cheng, Dan Lu, Kexin Li, Yan Wang, Xiwen Tong, Xiaolong Qi, Chuanzhu Yan.

Authors and Affiliations

  1. State Key Laboratory of Experimental Hematology, Haihe Laboratory of Cell Ecosystem, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

    Man Cheng, Kexin Li, Yan Wang & Xiaorong Zhang

  2. State Key Laboratory of Respiratory Health and Multimorbidity, National Center of Technology Innovation for Animal Model and National Human Diseases Animal Model Resource Center, NHC Key Laboratory of Comparative Medicine, Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Dan Lu, Xiaolong Qi, Huijiao Lv, Xu Zhang, Keru Li, Yaheng Wang & Yuanwu Ma

  3. Key Laboratory for Nucleic Acid Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Xiwen Tong, Wei Wang, Jian Zhang, Jingyu Feng, Panpan Wei, Qiushuang Li & Xiaorong Zhang

  4. Research Institute of Neuromuscular and Neurodegenerative Diseases and Department of Neurology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China

    Chuanzhu Yan & Kunqian Ji

  5. Department of Neurology, Xiangya Hospital, Central South University, National Regional Center for Neurological Diseases, Nanchang, China

    Junlin Wang

  6. Medical Primate Research Center and Neuroscience Center, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Beijing, China

    Huijiao Lv, Weining Kong, Jiaxin Ma & Yuanwu Ma

  7. Department of Neurobiology of the First Affiliated Hospital, Institute of Translational Medicine, School of Medicine, MOE Frontier Science Center for Brain Research and Brain-Machine Integration, Zhejiang University, Hangzhou, China

    Chengyong Shen

  8. Westlake Laboratory of Life Sciences and Biomedicine, School of Life Sciences, Westlake University, Hangzhou, China

    Xiang-Dong Fu

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Contributions

X.R.Z. and Y.W.M. planned the studies and analyzed all the data. M.C., D.L. and Y.W. performed all the experiments except for the bioinformatics analysis. X.W.T. and Y.W. performed all the RNA-seq and proteomics. K.X.L. and J.Z. performed the bioinformatics analysis. X.L.Q. carried out the gene editing of rats. C.Z.Y., K.Q.J. and W.W. performed the patient studies. J.L.W. provided the blood sequencing data for the patients with ALS. J.Y.F. performed the electromyography analysis. X.Z., W.N.K., J.X.M. and H.J.L. generated the mutant rats and performed the rat genotyping. J.X.M., K.R.L. and Y.H.W. conducted the animal behavior tests. P.P.W. and Q.S.L. performed the BN-PAGE analysis. C.Y.S. conducted the human brain analyses. X.R.Z. and X.-D.F. wrote the paper. All authors discussed the paper.

Corresponding authors

Correspondence to Yuanwu Ma or Xiaorong Zhang.

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Nature Neuroscience thanks Xiongwei Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Defective CIV in ALS patients.

a, Three representative sALS patients with frameshift mutations in their COXI and COXIII subunits. Patient 2 carried a 6289 C insertion (as illustrated) besides a 6692 A deletion as in patient 1 (not shown). Patient 3 carried a 9487 T deletion. b, Section of formalin-fixed cerebellar tissues stained for COXI from six non-demented controls, four C9-ALS/FTD, and two sALS patients. Scale bar, 50 μm. c, Section of formalin-fixed cerebellar tissues stained for COXII from seven non-demented controls, five C9-ALS/FTD, and two sALS patients. Scale bar, 50 μm.

Extended Data Fig. 2 Neuronal-specific targeting of mtDNA.

a, Illustration of the strategy for generating neuronal-specific COXII and COXIII mutant rats. Crossing COXIII/IILSL rats with NeuN-Cre rats results in DdCBE expression in mature neurons to target mtCOXs. The conversion of the targeted nucleotide to ‘TAA’ creates a premature termination codon for COXII or COXIII. LSL: loxp-stop-loxp, MTS: mitochondrial targeted sequence. b, Design overview of DdCBEs for targeting rat COXI G5396, COXII C7021 and COXIII G8645. The green text highlights the mitochondrial sequences targeted by the base editors for specific editing. c, Onset and progression of disease phenotype with COXIILSL/COXIINeuN-Cre, and COXIIILSL/COXIIINeuN-Cre rats. Data are presented as mean ± SEM. The statistical test used was Log-rank test. d, The survival curve of COXILSL/COXINeuN-Cre, COXIILSL/COXIINeuN-Cre and COXIIILSL/COXIIINeuN-Cre rats. The statistical test used was Log-rank test. e, Design overview of DdCBEs for targeting rat ND1 G2996, ATP8 G7783. The green text highlights the mitochondrial sequences targeted by the base editors for specific editing. f, Survival curves of ND1LSL/ND1NeuN-Cre and ATP8LSL/ATP8NeuN-Cre rats. Data are presented as mean ± SEM. The statistical test used was Log-rank test.

