Abstract
Neuroinflammatory microglia secrete cytokines to induce neurotoxic reactive astrocytes, which are one of the major causes of neuronal death. However, the intrinsic key regulators underlying neurotoxic reactive astrocytes induction are unknown. Here we show that the transmembrane protein 164 (TMEM164) is an early-response intrinsic factor that regulates neurotoxic astrocyte reactivity. TMEM164 overexpression inhibits the induction of neurotoxic reactive astrocytes, maintains normal astrocytic functions and suppresses neurotoxic reactive astrocyte-mediated neuronal death by decreasing the secretion of neurotoxic saturated lipids. Adeno-associated virus-mediated, astrocyte-specific TMEM164 overexpression in male and female mice prevents the induction of neurotoxic reactive astrocytes, dopaminergic neuronal loss and motor deficits in a Parkinson’s disease model. Notably, brain-wide astrocyte-specific TMEM164 overexpression prevents the induction of neurotoxic reactive astrocytes, amyloid β deposition, neurodegeneration and memory decline in the 5XFAD Alzheimer’s disease mouse model, suggesting that TMEM164 could serve as a potential therapeutic target for neurodegenerative disorders.
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Data availability
All next-generation sequencing data have been deposited in the National Genomics Data Center (https://ngdc.cncb.ac.cn/) database (project ID: PRJCA011982). We selected snRNA-seq datasets of neurodegenerative diseases based on three criteria: (1) the sampling area should be the recognized core area of disease onset, for example, PD should be sampled in the substantia nigra48 and MS should be sampled in the white matter49; (2) there should be at least ten samples in the disease and healthy groups; (3) the datasets should be published in top-rated journals. The links to the publicly available datasets are as follows: AD snRNA-seq (from both the RADC Research Resource Sharing Hub (project IDs: syn18485175 and syn21125841) and the Gene Expression Omnibus (GEO) repository (accession nos. GSE174367, GSE138852, GSE129308, GSE163577, GSE185553, GSE185277 and GSE198323); MS snRNA-seq (accession no. GSE118257); ALS snRNA-seq (accession no. GSE174332); FTLD snRNA-seq (accession no. GSE174332); PD snRNA-seq (accession no. GSE184950); and HD snRNA-seq (accession nos. GSE152058 and GSE173731). Source data are provided with this paper.
Code availability
All original code used in this study is available from the corresponding authors upon reasonable request.
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Acknowledgements
We thank Dr. M. Poo for helpful comments. We thank the Optical Imaging (Y. Wang, Y. Zhang and Q. Hu) and FACS facilities (H. Wu and L. Quan) for their technical assistance. We thank the National Health and Disease Human Brain Tissue Resource Center for providing the samples. This study was supported by the STI2030-Major Projects of China (no. 2021ZD0200900 to H.Z.), the National Key Research and Development Program of China (no. 2022YFC3400100 to H.Z.), the Shanghai Municipal Science and Technology Major Project (no. 2018SHZDZX05 to H.Z.), the Shanghai 2023 Special Biopharmaceutical Science and Technology Support Projects (no. 23S41900300 to H.Z.) and the State Key Laboratory of Neuroscience of China.
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Authors and Affiliations
Contributions
L.Z., Z.X. and H.Z. conceived the study. L.Z., Z.X., Z.J. and Q.W. carried out the study. L.Z. and Z.J. performed the experiments, analysed the data and wrote the manuscript with valuable revision from all authors. Z.X. performed the bioinformatics data analysis. Q.W. and X.L. conducted the AAV injection and the LPS-induced model of PD. T.B. analysed LPS-induced neurotoxicity and AAV expression. X.H. contributed to the in vivo delivery. B.W. contributed to AAV testing. T.L. contributed to the enzyme-linked immunosorbent assay, lentiviral packaging and plasmid construction. Y.C. and X.D. provided the human ESCs. J.F. and Z.L. prepared the human ESC-derived neurons. H.Z. supervised the project and wrote the manuscript.
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Competing interests
H.Z., L.Z., Z.J. and Z.X. are mentioned as inventors on a patent application (no. CN202310486745.2) related to this work. H.Z. is a founder and scientific adviser of Genemagic Biosciences.
