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Neurotoxic reactive astrocytes induce cell death via saturated lipids

Abstract

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1,2,3,4,5,6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system.

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Fig. 1: Proteins upregulated in reactive ACM.
Fig. 2: Differentially regulated lipids in reactive astrocytes.
Fig. 3: Mechanism of cell death from reactive ACM.
Fig. 4: Conditional knockout of the long-chain saturated lipid synthesis gene Elovl1 reduces reactive astrocyte toxicity.

Data availability

Mass spectrometry data in Figs. 1, 2, 4 are publicly available at http://gliaomics.com/ as well as the raw data in Supplementary Tables 1, 2. The accession information for raw protein mass spectrometry data is MassIVE MSV000087805 (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=5f39b6cde74c4911951dbef1f2dac443), and the accession information for raw lipid and metabolite mass spectrometry data is MassIVE MSV000087832 (http://massive.ucsd.edu/ProteoSAFe/status.jsp?task=ca311727ff524e32868736d3d1b3cc0a). Any other data are available from the corresponding author upon reasonable request.

Code availability

All mass spectrometry analysis code is available on the Chopra Lab Github (https://github.com/chopralab/reactive_astrocytes_omics).

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Acknowledgements

On behalf of all his trainees and colleagues, this paper is dedicated to the memory of Ben Barres. We acknowledge Merck for allowing us to use the Elovl1flox/flox mouse line for these experiments. This work used the Stanford Neuroscience Microscopy Service, which is supported by the grant award NIH NS069375. The Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University Mass Spectrometry contributed to this work. This work was supported in part by NIH P30 CA124435, using the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource. This work used the LTQ-Orbitrap mass spectrometer system that was purchased with funding from NIH Shared Instrumentation grant S10RR027425. Additional support was provided by the JPB Foundation, and Vincent and Stella Coates (to B.A.B.). K.A.G. was supported by the Wu Tsai Institute Interdisciplinary Scholar Award. S.A.L. was supported by the Cure Alzheimer’s Fund, Anonymous Donors, the Blas Frangione Foundation and the MD Anderson Neurodegenerative Consortium. This work was also supported in part by an unrestricted grant from Research to Prevent Blindness (RPB). We thank C. Ferreira at the Purdue Metabolite Profiling Facility for assistance with lipid mass spectrometry; U. K. Aryal at the Purdue Proteomics Facility for help with protein mass spectrometry and deposition of data; and Agilent Technologies for their gift of the Triple Quadrupole LC/MS to the Chopra Laboratory. This work was supported, in part, by the United States Department of Defense USAMRAA award W81XWH2010665, NIH National Center for Advancing Translational Sciences ASPIRE Design Challenge awards, Purdue Integrative Data Science Institute award and a start-up package from the Department of Chemistry at Purdue University to G.C. Additional support, in part, by the Stark Neurosciences Research Institute; the Indiana Alzheimer Disease Center; Eli Lilly and Company; the Indiana Clinical and Translational Sciences Institute grant UL1TR002529 from the NIH, National Center for Advancing Translational Sciences; and the Purdue University Center for Cancer Research funded by NIH grant P30 CA023168 is also acknowledged. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Select illustrations in figure subpanels were made using BioRender.

Author information

Affiliations

Authors

Contributions

K.A.G., S.A.L., A.D.G. and B.A.B. designed the experiments. K.A.G. and S.A.L. wrote the paper. K.A.G., M.K.W. and A.E.M. performed experiments and analysed data. P.P., P.R.W., J.F. and G.C. designed, performed and analysed mass spectrometry proteomics, lipidomics and metabolomics experiments and developed the web application. P.H., U.R.-B., J.A.B. and S.A.L. performed Elovl1 cKO validation experiments. M.C.N. and K.D.B. performed HPLC and lipoparticle analysis experiments.

Corresponding author

Correspondence to Shane A. Liddelow.

Ethics declarations

Competing interests

A.D.G. has served as a consultant for Aquinnah Pharmaceuticals, Prevail Therapeutics and Third Rock Ventures, and is a scientific founder of Maze Therapeutics. S.A.L. is an academic founder of AstronauTx. B.A.B. is a co-founder of Annexon Biosciences, a company working to make new drugs for the treatment of neurological diseases. G.C. is the Director of the Merck-Purdue Center for Measurement Science funded by Merck Sharp & Dohme, a subsidiary of Merck. The remaining authors declare no competing interests.

