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TDP-43 aggregation induced by oxidative stress causes global mitochondrial imbalance in ALS

Nature Structural & Molecular Biology volume 28, pages 132–142 (2021)Cite this article

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Abstract

Amyotrophic lateral sclerosis (ALS) was initially thought to be associated with oxidative stress when it was first linked to mutant superoxide dismutase 1 (SOD1). The subsequent discovery of ALS-linked genes functioning in RNA processing and proteostasis raised the question of how different biological pathways converge to cause the disease. Both familial and sporadic ALS are characterized by the aggregation of the essential DNA- and RNA-binding protein TDP-43, suggesting a central role in ALS etiology. Here we report that TDP-43 aggregation in neuronal cells of mouse and human origin causes sensitivity to oxidative stress. Aggregated TDP-43 sequesters specific microRNAs (miRNAs) and proteins, leading to increased levels of some proteins while functionally depleting others. Many of those functionally perturbed gene products are nuclear-genome-encoded mitochondrial proteins, and their dysregulation causes a global mitochondrial imbalance that augments oxidative stress. We propose that this stress−aggregation cycle may underlie ALS onset and progression.

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Fig. 1: Distinct gene expression programs induced by knockdown of TDP-43 and overexpression of an aggregation-prone TDP-43 C-terminal fragment.
Fig. 2: TDP-43 selectively binds a large subset of miRNAs in N2a cells.
Fig. 3: Functional impact of TDP-43-sequestered miRNAs.
Fig. 4: Effects of ALS-linked mutations and oxidative stress on TDP-43 aggregation.
Fig. 5: Oxidative stress−induced TDP-43 aggregation.
Fig. 6: TDP-43 co-aggregation of RBPs and nDNA-encoded mitochondrial proteins.
Fig. 7: Mitochondrial imbalance induced by TDP-43 proteinopathy.

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Data availability

All deep sequencing data from this study have been deposited in the Gene Expression Omnibus (GEO) under series accession number GSE126801. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE80 partner repository with the dataset identifier PXD021880. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (91640115, 31670827, and 31871316 to Y.Z.; 31770833 and 31570779 to Y. Liang), and the Ministry of Science and Technology of China (2017YFA0504400 to Y.Z.; 2017YFA0504600 to X. Zhang). We thank W. Xu and Y. Liang (Wuhan University) for providing the Sarkosyl reagent and the related protocol. Part of the computation in this work was done on the supercomputing system in the Supercomputing Center of Wuhan University.

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  1. These authors contributed equally: Xinxin Zuo, Jie Zhou, Yinming Li.

Authors and Affiliations

  1. State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China

    Xinxin Zuo, Jie Zhou, Yinming Li, Kai Wu, Zonggui Chen, Yi Liang & Yu Zhou

  2. Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China

    Xinxin Zuo, Yinming Li, Kai Wu & Yu Zhou

  3. Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Zhiwei Luo & Miguel A. Esteban

  4. Key Laboratory for Nucleic Acid Research, Institute of Biophysics, Chinese Academy of Science, Beijing, China

    Xiaorong Zhang

  5. Department of Cellular and Molecular Medicine, Institute of Genomic Medicine, University of California, San Diego, USA

    Xiang-Dong Fu

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Contributions

X. Zuo, X.-D.F. and Y.Z. designed the experiments. X. Zuo and Y. Li carried out the majority of the experiments. J.Z., Z.C. and Y.Z. analyzed the sequencing data. K.W. performed mass spectrometric analysis. X. Zhang and Y. Liang contributed to critical experimental information. M.A.E. and Z.L. provided sodium arsenite and human iPSCs with related protocols. X. Zuo, Y.Z. and X.-D.F. wrote the paper. All authors discussed the results and approved the manuscript.

Corresponding authors

Correspondence to Yu Zhou or Xiang-Dong Fu.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Defne Amado and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Transcriptomic comparison between TDP-43 knockdown and expression of the aggregation-prone CTF35.

a, Confirmation of the knockdown effect of multiple siRNAs against TDP-43 in N2a cells by Western blotting analysis. GAPDH served as loading control. The targeting regions in mouse TDP-43 are indicated with individual siRNAs. Note that siTDP-43-2290 corresponds to the siRNA against TDP-43-3′UTR. For RNA-seq analysis, total RNAs were extracted from N2a cells transfected with siTDP-43-1078 or control siRNA. b, Expression of wt TDP-43 or CTF35 in N2a cells depleted of endogenous TDP-43, analyzed by Western blotting. c, Reproducibility of duplicated RNA-seq libraries from siNC-treated (left) or siTDP-43 treated (right) N2a cells. d, Reproducibility of duplicated RNA-seq libraries from TDP-43 depleted N2a cells complemented with either wt full-length TDP-43 (left) or with CTF35 (right). e, Percentages of commonly affected genes in siTDP-43 treated N2a cells relative to three different sources of ALS patients. For a and b, a representative example of three independent experiments is shown. Uncropped images for panels a and b are available as source data online.