Source data

Extended Data Fig. 3 Characterization of neuronal-specific targeting of COXII/III.

a-b, DdCBE-mediated G-to-A conversion frequency at COXII C7021 and COXIII G8645 analyzed by deep sequencing. n = 3 rats/group. Data were presented as means ± SEM. c, DdCBE-mediated G-to-A conversion frequency at COXIII G8645 analyzed by deep sequencing. n = 3 rats. Data were presented as means ± SEM. d, BN-PAGE followed by Western blotting analysis to analyze the abundance of complex II, V and IV in the brain of COXIIILSL/COXIIINeuN-Cre rats, n = 3 rats/group. e, SDS-PAGE followed by Western blotting to analyze the protein level of CIV subunits in the brain, n = 3 rats/group. f, BN-PAGE followed by Western blotting to analyze the abundance of complex II, V and IV in thoracic spinal cord of COXIIILSL/COXIIINeuN-Cre rats, n = 3 rats/group. g-h, SDS-PAGE followed by Western blotting to analyze the protein level of CIV subunits in the thoracic spinal cord and lumbar spinal cord of COXIIILSL/COXIIINeuN-Cre rats, n = 3 rats/group. i, SDS-PAGE followed by Western blotting to analyze the protein level of CIV subunits in the lumbar spinal cord of COXIILSL/COXIINeuN-Cre rats, n = 3 rats/group. j-k, Body weight changes in male and female COXIILSL/COXIINeuN-Cre and COXIIILSL/COXIIINeuN-Cre rats. Data are presented as mean ± SEM. The statistical test used was Log-rank test.

Source data

Extended Data Fig. 4 Motor-related behavior of neuronal CIV deficient rats.

a-b, Behavioral tests conducted on COXIIILSL and COXIIINeuN-Cre rats to evaluate the locomotor ability at the age of 3 weeks and 1 month. Data are presented as mean ± SEM. The statistical test performed was a two-sided unpaired Student’s t-test.

Source data

Extended Data Fig. 5 Deficient COXII or COXIII impairs spinal cord structures in adult rats.

a-b, Electron microscopic images of the ventral lumbar roots in comparison with dorsal roots from COXIIINeuN-Cre/ COXIIILSL and COXIINeuN-Cre/COXIILSL rats at the end-stage disease. Scale bars: (a) Ventral root 10 µm and 2 µm, Dorsal root 5 µm and 1 µm (b) Ventral root 10 µm and 2 µm, Dorsal root 5 µm and 2 µm. All experiments were performed independently three times with similar results.

Extended Data Fig. 6 Denervation and atrophy of skeletal muscle in neuronal specific COXII deficient rats.

a-b, Representative images of gastrocnemius (GA) muscle and tibialis anterior (TA) muscle of 5-week COXIILSL and COXIINeun-Cre rats and quantified the muscle weights are presented on the right. Scale bar, 1 cm. Data are presented as mean ± SEM. The statistical test performed was a two-sided unpaired Student’s t-test. c, Representative cross sections of WGA-stains transverse sections of tibialis anterior (TA) muscle in COXIILSL and COXIINeun-Cre rats. scale bar, 200 μm. All experiments were performed independently three times with similar results. d, Quantification of averages cross-section area (CSA) of tibialis. Data are presented as mean ± SEM. The statistical test performed was multiple unpaired Student’s t-tests. e, The representative image shows the neuromuscular junction (NMJ) in hindlimb skeletal muscles of COXIILSL and COXIINeuN-Cre. Motor endplates were stained with a-bungarotoxin to visualize AchR and SV2 was used to label neuromuscular synapses. Scale bar, 20 μm. All experiments were performed independently three times with similar results. f, Quantification of denervated neuromuscular junction (NMJ) in COXIINeuN-Cre rats. Data are presented as mean ± SEM. The statistical test performed was multiple unpaired Student’s t-tests. g-i, Representative images and quantification of CMAPs evoked by supramaximal stimulation of sciatic nerves and the quantification of MUNEs for COXIILSL and COXIINeuN-Cre. Data are presented as mean ± SEM. The statistical test performed was a two-sided unpaired Student’s t-test.

