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Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. Here, we applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1G93A mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

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Fig. 1: Differentiation of PHOX2B::GFP+ and HB9::GFP+ neurons.
Fig. 2: Genome-wide RNA-seq analysis reveals aberrant lipid metabolism after comparison between post-sorted HB9::GFP+ and PHOX2B::GFP+ cells in SOD1A4V and C9ORF72 ALS lines.
Fig. 3: Metabolomics analysis indicates upregulation of lipid metabolism in post-sorted HB9::GFP+ cells of SOD1A4V and C9ORF72 ALS lines.
Fig. 4: Metabolomics analysis in unsorted sMN differentiation confirms upregulation of lipid metabolism and provides lipid-related metabolic candidates in TDP43Q343R, C9ORF72, SOD1A4V and sporadic ALS lines.
Fig. 5: 5-LOX inhibitors rescue motor neuron degeneration in vitro.
Fig. 6: 5-LOX inhibitors rescue the phenotype of Drosophila model.
Fig. 7: Caffeic acid alleviates disease pathogenesis in SOD1G93A mice.

Data availability

The raw bulk RNA-seq data (Figs. 2 and 3d, Extended Data Figs. 4 and 5 and Supplementary Fig. 4) are available at the Gene Expression Omnibus under accession codes GSE132972 and GSE173115. Source data are provided with this paper.

Code availability

The R code used to analyze RNA-seq datasets is available from the corresponding authors on reasonable request.

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Acknowledgements

The authors thank H. Zhang at the Flow Cytometry Core Facility (JHSPH) and H. Hao and C. Talbot at the Deep Sequencing Core Facility (JHU) for RNA-seq data analysis. We also thank the Developmental Studies Hybridoma Bank for antibodies and K. Eggan (Harvard university) for sharing the isogenic control line. This work was supported by grants from the Donald E. & Delia B. Baxter Foundation (to H.E.), Department of Molecular Microbiology and Immunology KSOM, USC (to H.E.), Robert Packard Center for ALS Research (to G.L.), the (R01NS093213 to G.L.), the Global Research Development Center Program from the National Research Foundation (NRF) of Korea (2017K1A4A3014959 to G.L.), NIH/NINDS (1R01NS117604-01 to N.J.M., Basic Research Program of the NRF (2021R1A2C1007793 to Y.B.H) and the Medical Research Center program (2016R1A5A2007009 to Y.B.H.) from the NRF.

Author information

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Authors

Contributions

H.L. performed the experiments, analyzed the data and wrote the manuscript with input from all authors. J.J.L., N.Y.P., S.D., T.K., K.R., S.B.L., S.-H.P., S.H., I.K., K.-t.K., S.K. and Y.O. performed the experiments and/or analyzed the data. Y.O., H.K., S.-U.K., M.-R.S., T.E.L., N.J.M., Y.B.H. and H.E. contributed to the design, supervised the study, and aided in analysis and interpretation of data. G.L. designed the study, supervised the study/experiments, data analysis and interpretation and wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Young Bin Hong, Hyungjin Eoh or Gabsang Lee.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of transcripts in hiPSC-derived PHOX2B::GFP+ oMN-like cells.

(A-C′) Characterization of post-sorted PHOX2B::GFP+ cells using ISL1, NKX6.1 and PHOX2B (red), and TUJ1 (green) antibodies. (D-I) qRT-PCR analysis shows enrichments of oMN specification transcripts (ISL1, PHOX2A, NKX6.1 and CHAT) and midbrain regional transcript (EN1), but not mDA specification transcript (NURR1) after sorting (D14) (n=10 for each group from 3 independent batches; technical replicates; n.s.: not significant, ****P<0.0001 (p-value:<0.0001), two-tailed; unpaired student’s t-test). (J) Heatmap presents characteristic marker expression of ES (OCT4::GFP), oMN-like (PHOX2B::GFP), sympathetic autonomic neuron (PHOX2B::GFP), mDA (unsorting) and sMN (unsorting) (n=3 for each group, n means independent batches of differentiation; technical replicates). (K-L) Different schematic protocols to optimize oMN-like cell differentiation (K) and FACS results of PHOX2B::GFP+ (L) (n=3 for each groups (3 independent differentiation) from 1 batch; technical replicates; n.s.: not significant, **P<0.01 (0.0029), two-tailed; unpaired student’s t-test). Scale bar: 100 μm, Error bars, mean ± SEM., PHX2B: PHOX2B.