Source data

Extended Data Fig. 2 Characterization of TDP-43 and miRNA interactions.

a, Western blotting analysis of TDP-43 immunoprecipitation in N2a cells by probing TDP-43, Ago2 and GAPDH. b, Reproducibility of duplicated small RNA-seq libraries generated with total input (left) or TDP-43 IPed (right) miRNAs. c, Profile of total miRNAs versus those enriched with TDP-43 IP. Heat map show the expression miRNAs levels (left) and fold-enrichment compared to Input (right). d, Mass spectrometric analysis of proteins pulled down with biotin-labeled let-7c, biotin-labeled control RNA and naked beads without RNA bait. The data are presented in a triangular plot with yellow dots highlighting let-7c enriched proteins that include TDP-43 (top), which was further validated by Western blotting (bottom). e, RT-qPCR analysis of miRNAs enriched by myc-tag IP from N2a cells expressing myc-tagged full-length TDP-43 versus RRM1 and 2-deleted mutant. Red and Gray: target and non-target miRNAs analyzed. f, Biotin-let-7c capture of individually expressed wt and RRM1 or RRM2 deleted TDP-43 in N2a cells. Data in e (lower panel) are shown as mean ± s.d. (n = 3 independent experiments). For western blot in a and d-f, a representative example of three independent experiments is shown. Data for graph in e are available as source data online. Uncropped images for panels a and d-f are available as source data online.

Source data

Extended Data Fig. 3 TDP-43 modulation of miRNA target gene expression.

a, Western blotting analysis of IPed Ago2 under mock-treated (left) and TDP-43 overexpression (right) conditions. GAPDH served as loading control. b, RT-qPCR analysis of the expression of targets for TDP-43 bound miRNAs (red) and for non-bound miRNA (gray) in response to TDP-43 depletion. c, Dual luciferase reporter assay on individual target reporters. In each, the MRE sequence is shown with the seed region base-paired with the miRNA. Three copies of each MRE was inserted in each reporter. Corresponding to each wt reporter is the mutant one in which the seed region was mutated. Each pair of reporters was assayed in response to specific miRNA mimics or inhibitors. d, and e, Cumulative distribution plots of siTDP-43 induced expression changes among up-regulated genes with or without predicted target sites for TDP-43 sequestrated miRNAs (d), and of siTDP-43 induced expression changes among down-regulated genes with or without predicted target sites for TDP-43 non-bound miRNAs (e). Different colored lines indicate non-targets (gray), all predicted targets (blue), genes that contain more than one miRNA target (orange), and genes that contain more than two predicted miRNA target sites (red). The significance for each shift from non-target genes is indicated by P value determined by Wilcoxon signed-rank test. f, Boxplots of TDP-43 depletion-induced expression changes for genes with (n = 9,217) or without (n = 2,140) TDP-43 binding peaks. g, Boxplots of expression for genes with (n = 3,974) or without (n = 2,140) TDP-43 binding peaks in 3′UTRs. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are outlying points that are plotted individually. Data in b and c are presented as mean ± s.d. (n = 3 independent experiments). For b, c, f, and g, the P values are based on a two-tailed unpaired t test: *p < 0.05, **p < 0.01, ***p < 0.001; N.S., non-significant. For a, a representative example of three independent experiments is shown. Data for graphs in b and c are available as source data online. Uncropped images for panels a are available as source data online.

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Extended Data Fig. 4 Characterization of isolated and induced neurons.

a, Western blotting analysis of isolated primary mouse neurons and N2a cells by probing both pan-neuron (Tuj1) and mature neuron (SYN1 and MAP2) markers. The non-neuronal MEF cells served as control. b, Immunostaining with Tuj1 (red) and SYN1 (green) on isolated primary mouse neurons. Scale bar: 100 μm. c,d,e, Characterization of iPSCs with the stem cell markers Oct-4A and SOX2 (c), induced NPCs with Nestin, Vimentin, SOX2, and Pax6 (d), and differentiated neurons with Tuj1, SYN1, and MAP2 (e). Scale bar: 100 μm. For a-e, a representative example of three independent experiments is shown. Uncropped images for panel a are available as source data online.

