Abstract
Astrocytes promote neuroinflammation and neurodegeneration in multiple sclerosis (MS) through cell-intrinsic activities and their ability to recruit and activate other cell types. In a genome-wide CRISPR-based forward genetic screen investigating regulators of astrocyte proinflammatory responses, we identified the C-type lectin ___domain-containing 16A gene (CLEC16A), linked to MS susceptibility, as a suppressor of nuclear factor-κB (NF-κB) signaling. Gene and small-molecule perturbation studies in mouse primary and human embryonic stem cell-derived astrocytes in combination with multiomic analyses established that CLEC16A promotes mitophagy, limiting mitochondrial dysfunction and the accumulation of mitochondrial products that activate NF-κB, the NLRP3 inflammasome and gasdermin D. Astrocyte-specific Clec16a inactivation increased NF-κB, NLRP3 and gasdermin D activation in vivo, worsening experimental autoimmune encephalomyelitis, a mouse model of MS. Moreover, we detected disrupted mitophagic capacity and gasdermin D activation in astrocytes in samples from individuals with MS. These findings identify CLEC16A as a suppressor of astrocyte pathological responses and a candidate therapeutic target in MS.
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Data availability
Data supporting the findings of this study are available as supplementary and source data files. All raw and processed deep-sequencing data have been deposited into the Gene Expression Omnibus under SuperSeries accession numbers GSE189030 and GSE283395. Source data are provided with this paper.
Code availability
No custom software code was used in this research.
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Acknowledgements
A.K. was supported by a Uehara Memorial Foundation Overseas Research Fellowship, the Japan Society for the Promotion of Science (JSPS) Overseas Research Fellowship, the JSPS PD Research Fellowship and Grant-in-Aids for JSPS fellows (grant title: ‘Investigation of the role of mitophagy-regulating molecules in astrocytic CNS inflammation’). This work was supported by grants NS102807, NS087867, ES02530, AI126880 and AI093903 from the National Institutes of Health (NIH), RG4111A1 from the National MS Society (NMSS) and PA-1604-08459 from the International Progressive MS Alliance and the Chan Zuckerberg Initiative to F.J.Q. M.A.W. was supported by R00NS114111, R01MH130458 and R01MH132632. B.M.A. was supported by the Training Program in Nervous System Tumors (K12CA090354) from the National Cancer Institute (NCI)/NIH, the Career Enhancement Program (CEP) for the SPORE at the Harvard Cancer Center (P50CA165962) from the NCI/NIH and the Postdoctoral Fellowship in Translational Medicine from the PhRMA Foundation. H.-G.L. was supported by the Basic Science Research Program through the National Research Foundation of Korea (2021R1A6A3A14039088). J.-H.L. was supported by the Basic Science Research Program funded by the National Research Foundation of Korea (2022R1A6A3A03071157) and a long-term postdoctoral fellowship funded by the Human Frontier Science Program (LT0015/2023-L). S.A.S. was supported by the JDRF (CDA-2016-189, COE-2019-861), the NIH (R01 DK108921, U01 DK127747) and the Department of Veterans Affairs (I01 BX004444). A.N. was supported by a Max Kade Fellowship. I.C. was supported by the NIH (5R01DK127257, 5R01AI130019), Burroughs Wellcome Trust, Chan Zuckerberg Initiative and Kenneth Rainin Foundation. A.P. holds a T1 Canada Research Chair in MS and was supported by the Canada Institute of Health Research, MS Canada, the NMSS and the Canadian Foundation for Innovation. V.K. is a New York Stem Cell Foundation Robertson Stem Cell Investigator, and this work was additionally supported by NIH grant NS109209-01A1. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank all members of the Quintana laboratory for helpful advice and discussions, as well as L. Ding for assistance with live-cell imaging and quantification, T. Yamamura (National Institute of Neuroscience, Tokyo, Japan) and H. Mochizuki (Osaka University, Osaka, Japan) for support, and R. Saga (BWH) for advice on histology and support.
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A.K. conceptualized and designed the study, performed most of the studies, analyzed and interpreted data, wrote the manuscript and secured funding. M.A.W. designed and performed the CRISPR screen and helped with single-cell data analysis. S.E.J.Z., W.K. and A.P. provided human brain tissues and analyzed them by IHC. A.K., H.B., D.N., M.B. and I.C. performed IHC analysis of mouse CLEC16A, LC3 and GSDMD. A.N. generated hES cell-derived astrocytes and helped with related studies under the supervision of V.K. C.-C.C. offered technical advice, discussion and data interpretation. J.V.M. assisted with EAE and histology. Z.L. performed bioinformatics analyses. B.M.A. assisted with single-cell and mtDNA studies. H.-G.L. quantified IL-1β in EAE. T.I. helped with mitophagy-related protein analysis. J.-H.L. helped with live-cell imaging. G.L. and L.S. kept mouse colonies and assisted with astrocyte cultures. S.A.S. provided Clec16afl/fl mice and CLEC16A-specific antibodies. V.R. participated in the early project stages. F.J.Q. conceptualized and designed the study, interpreted data, wrote the manuscript, secured funding and supervised the project.
