Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call ‘lipid-droplet-accumulating microglia’ (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR–Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.
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The authors thank the members of the Wyss-Coray laboratory for feedback and support throughout the study, W. Stoiber (University of Salzburg, Austria) and the Division of Optical and Electron Microscopy, University of Salzburg, Austria, for excellent assistance with EM images, and D. Channappa from the Department of Neurology and E. Plowey from the Department of Pathology, Stanford University, for providing human postmortem brain samples. CARS imaging was performed at the Microscopy Core Facility of the Institute of Molecular Biosciences, University of Graz, Austria. They also thank A. Enejder, the Heilshorn Biomaterials Group, Stanford University, for discussion and for reviewing the manuscript. This work was supported by the FWF Hertha-Firnberg Postdoctoral program no. T736-B24 (to J.M.), the PMU-FFF E-16/23/117-FEA (to T.K.F), the NIH grant R37 GM058867 (to C.R.B), the Stanford Neuroscience Institute Brain Rejuvenation Project Award and NIH Director’s New Innovator Award (1DP2HD084069-01) to M.C.B., the Department of Veterans Affairs (to T.W.-C.), the National Institute on Aging (DP1-AG053015 to T.W.-C.), the NOMIS Foundation (to T.W.-C.), the Glenn Foundation for Medical Research (to T.W.-C.), the Cure Alzheimer’s Fund (to T.W.-C.) and the Nan Fung Life Sciences Aging Research Fund (to T.W.-C.).
T.W.-C., J.M., C.R.B. and M.S.H. are co-inventors on a patent application related to the work published in this paper. All other authors have no competing interests.
Peer review information Nature Neuroscience thanks Staci Bilbo, Serge Rivest, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Lipid droplet accumulating microglia are abundant in the hippocampus but rare in other brain regions of aged mice.
a–d, Representative confocal images of the cortex (a), thalamus (b), corpus callosum (c) and hippocampal dentate gyrus (d) from 20-month old male mice stained for BODIPY+ (lipid droplets) and Iba1+ (microglia). Scale bar: 20 μm. Arrows point towards BODIPY+ lipid droplets. e, Quantification of BODIPY+/Iba1+ cells. n = 4 mice per group. One-way ANOVA followed by Tukey’s post hoc test. Error bars represent mean ± SD. ***P< 0.001.
Extended Data Fig. 2 LDAM have a unique transcriptional signature that minimally overlaps with published gene expression profiles of microglia in aging and neurodegeneration.
a,b, IPA pathway analysis of genes that are significantly upregulated (a) or downregulated (b) in LD-hi microglia in aging. Analysis based on top 100 down- and up-regulated genes (Fisher’s exact test, Benjamini-Hochberg FDR). c-g, Expression plots comparing RNA-Seq data of LDAM (see Fig. 2) with published RNA-Seq data of microglia in aging (c), AD (d), ALS (e), disease-associated microglia (DAM) (f) and neurodegenerative microglia (MGnD) (g). Data are expressed as signed fdr, i.e the product of log2 FC and log10 fdr. h, Paired dot plot showing FPKM values of LD-lo and LD-hi microglia for ApoE (paired Student’s t-test; P= 0.423). Dotted lines connect LD-lo and LD-hi microglia sorted from the same samples. i, Heatmap showing expression changes of LDAM genes (genes differentially expressed in LD-hi microglia in aging) in LD-hi microglia from GRN-/- mice, from LPS treated mice, and in microglia clusters revealed by Li et al. (2019) and Hammond et al. (2019)15,16. Sample size in a,b,h: n = 3 samples per group. Each sample is a pool of microglia from the hippocampi of 3 mice. LD, lipid droplet.
a,b, 3-month-old male mice were given intraperitoneal (i.p.) injections of LPS (1 mg/kg BW) for four days. Representative confocal images of BODIPY+ and Tmem119+ in the hippocampus (a) and of BODIPY and Iba1 staining in the cortex, corpus callosum, and thalamus (b). c-e, Lipidome profiling of lipid droplets from LPS-treated BV2 cells, primary microglia, and liver tissue. c, Pie charts showing that the lipid composition of lipid droplets from young and aged microglia is highly similar, but differs between young and aged liver tissue. d,e, Distribution of MAG chain lengths (d) and TAG saturation levels (e) of lipid droplets isolated from LPS-treated BV2 cells and from microglia and liver tissue from aged mice. young= 5-month-old male mice, old= 20-month-old male mice; n = 4 mice per group. Data in a-b were replicated in at least two independent experiments. Error bars represent mean ± s.e.m. Scale bars, 20 μm.
a, Representative micrographs of BODIPY+ staining and of phagocytosis of of pHrodo red Zymosan in BV2 cells treated with 5% plasma from young (3-months) and aged (18-months) mice for 12 hours. Scale bars, 5 μm. b, Quantification of BODIPY+ staining in BV2 cells treated with young and aged plasma. c,d, Quantification of Zymosan uptake in BV2 cells treated with young and aged plasma (c), and in aged plasma treated BODIPY-low and BODIPY-high cells (d). Statistical tests: two-sided Student’s t-test. Error bars represent mean ± SD. *P< 0.05, ***P< 0.001.
Extended Data Fig. 5 Lipid droplet containing microglia in the cortex, corpus callosum, and thalamus of GRN-/- mice.
a-c, Representative confocal images of BODIPY+ (lipid droplets) and Iba1+ (microglia) in the cortex (a), corpus callosum (b), (c) and thalamus from 9-month-old male GRN-/- mice. BODIPY+/Iba1+ cells were frequently found in the thalamus and were detected to a lesser extent in cortex and corpus callosum. Data were replicated in at least three independent experiments.
Extended Data Fig. 6 Expression changes of LDAM genes in lipid droplet-rich microglia from normal aging, GRN-/- and LPS-treated mice.
a, Heatmap showing expression changes of LDAM genes (genes differentially expressed in LD-hi microglia in aging; 692 genes) in LD-hi microglia from GRN-/- mice and from LPS treated mice.
a, Paired dot plot showing FPKM values of LD-lo and LD-hi microglia for ACLY (data obtained from RNA-Seq analysis, see Fig. 2). Dotted lines connect LD-lo and LD-hi microglia sorted from the same samples. P=b, NAD colorimetric assay showing the NAD+/NADH ratio of primary hippocampal microglia from 3-month old mice (young) and of LD-lo and LD-hi primary microglia from 20-month old male mice. Experiments were performed two times in technical triplicates. n=3 mice per group per experiment. Statistical tests: paired two-sided Student’s t-test (a) one-way ANOVA (b) followed by Tukey’s post hoc test. Horizontal lines in the box plots indicate medians, box limits indicate first and third quantiles, and vertical whisker lines indicate minimum and maximum values. *P< 0.05, ***P< 0.001.
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Marschallinger, J., Iram, T., Zardeneta, M. et al. Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nat Neurosci 23, 194–208 (2020). https://doi.org/10.1038/s41593-019-0566-1
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