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Extended Data Fig. 5 Partial purification of TDP-43 aggregates for global analysis.

a, The fractionation scheme for purifying TDP-43 aggregates. b, and c, Western blotting analysis of TDP-43 in N2a cells in response to sodium arsenite (b) and heat shock (c). d, and e, Western blotting analysis of TDP-43 in soluble and pellet fractions of two indicated neuronal (d) and two non-neuronal (e) cells. (f) Reproducibility of duplicated small RNA-seq on total (left) versus pellet fraction (right) miRNAs. g, RT-qPCR analysis of TDP-43 target and non-target miRNAs in the pellet. Data in g are shown as mean ± s.d. (n = 3 independent experiments). For b-e, a representative example of three independent experiments is shown. Data for graph in g are available as source data online. Uncropped images for panels b-e are available as source data online.

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Extended Data Fig. 6 Analysis of the proteome in H2O2-induced TDP-43 pellet.

a, SDS-PAGE analysis of total versus pellet from mock-treated and H2O2-treated N2a cells. Proteins were stained with Coomassie brilliant blue. b, Western blotting validation of specific nDNA-encoded mitochondrial proteins in H2O2-induced pellet in N2a cells. c, and d, Immunofluorescence of N2a cells treated with DMSO or H2O2 and stained with antibodies against TDP-43 C-terminal, representative RBPs hnRNP A0 and hnRNP M (c), and against TDP-43 C-terminal, representative mitochondrial proteins LRPPRC and ATP5b (d). Arrows indicate colocalization of TDP-43 with indicated proteins in granules. Scale bar: 10 μm. e, The STRING DATABASE was used to connect the proteins identified in H2O2-induced pellet using a cutoff score > 0.15. The resulting protein interaction network contains 69 nodes and 456 non-redundant edges. Of these, 5 proteins do not interact with any other proteins, and 3 other proteins cannot be recognized by STRING. For panel a, a representative example of five independent experiments is shown. For b-d, a representative example of three independent experiments is shown. Uncropped images for panel b are available as source data online.

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Extended Data Fig. 7 Mimicking mitochondrial imbalance by RNAi and overexpression.

a, Overexpression of myc-tagged CTF35 in N2a cells, validated with the anti-myc antibody. GAPDH served as loading control. b, Western blotting validation of the siRNA effects on indicated nDNA-encoded mitochondrial proteins in N2a cells. c, Western blotting validation of the efficacy and specificity in double siRNA-treated N2a cells. d, and e, Western blotting of single and double expression of indicated nDNA-encoded mitochondrial proteins. f, Western blotting analysis of the effect of combinatory siRNA treatment (LRPPRC) and overexpression (Bcl2l2) of separate nDNA-encoded mitochondrial proteins in N2a cells. g, Quantification of ROS production upon knockdown of two representative “up-regulated” genes (Fam173b and Timm9; ATP5b as a positive control) or overexpression of two representative “down-regulated” genes (Ndufa9 and ATP5C1; Bcl2l2 as a positive control). Data in g were presented as mean ± s.d. (n = 3 independent experiments). The P values are based on a two-tailed unpaired t test: **p < 0.01, ***p < 0.001. For a-f and g (lower panel), a representative example of three independent experiments is shown. Data for graphs in g are available as source data online. Uncropped images for panels a-f and g are available as source data online.

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Supplementary information

Reporting Summary

Supplementary Table 1

Differentially expressed genes in TDP-43-knockdown and CTF35-overexpression N2a cells.

Supplementary Table 2

TDP-43 RIP-enriched miRNAs and a subset of enriched miRNAs contributing to the shift of downregulated targets.

Supplementary Table 3

Enriched miRNAs in H2O2-induced TDP-43 pellet.

Supplementary Table 4

Enriched proteins in H2O2-induced TDP-43 pellet.

Supplementary Table 5

Mitochondrial proteins co-aggregated with TDP-43 and upregulated mitochondrial genes induced by CTF35 overexpression.

Supplementary Table 6

Primers for vector construction and small RNA oligos.

Supplementary Table 7

Primers for quantifying miRNA expression.

Supplementary Table 8

Oligos used for small RNA library preparation.

Supplementary Table 9

Modified RNA probes.

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Zuo, X., Zhou, J., Li, Y. et al. TDP-43 aggregation induced by oxidative stress causes global mitochondrial imbalance in ALS. Nat Struct Mol Biol 28, 132–142 (2021). https://doi.org/10.1038/s41594-020-00537-7

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