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Extended data
Extended Data Fig. 1 Analysis of the mitophagy-related and mitochondrial proteins.
(a) PINK1 expression in Clec16a siRNA KD primary astrocytes stimulated with TNF/IL-1β. Representative of 3 experiments. (b) Parkin and mitophagy-specific receptor expression in Clec16a siRNA KD primary astrocytes. Representative blots and quantification of mean fold change from baseline (time 0) of normalized mitophagy-specific receptor expression from 3 experiments are shown. *p < 0.05 by paired t-test, two-sided (Calococo2 16Hours p 0.0131). (c) Expression of mitochondrial protein TOM20. Representative blots and quantification from 3 experiments are shown. Mean and s.e.m. p < 0.05 by two-way ANOVA, Sidak post-test (TNFα/IL-1β p 0.0123).
Extended Data Fig. 2 Role of CLEC16A in the control of mitophagy in astrocytes.
(a) Live cell imaging of siRNA KD FCCP or TNF/IL-1β-stimulated murine astrocytes, stained using Mitophagy dye (red) and Lyso-dye (green). Merged area indicate mitochondria undergoing mitophagy. Scale bars 100 μm. Regions of interest are highlighted with white squares and displayed in Fig. 2c. Representative of two experiments. (b, c) Live cell imaging of Control, Atg7 or Clec16a siRNA KD and TNFα/IL-1β-stimulated murine astrocytes, stained using Mitophagy dye (red) and Lyso-dye (green). The astrocytes were also pre-stimulated with TNFα/IL-1β for 13 hrs. (b) Representative images are shown. High Mitophagy dye intensity area generated from the original image by cutting off the low intensity signal area is also shown. Scale bars 30 μm. (c) Quantification of the overlay puncta numbers or area size of the high mitophagy-dye intensity area and lyso-dye are calculated from 9 splitted fields per each time point in each condition. Mean and s.e.m. p** < 0.01, p*** < 0001, p**** < 0.0001 by One-way ANOVA Dunnett’s post-test. (No. overlay puncta siControl versus siAtg7 P = 0.0090, overlay puncta area size siControl versus siAtg7 P = 0.0002).
Extended Data Fig. 3 Cytoplasmic mitochondrial DNA in unstimulated CLEC16A KD astrocytes.
qPCR quantification of mitochondrial gene mt-Co1 DNA extracted from the cytoplasmic fractions of Clec16a KD unstimulated or TNFα/IL-1β-stimulated murine astrocytes. Relative copy numbers to control are displayed. n = 3 samples per condition. Representative of two experiments. Mean and s.e.m. *p < 0.05 by two-way ANOVA, Sidak post-test (TNFα/IL-1β p 0.0189).
Extended Data Fig. 4 Nlrp3 expression in CLEC16A KD astrocytes.
qPCR in Clec16a KD astrocytes. Combination of two experiments, each containing 3 samples per condition, n = 6 in total. Mean and s.e.m. ** p < 0.01 by two-way ANOVA, Sidak post-test (TNFα/IL-1β p 0.0010).
Extended Data Fig. 5 RNA-seq of astrocytes and microglia from CLEC16AGFAP EAE mice.
(a) Clec16a and Ppargc1a expression determined by qPCR in astrocytes and microglia from Control (n = 13) and CLEC16AGFAP (n = 12) EAE mice (day 30). Sorting strategy in Supplementary Fig. 3. Mean and s.e.m. **p < 0.01, *p < 0.05 by two-way ANOVA Sidak post-test (Ppargc1a astrocytes p 0.0032, Clec16a astrocytes p 0.0006). (b) IPA of RNA-Seq data of astrocytes from Control (n = 5) and CLEC16AGFAP (n = 6) EAE mice (day 30). P values were determined using Fisher’s exact test. (c) Differentially expressed genes (DEGs) detected by RNA-seq analysis of microglia from Control (n = 5) or CLEC16AGFAP (n = 6) EAE mice on day 30. (d) IPA of RNA-Seq data of microglia from Control (n = 5) and CLEC16AGFAP (n = 6) EAE mice (day 30). P values were determined using Fisher’s exact test.
Extended Data Fig. 6 Astrocyte-specific CLEC16A deletion in CLEC16AGFAP mice.