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Nature Metabolism thanks Liliane Tenenbaum, Marc Freeman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 Identification of potential regulators of neurotoxic reactive astrocytes via bulk RNA-seq analysis. (Related to Fig. 1).
a, Volcano plot and Venn diagram showing the RNA-seq analysis of 36 upregulated regulators in neurotoxic reactive astrocytes. b, Volcano plot and Venn diagram showing the RNA-seq analysis of 69 downregulated regulators in neurotoxic reactive astrocytes. The DESeq2 R package, along with the Wald test, was utilized to identify differentially expressed genes (DEGs). Genes were categorized as upregulated DEGs when they exhibited a p-value < 0.05 and a log2Foldchange > 0. Conversely, genes were classified as downregulated DEGs when they displayed a p-value < 0.05 and a log2Foldchange < 0. Genes with a p-value ≥ 0.05 were considered stable. Schematic illustrations were created with BioRender.com.
Extended Data Fig. 2 Unique 6 h DEGs in bulk RNA-seq. (Related to Fig. 1).
Heatmap displaying 69 downregulated and 36 upregulated factors in the unique 6 h DEGs dataset from primary astrocyte RNA-seq results. Schematic illustration was created with BioRender.com.
Extended Data Fig. 3 Expression of Tmem164 in different cell types of human brains. (Related to Fig. 1).
a-c, Analysis for the expression levels of Tmem164 in different brain cell types of post-mortem tissue from individuals without neurological disorders in AD snRNA-seq datasets (n = 3194 astrocytes, 1559 microglia, 10911 neurons, 16552 oligodendrocytes, and 1091 OPCs from 11 heath cases for the dataset of Zhou et al., 2020; n = 1562 astrocytes, 965 microglia, 21923 neurons, 9200 oligodendrocytes, and 1337 OPCs from 24 heath cases for the dataset of Mathys et al., 2019; n = 5610 astrocytes, 1603 microglia, 768 neurons, 4183 oligodendrocytes, and 56 OPCs from the cortex of 4 heath cases for the dataset of Yang et al., 2022), showing higher expression level of Tmem164 in astrocytes than other cell types. All data were presented as minima, maxima, median, 25th and 75th percentiles. p < 0.0001 for three datasets. ***p < 0.001, Kruskal−Wallis test.
Extended Data Fig. 4 Expression of Tmem164 is reduced in reactive astrocytes. (Related to Fig. 1).
a-c, Comparison of Tmem164 expression levels between non-reactive astrocytes and reactive astrocytes (see Methods for the definitions) in the snRNA-seq datasets of AD patients (n = 5568 non-reactive astrocytes and 901 reactive astrocytes from 21 AD patients for the dataset of Zhou et al., 2020, p = 0.01; n = 2007 non-reactive astrocytes and 965 reactive astrocytes from 11 AD patients for the dataset of Morabito et al., 2021, p < 0.0001; n = 9358 non-reactive astrocytes and 1466 reactive astrocytes from 4 AD patients for dataset of Yang et al., 2022, p < 0.0001), 7-month-old 5XFAD mice (n = 585 non-reactive astrocytes and 67 reactive astrocytes from 3 mice, p = 0.031), and PD patients (n = 3988 non-reactive astrocytes and 1022 reactive astrocytes from 20 PD patients, p = 0.00019), respectively. All data were presented as minima, maxima, median, 25th and 75th percentiles. *p < 0.05, ***p < 0.001, two-sided Wilcoxon test.
Extended Data Fig. 5 CAPN15 is the downstream transcriptional factor of TMEM164. (Related to Fig. 3).
a, Protein concentrations of CXCL1, CXCL2, and CXCL10 in the culture medium of EGFP-PBS, EGFP-ITC, and TMEM164-ITC astrocytes were measured by ELISA, n = 3 replicates per group. b, Relative mRNA expression levels of C3 among the groups of astrocytes with TMEM164 overexpression and individual transcriptional factor co-overexpression, n = 3 replicates per group. c, Relative mRNA expression levels of Tmem164, Capn15, and Elovl1 in primary mouse astrocytes treated with PBS or ITC, n = 3 replicates per group. d, Relative mRNA expression levels of Capn15 when knocking down Capn15 using CasRx/gRNA system, n = 5 replicates per group. e, Schematic illustration of the possible mechanism underlying TMEM164 overexpression-mediated suppression of neuronal death. Created with BioRender.com. All data were presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, p values were provided in Source data, one-way ANOVA and Tukey’s multiple comparison test in panels a, b, and d; unpaired two-tailed student’s t-test in panel c.