Additional information

Peer review information Nature thanks Jeremy Kay, Robert Zorec and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Principal component analysis of protein mass spectrometry data.

a, Number of significant proteins and PCA variation based on number of replicates of protein mass spectrometry that were required to have a non-zero spectral count to be considered for analysis. 3660 total unique proteins detected in astrocytes and 183 total unique proteins detected in ACM. 4 of 10 (4x) was chosen for final analysis. b, PCA plots of cellular and ACM protein mass spectrometry of all proteins detected in at least 4 of 10 astrocytes samples see shows clear separation of the proteome and secretome of reactive versus control astrocytes. c, Quantification of differentially regulated proteins in reactive astrocytes and ACM (FDR < 0.1). d, 10 most upregulated and downregulated proteins in reactive versus control astrocytes (bold = known reactivity markers).

Extended Data Fig. 2 Testing the toxicity of various candidate toxic proteins.

a, Oligodendrocytes were treated with various doses of candidate toxic proteins found in our proteomics analysis or from previous literature but were not found to be toxic in our culture conditions. b, Reactive ACM, but not Lcn2, Lgals1, or complement component C3 family members, is toxic to retinal ganglion cell (RGC) neuron cultures. (all data represents N=3/4 independent samples from 2 separate primary cell isolations; presented as mean ± SEM).

Extended Data Fig. 3 Toxic factor enrichment.

a, Diagram of sequential toxic factor enrichment by various biochemical purification columns. b, Validation that sequentially enriched reactive ACM is more toxic than sequentially enriched control ACM. (data represents 3 independent samples from 3 separate primary cell isolations).

Extended Data Fig. 4 Astrocyte lipoparticle analysis.

a, Example control and reactive protein abundance traces for HPLC size exclusion column. b, ELISA shows an increase in APOJ concentration within fractions associated with astrocyte HDL. (individual data points represent independent samples from a single primary cell isolation; presented as mean ± SEM) c, ELISA on concentrated control versus reactive HPLC fractions associated with HDL shows more APOE in reactive versus control. Control and reactive HDL fractions were combined and concentrated to achieve sufficient signal for APOE ELISA so only one sample for control versus reactive was analysed.

Extended Data Fig. 5 Reconstituted HDL incorporation into cells.

Example images of fluorescently labelled reconstituted HDL incorporation into oligodendrocyte, microglia, endothelial cells, oligodendrocyte precursor cells (OPCs), retinal ganglion cell neurons (RGCs), and astrocytes in vitro. Note that all cells incorporate reconstituted lipoparticles except for endothelial cells. (experiment performed on 2 separate primary cell isolations for each cell type; scale bar = 100 µm).

Extended Data Fig. 6 Astrocyte metabolomics and lipidomics.

a, Reactive (red) versus control (grey) astrocytes and ACM are somewhat separable in PCA space based on their metabolome, but less so than by their lipidome (Fig. 2). AFU, arbitrary fluorescence units. b, Distribution of MRM transitions selected for screening lipids. A total of 1547 transitions (used to ID lipid species) were organized into 11 MRM-based mass spectrometry methods (for lipid classes). c, Quantification of differentially regulated lipids and metabolites in reactive astrocytes and ACM (FDR < 0.1). d, Scatterplot of differentially regulated lipids in reactive versus control astrocytes and ACM highlights the overall abundance of differentially regulated lipids.

Extended Data Fig. 7 Saturated free fatty acids and phosphatidylcholines are toxic to oligodendrocytes.

a, Cultured oligodendrocytes (phase) incorporate fluorescent C16:0 free fatty acids (FFAs, green) upon treatment (0.5 µM; scale bar = 150 µm). b, Dose curve of oligodendrocyte survival following treatment with C16:0 and C18:0 saturated FFAs shows that saturated FFAs are toxic to oligodendrocytes with longer chain lengths leading to greater toxicity (curve fits performed using one-phase decay model). c, Long-chain saturated phosphatidylcholines (PC, 20:0) are toxic to oligodendrocytes in a dose-dependent fashion. (data, including representative image in subpanel a, represents N=4 independent samples from 3 separate primary cell isolations; presented as mean ± SEM).