(a,b) IHC analysis of CLEC16A (Green), GFAP (purple) in spinal cord sections from Control EAE or CLEC16AGFAP EAE mice (day 21); representative maximum intensity images of Z-stacks are shown. Scale bars 200 μm (upper images) and 30 μm (lower images). (c) Quantification of CLEC16A expression in astrocytes (GFAP), neurons (Neurofilament-L), and microglia (IBA1) are shown. Each condition is quantified from 6 images, containing 2 independent images of whole spinal cord sections from 3 mice. **** p < 0.0001, n.s. not significant by Sidak’s test.
Extended Data Fig. 7 Peripheral immune cells in CLEC16AGFAP EAE mice.
(a) Flow cytometry analysis of CD4+ T cells (CD4+CD3+) and monocytes (CD45hiCD11b+Ly6chi) in CNS and spleen of Control and CLEC16AGFAP mice 30 days after EAE induction. (b) Flow cytometry analysis of IL-17A for Th17 cells, IFNγ for Th1 cells, Foxp3 for regulatory T cells, and IL-10 for type 1 regulatory T cells in CNS and spleen of EAE mice (day 30). (c) Recall cytokine response of splenocytes from Control and CLEC16AGFAP mice (day 30) to MOG(35-55). (a-c) Mean and s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 by Mann Whiney test, two-sided (CD4+ T cells p 0.0022, monocytes p 0.0022) (a) and two-way ANOVA Sidak post-test (IL-17 + IFNγ- p 0.0007, IL-17-IFNγ + p 0.0353) (b). Representative of two experiments. (a, b) In the box plots, the data are presented as the median and interquartile range and whiskers represent minimum to maximum.
Extended Data Fig. 8 Role of CLEC16A in autophagy in vivo.
(a) Immunohistochemical analysis of LC3 (Green) and GFAP (red) and merged images in spinal cord (SC) sections from Control EAE or CLEC16AGFAP EAE mice (day 21); representative maximum intensity images of Z-stacks are shown. (b) Region 1, 2, and 3 (Fig. 5e) in (a) are depicted in larger images. White scale bars 100 μm, gray scale bars 25 μm. Representative of two experiments.
Extended Data Fig. 9 Proinflammatory gene expression in non-immunized CLEC16AGFAP mice.
(a) qPCR performed in sorted astrocytes from non-immunized Control (n = 6) and CLEC16AGFAP mice (n = 6). Comibination of two experiments. Mean and s.e.m. n.s. not significant by unpaired t-test, two-sided. (b) scRNA-seq analysis of astrocytes in naïve Control and CLEC16AGFAP mice; ingenuity pathway analysis shown. P values were determined using Fisher’s exact test.
Extended Data Fig. 10 scRNA-seq analysis of astrocytes in CLEC16AGFAP EAE mice.
(a) Feature plots depicting astrocyte marker genes. (b) Gene set enrichment analysis (GSEA) was performed in cluster 2 astrocytes. Indicated gene sets were used, normalized enrichment score (NES) and p values are depicted. Nominal p-value was one-tailed test on the appropriate side of the null distribution. (c) IPA analysis of cluster 1 and 3 astrocytes. Representative pathways are shown. P values were determined using Fisher’s exact test.
Supplementary information
Supplementary Information
Supplementary Figs. 1–5 and unprocessed western blots of Supplementary Fig. 2.
Supplementary Tables
Supplementary Table 1. Genes detected in the astrocyte CRISPR–Cas9 screen using NF-κB–EGFP reporter mice. Genes are ranked according to log(fold change), which was determined using MAGeCK to compare the number of reads enriched in the EGFP+ astrocyte fraction versus the EGFP− astrocyte fraction, signifying negative regulators of NF-κB (EGFP) activation. P value was determined by MAGeCK and corresponds to the statistical enrichment of a particular guide within its corresponding fraction. Supplementary Table 2. DEGs detected by RNA-seq in astrocytes from control (n = 5) or CLEC16AGFAP (n = 6) EAE mice on day 30 after EAE induction. Genes are ordered in accordance with the multiplicity-adjusted P value (Padj). Genes with a P value smaller than 0.05 are considered DEGs. Supplementary Table 3. Demographic features of patients with MS and control individuals studied in Fig. 8d–g. PM, postmortem; NA, not applicable.
Supplementary Data
Marker gene lists of astrocyte clusters (1–10) in scRNA-seq experiments (a total of ten Excel files). Files are named ‘Cluster.(# of cluster).markers’.
Source data
Source Data Fig. 2
Unprocessed western blots.
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 4
Unprocessed western blots.
Source Data Fig. 7
Unprocessed western blots.
Source Data Extended Data Fig. 1
Unprocessed western blots.
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Kadowaki, A., Wheeler, M.A., Li, Z. et al. CLEC16A in astrocytes promotes mitophagy and limits pathology in a multiple sclerosis mouse model. Nat Neurosci 28, 470–486 (2025). https://doi.org/10.1038/s41593-025-01875-9
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DOI: https://doi.org/10.1038/s41593-025-01875-9
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