Source data

Extended Data Fig. 7 RNA-Seq and quantitative-mass spectrometry analysis of COXIIINeuN-Cre rats.

a, GO term enrichment analysis of significantly up-regulated genes in lumber spinal cord of COXIIINeuN-Cre rat. Padj<0.05 and |log2FC | >1. b & d, GSEA plots showing the enrichment profiles of cell death genes and immune response genes.Padj<0.05. c, Heatmap showing upregulated DEGs related to the cellular response to oxidative stress and downregulated DEGs related to neuroactive ligand receptor interactions in COXIIINeuN-Cre rats. padj<0.05 and |log2FC | >1. e-f, Volcano plots showing the proteins with significantly altered abundance in lumbar spinal cord of COXIIINeuN-Cre rats relative to scrambled controls. P < 0.05 and |log2FC | >1.

Extended Data Fig. 8 ATP and development progression analysis of COXIIINeuN-Cre rats.

a-b,The representative image shows the TDP-43 at 2, 4 and 6 weeks in spinal cords of COXIIILSL and COXIIINeuN-Cre rats. Scale bar, 20 μm. c, ATP production in lumber spinal cord of COXIIILSL and COXIIINeuN-Cre rats. Data are presented as mean ± SEM. The statistical test performed was a two-sided unpaired Student’s t-test. d, Representative confocal fluorescence microscopic analysis showing the expression of GFAP and IBA1 in the lumbar spinal cord of COXIIILSL and COXIIINeuN-Cre rats. All experiments were performed independently three times with similar results.Scale bar, 500 μm. e, Electron microscopic images showing demyelination in the lumbar spinal cord. All experiments were performed independently three times with similar results. Scale bar, 2 μm. f, The temporal map of development progression in ALS rat model.

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Extended Data Fig. 9 Changes in ALS-related proteins of COXIINeuN-Cre and COXIIINeuN-Cre rats.

a-b, SDS-PAGE followed by Western blotting to analyze the protein level of ALS-related protein in COXIINeuN-Cre and COXIIINeuN-Cre rats, n = 3 rats/group.

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Supplementary information

Supplementary Information (download PDF )

Supplementary Tables 1 and 2. Table 1: Human sample information. Table 2: Primers used for genotyping, Sanger sequencing, deep sequencing and qPCR.

Reporting Summary (download PDF )

Supplementary Video 1 (download MP4 )

Video for COXIINeuN-Cre rat and COXIILSL control rat at 3 weeks of age in a horizontal view angle.

Supplementary Video 2 (download MP4 )

Video for COXIINeuN-Cre rat and COXIILSL control rat at 1 month of age in a horizontal view angle.

Supplementary Video 3 (download MP4 )

Video for COXIINeuN-Cre rat and COXIILSL control rat at 1.5 month of age in a horizontal view angle.

Supplementary Video 4 (download MP4 )

Video for COXIIINeuN-Cre rat and COXIIILSL control rat at 3 weeks of age in a horizontal view angle.

Supplementary Video 5 (download MP4 )

Video for COXIIINeuN-Cre rat and COXIIILSL control rat at 1 month of age in a horizontal view angle.

Supplementary Video 6 (download MP4 )

Video for COXIIINeuN-Cre rat and COXIIILSL control rat at 2 months of age in a horizontal view angle.

Supplementary Video 7 (download MP4 )

Video for COXINeuN-Cre rat and COXILSL control rat at 2 weeks of age in a vertical view angle.

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Source Data Figs. 1–5 and Extended Data Figs. 2–4, 6, 8 and 9 (download ZIP )

Source data files for the main and extended data figures.

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Cheng, M., Lu, D., Li, K. et al. Mitochondrial respiratory complex IV deficiency recapitulates amyotrophic lateral sclerosis. Nat Neurosci 28, 748–756 (2025). https://doi.org/10.1038/s41593-025-01896-4

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