Source data

Extended Data Fig. 2 Generation of PHOX2B::GFP reporter line and oMN-like cell specification in SOD1A4V and C9ORF72 ALS lines.

(A-C′) Representative images and karyotype results of control hiPSC, SOD1A4V and C9ORF72 PHOX2B::GFP reporter lines. (D-E) Time course GFP expression of oMN-like differentiation in SOD1A4V and C9ORF72 by FACS analysis (SOD1A4V: n=3, C9ORF72: n=4 for each group; technical replicates; n means independent batches of differentiation). (F-K) Enrichment of transcripts in post-sorted ES-derived and both ALS-derived PHOX2B::GFP+ is comparable for oMN (ISL1, PHOX2A/B and NKX6.1), midbrain specification (EN1) and mDA specification (NURR1) by qRT-PCR analysis (D14) (at least n=3 for each group, n means independent batches of differentiation (independent sorted samples); technical replicates; n.s.: not significant, #P < 0.05 (0.0104), **P<0.01 (0.0097), two-tailed; unpaired student’s t-test). Scale bar: 100 μm, Error bars, mean ± SEM.

Source data

Extended Data Fig. 3 Generation of HB9::GFP reporter in SOD1A4V and C9ORF72 ALS lines.

(A-B′) Wholemount GFP expression of Hb9 and Isl1 transgenic mouse at E11.5 embryo. Magnified view of images as indicated in (A′) and (B′). (C-D) Representative images and karyotypes of SOD1A4V and C9ORF72 HB9B::GFP reporter lines. (E) Illustration of HB9 gene targeting using CRISPR-Cas9 homologous recombination. (F) Schematic protocol of sMN cell differentiation. (G) Time course GFP expression of sMN differentiation in SOD1A4V / C9ORF72 and HB9 antibody-stained cells of control hiPSC line by FACS analysis (at least n=3 for each group, n means independent batches of differentiation; technical replicates; n.s.: not significant, *P<0.05, **P<0.01, ***P<0.001, two-tailed; unpaired student’s t-test; p-values are indicated in each graph). (H) Heatmap presents characteristic marker expression of ES (OCT4 and NANOG) and sMN (HB9, ISL1, LHX3, CHAT and FOXP1) in post-sorted OCT4::GFP+, SOD1A4V HB9::GFP+ and C9ORF72 HB9::GFP+ cells by qRT-PCR (n=3 for each group, n means independent batches of differentiation; technical replicates). (I) A heatmap presents the gene expression levels of different spinal axis region markers (HOXA2, A5, A7 and A10) and cell type specific makers (HB9 for sMN and PHOX2A and TBX20 for oMN-like) in post-sorted PHOX2B::GFP+ and HB9::GFP+ cells of SOD1A4V and C9ORF72 ALS hiPSCs by qRT-PCR (n=3 for each group, n means independent batches of differentiation; technical replicates). Scale bars: 2000 μm (A-B′) or 100 μm (C-D), Error bars: mean ± SEM.

Source data

Extended Data Fig. 4 Validation of oMN and sMN population by comparing transcriptome profile with reference dataset.

(A-B) Heatmap shows differential expression levels of oMN- or sMN-specific genes in sorted HB9::GFP+ and PHOX2B::GFP+ of SOD1A4V and C9ORF72 ALS hiPSC lines (A), or reanalyzed mouse dataset from a previous literature (B). (C) Combined heatmap shows relative expression levels of oMN- or sMN-specific genes in sorted HB9::GFP+ and PHOX2B::GFP+ of SOD1A4V and C9ORF72 ALS hiPSC lines, and reanalyzed mouse dataset from a previous literature.