Extended Data Fig. 6 Expression of TMEM164 is decreased during the induction of human neurotoxic reactive astrocytes. (Related to Fig. 4).
a, Predicted structure of human TMEM164 with seven transmembrane motifs by AlphaFold. b, Representative staining of TMEM164, the cell junction marker ZO1, and α-tubulin in human non-reactive astrocytes, showing that TMEM164 is mainly distributed in the cytoplasm and cell membrane, especially at cell junctions. Scale bar, 10 μm. Experiments were independently repeated twice with similar results. c, Representative staining of TMEM164, ZO1, and α-tubulin in human astrocytes treated with ITC for a time course, showing a decreased protein level of TMEM164 during the induction of human neurotoxic reactive astrocytes. Scale bar, 10 μm. Experiments were independently repeated twice with similar results.
Extended Data Fig. 7 AAV-mediated TMEM164 overexpression inhibits the induction of reactive astrocytes but not inflammatory microglia. (Related to Fig. 5).
a, Double staining of reactive astrocyte markers of GFAP and C3 in the SNc of untreated control and AAV-injected PD mice. The astrocytes infected with TMEM164-overexpressed AAV showed downregulated GFAP and C3 signals (the area below the white dashed line in the AAV-Tmem164 + LPS group). Scale bar, 100 μm. b, Representative images of Iba1 staining in the LPS-induced PD model. Scale bar, 50 μm. c, Quantification for the area covered by Iba1-positive microglia in the SNc, showing similar microglial activation between the AAV-mCherry + LPS and AAV-Tmem164 + LPS groups, n = 6 mice per group. Data were presented as mean ± SEM., p value was provided in Source data, unpaired two-tailed student’s t-test.
Extended Data Fig. 8 LPS-induced neurotoxicity in different brain regions. (Related to Fig. 5).
a, Representative images of NeuN and GFAP staining in the cortex of mice injected with AAV and LPS. Scale bar, 50 μm. b, Quantification for the neuron number in the cortex, n = 3 mice per group. c, Representative images of NeuN and GFAP staining in the striatum of mice injected with AAV and LPS. Scale bar, 50 μm. d, Quantification for the neuron number in the striatum, n = 3 mice per group. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, p values were provided in Source data, one-way ANOVA and Tukey’s multiple comparison test.
Extended Data Fig. 9 Evaluation of AAV expression in the brain and peripheral organs of 5XFAD mice. (Related to Fig. 6).
a, Representative images showing the expression of AAV-sGFAP-Tmem164-P2A-mCherry-WPRE in different brain regions of 9-month-old 5XFAD mice at 6 months after AAV injection. Scale bar, 20 μm. b, Representative images showing the expression of AAV-sGFAP-Tmem164-P2A-mCherry-WPRE in the peripheral organs of 9-month-old 5XFAD mice at 6 months after AAV injection. Scale bar, 20 μm. c, Percentage of mCherry-positive astrocytes in different brain regions or mCherry-positive cells in the peripheral organs of 5XFAD mice, n = 3 mice per group. Data were presented as mean ± SEM.
Extended Data Fig. 10 Effects of TMEM164 overexpression on neurodegeneration and Aβ deposition in 6-month-old 5XFAD mice at 3 months after AAV injection. (Related to Fig. 6).
a, Representative images showing Fluoro-Jade C-labeled degenerated neurons in the cortex and hippocampus of 6-month-old 5XFAD mice at 3 months after AAV injection. Scale bar, 50 μm. b, Quantification of the number of degenerated neurons per field in the cortex and hippocampus in panel a. c, Representative images showing Iba1 and Aβ staining in the cortex and hippocampus of 6-month-old 5XFAD mice at 3 months after AAV injection. Scale bar, 50 μm. d, Quantification of the area covered by Aβ in the cortex and hippocampus, n = 4 mice per group. e, Quantification of the area covered by Iba1-positive microglia in the cortex and hippocampus, n = 4 mice per group. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, p values were provided in Source data, unpaired two-tailed student’s t-test.
Supplementary information
Supplementary Information
Supplementary Figs. 1–3.
Supplementary Table 1
List of DEGs in the RNA-seq data of the primary astrocytes
Supplementary Table 2
List of DEGs and KEGG pathway in the RNA-seq data of 5XFAD model
Supplementary Table 3
Oligonucleotides used in this study
Supplementary Data 1
Raw gas chromatography–mass spectrometry data
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Zhang, L., Jia, Z., Wu, Q. et al. Alleviating symptoms of neurodegenerative disorders by astrocyte-specific overexpression of TMEM164 in mice. Nat Metab 5, 1787–1802 (2023). https://doi.org/10.1038/s42255-023-00887-8
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DOI: https://doi.org/10.1038/s42255-023-00887-8