Extended Data Fig. 8 Further analysis of the mechanism of toxic-factor-induced cell death.

a, Various doses of ethoxyquin in DMSO was added to oligodendrocytes with or without 30 μg/ml reactive ACM. Simple linear regression analysis on increasing doses of ethoxyquin without reactive ACM (Slope = −0.0000025, P value [slope ≠ 0] = 0.1932) and with reactive ACM (Slope = −0.000001788, P value [slope ≠ 0] = 0.4194) failed to show a significant relationship between ethoxyquin concentration and survival, suggesting that the free radical scavenger did not affect cell survival when treated in isolation and did not affect the toxicity of reactive ACM. This data, in addition to the data that Ferrostatin-1 has no effect on astrocyte toxicity2, suggests that lipid peroxidation may not mediate the ACM toxicity. b, siRNAs potently knock down the lipoapoptosis sensitivity modulated genes Scd1 and Insig1 in oligodendrocytes in vitro. c, Knockdown of SCD and INSIG1, which bidirectionally modulate sensitivity to lipoapoptosis, bidirectionally modulate sensitivity of oligodendrocytes to toxic ACM. (data represents n=3 independent samples from 2 separate primary cell isolations; presented as mean ± SEM).

Extended Data Fig. 9 Elovl1 cKO validation.

a, GFP expression (green) from NuTrap mice crossed to Gfap-Cre line used in this study shows efficient recombination in Slc1a3+ astrocytes (red, as identified by RNAscope in situ hybridization) of the ganglion cell layer (GCL, identified by DAPI staining of nuclei, blue; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer). b, DNA gel following PCR amplification of Elovl1 and Gfap in the retina and optic nerve shows a decrease in Elovl1 expression relative to Gfap expression in the Elovl1 cKO mouse visual system. (white numbers indicate molecular weight markers) c, Quantification of the decrease in Elovl1 expression relative to Gfap expression in the Elovl1 cKO retina (top) and optic nerve (bottom) N=4 animals per group, bars represent s.e.m., two-tailed Student’s t-test). d, Targeted lipidomics of Elovl1 cKO ACM shows dampened upregulation of the long-chain saturated lipids normally upregulated in WT reactive ACM. (black line indicates equal upregulation; red dots indicate lipids less upregulated in Elovl1 cKO versus WT ACM; black dot indicates a lipid less upregulated in WT ACM versus Elovl1 cKO ACM). e, Separation of Elovl1 cKO and WT cell and ACM lipidomes in PCA space.

Extended Data Fig. 10 Toxicity of Elovl1 cKO versus wild-type ACM over time.

Toxicity of oligodendrocytes in response to Elvol1 cKO versus wt control, reactive, and concentrated reactive ACM over 96 h (data represents mean ± SEM of 6 experimental replicates each from 3 independent samples from 3 separate primary cell isolations; presented as mean ± SEM).

Supplementary information

Supplementary Information

This file contains Supplementary Fig. 1 and Supplementary Tables 2 and 3. Supplementary Fig. 1 shows the uncropped western blots from Fig. 3. Supplementary Table 2 contains mass spectrometry data for lipoprotein pull-downs. Spectral counts of proteins detected in all replicates of APOE and APOJ antibody pull-downs of control and reactive ACM show little specificity between APOE and APOJ antibodies for detected lipoproteins. Supplementary Table 3 contains statistical details for all experimental comparisons including exact p-values.

Reporting Summary

Supplementary Table 1

Mass spectrometry data of purified ACM. Spectral counts from protein mass spectrometry of n = 3 control and n = 6 reactive ACM samples sequentially purified according to Extended Data Fig. 3.

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Guttenplan, K.A., Weigel, M.K., Prakash, P. et al. Neurotoxic reactive astrocytes induce cell death via saturated lipids. Nature 599, 102–107 (2021). https://doi.org/10.1038/s41586-021-03960-y

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