Extended Data Fig. 5 Transcriptome profiling reveals differences between PHOX2B::GFP+ and HB9::GFP+ cells in both SOD1A4V and C9ORF72 ALS lines.

(A) Illustration of transcriptome profiling of HB9::GFP+ versus PHOX2B::GFP+. (B-C) Volcano plots indicate a substantial transcriptomic difference between HB9::GFP and PHOX2B::GFP in both SOD1A4V and C9ORF72 ALS lines (n=3 for each group, n means independent batches of differentiation; technical replicates) (see Methods section for details). (D) Principal component analysis (PCA) plot represents distinct clustering between HB9::GFP and PHOX2B::GFP cell types-derived from both SOD1A4V and C9ORF72 ALS lines (n=3 for each group, n means independent batches of differentiation; technical replicates). (E-F) Gene set enrichment analysis (GSEA) plots show commonly over-represented GO terms of HB9::GFP+ cells compared to PHOX2B::GFP+ cells in both SOD1A4V and C9ORF72 ALS lines (n=3 for each group, n means independent batches of differentiation; technical replicates). (G) Combined dataset of the two ALS lines consistently shows the same over-represented GO terms as observed in single ALS line datasets (n=3 for each group, n means independent batches of differentiation; technical replicates). p-value of the enrichment analysis was calculated using the Hypergeometric test (phyper function) which is equivalent to one-tailed Fisher’s Exact test.

Extended Data Fig. 6 Verification of abnormal expression of lipid related transcripts in SOD1A4V and C9ORF72 ALS lines by qRT-PCR analysis.

(A) Heatmap shows enriched transcripts in sorted HB9::GFP+ of SOD1A4V and C9ORF72, but not sorted control and PHOX2B::GFP+. (B-J) Individual plot indicates altered expression transcripts in post-sorted HB9::GFP+ of SOD1A4V and C9ORF72 (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (p-value:<0.0001), n.s.: not significant, two-tailed; unpaired student’s t-test, at least n=3 for each group, n means independent batches of differentiation; technical replicates; p-values are indicated in each graph). Error bars: mean ± SEM.

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Extended Data Fig. 7 Common alteration of C21H26O3 in multiple ALS lines and direct comparision of altered metabolic metabolites by metabolomics analysis in isogenic control of SOD1A4V and SOD1A4V ALS hiPSC lines.

(A-B) Ion count values present commonly down-regulated C21H26O3 metabolic candidate in multiple ALS lines (A) and direct comparison of isogenic control of SOD1A4V and SOD1A4V lines (B) (*P<0.05, ***P<0.001, ****P<0.0001, n.s.: not significant, two-tailed; unpaired student’s t-test; p-values are indicated in each graph; at least n=3 for each lines; technical replicates; n=1 hESC control, n=3 hiPSC control, n=1 isogenic control hiPSC of SOD1A4V, n=6 C9ORF72, n=3 SOD1, n=3 TDP43, n=5 sporadic ALS hiPSC; biological replicates). (C-D) Heatmap shows the lists of selective metabolite candidate in isogenic control of SOD1A4V and SOD1A4V ALS hiPSC lines (n=6 for each group, n means 6 independent differentiation from 2 batch; technical replicates). Error bars: mean ± SEM.

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Extended Data Fig. 8 Caffeic acid exclusively rescues HB9::GFP+ cells in SOD1A4V and C9ORF72.

(A-B) Experimental modification of media (conditioned media) shows enhanced HB9::GFP+ degeneration of SOD1A4V and C9ORF72 at D19 to D25 (complete media: n=3, conditioned media: n=4 for each ALS lines; technical replicates; n means independent batches of differentiation) (*P<0.05, **P<0.01, ****P<0.0001, two-tailed; unpaired student’s t-test; p-values are indicated in each graph). (C-D) Results of compound tests in SOD1A4V and C9ORF72 HB9::GFP+ cells indicate comparable effects to control vehicle for each compound (R-Deprenyl hydrochloride, Ajamaline, Creatine and ISP-1) by FACS analysis (Dot indicates different wells from at least 3 batches, at least n=6; technical replicates; *P<0.05, n.s.: not significant, unpaired student’s t-test, two-tailed). (E-F) FACS results of HB9::GFP+ between control (non-treated) and mitomycin C treated (1 µg/ml, 1hr; D17 to D19 (2 days)) in C9ORF72 and SOD1A4V ALS hiPSC lines (Dot indicates different wells from at least 2 batches, at least n=6; technical replicates; n.s.: not significant; unpaired student’s t-test, two-tailed). (G-H) CA elevates the levels of HB9::GFP expression in the sMN culture of C9ORF72 and SOD1A4V ALS hiPSC lines after mitomycin C treatment (n=3 for each group; n means 3 independent differentiation from 1 batch; technical replicates; n.s.: not significant; *,#, P < 0.05; **,##, P < 0.01 (*:compare with none treated vehicle, #: compare with mitomycin C treated vehicle), two-tailed; unpaired student’s t-test; P-values are indicated in each graph). Error bars: mean ± SEM.

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Extended Data Fig. 9 Caffeic acid alleviates disease pathogenesis in SOD1G93A mice.

(A) Experimental scheme illustrating the CA administration and assessment of the efficacy. CA or vehicle (PBS with 10% ethanol) was administered to SOD1G93A mice from D60 to D120 of age (5 days per week). (B) Changes of body weight monitored weekly (n=24 for each SOD1G93A mice group; and n=20 for WT mice group). (C) Grip strength analysis. n=24 for each group. (D) The ratio of gastrocnemius muscle to body weight (mg/g) at the indicated time points. n=14 at 16 wks and n=10 at 20 wks for each group. (E) Neuromuscular junction visualized by α-bungarotoxin (α-BTX, green) and neurofilament H/synapsin (NF/Syn, red) in gastrocnemius muscle at 16 wks (Scale bar: 20 µm). (F) The ratio of innervated neuromuscular junction (NMJ). n=8 for each group. (G) Cresyl violet staining (Nissl staining) to visualize the pyramidal neuron (layer V) in motor cortex at 20 wks (Scale bar: 50 µm). (H) The number of pyramidal neurons and dysmorphic neurons in the layer V of motor cortex (n=10 for each group). (WT, wild-type mice; Ctrl, vehicle administered SOD1G93A mice; CA, caffeic acid (30 mg/kg) administered SOD1G93A mice; *,#, P < 0.05; **, P < 0.01;***, P < 0.001 and ****, P < 0.0001., n.s.: not significant, two-tailed; unpaired student’s t-test; p-values are indicated in each graph). Error bars: mean ± SEM.

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Extended Data Fig. 10 Caffeic acid rescues aberrant levels of arachidonic acid in the sMN culture of multiple ALS hiPC lines.

(A-F) Ion count value shows arachidonic acid level is down-regulated in post-treatment with 25 μg/ml CA at D11 to D17 of sMN differentiation of control and CA treated of ALS hiPSC lines (at least n=3 for each group; the cell line names are listed; technical replicates; biological replicates; *P<0.05, **P<0.01, ***P<0.001, two-tailed; unpaired student’s t-test). (G) Schematic model of this study. Error bars: mean ± SEM.

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Supplementary information

Supplementary Information

Supplementary Figs.1–5 and Supplementary Tables 1 and 2

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Supplementary Data 1

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Lee, H., Lee, J.J., Park, N.Y. et al. Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS. Nat Neurosci 24, 1673–1685 (2021). https://doi.org/10.1038/s41593-021-00944-z

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