Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain
- Multiple Sclerosis & Neurorepair Research Unit, Biogen, United States
- Department of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, United States
- Emerging Neurosciences Research Unit, Biogen, United States
- Chemical Biology and Proteomics, United States
- Multiple Sclerosis Development Unit, Biogen, United States
- Center for Genomic Medicine, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, United States
- Third Rock Ventures, United States
Figures
Figure 1 with 1 supplement
Cellular autofluorescence identifies two discrete and novel microglia subsets.
(A) Representative flow cytometry scatterplot showing CD45 and CD11B surface levels in a brain single cell suspension and histogram of autofluorescence (AF) signal in DAPI− CD45dim CD11B+ microglia …
Figure 1—figure supplement 1
Autofluorescence identifies two discrete microglia subsets across brain regions and in both male and female mice.
Related to Figure 1. (A) Flow cytometry gating strategy used to identify microglia and AF subsets. (B) Percent AF+ microglia detected in 6-month-old mice across multiple independent experiments. …
Figure 2 with 1 supplement
Natural aging increases microglia AF signal intensity solely within AF+ cells.
(A) Representative imaging flow cytometry images and (B) corresponding quantitation of average AF area per cell in AF+ and AF− CD45dimCD11B+ microglia isolated from 3- and 10-month old mice. …
Figure 2—figure supplement 1
Microglial autofluorescence increases with age in multiple flow cytometry channels and is unique to microglia.
Related to Figure 2C,D. (A) Representative flow cytometry histograms of AF intensity in the entire microglia population for multiple cytometer excitation lasers and emission filter wavelengths …
Figure 3
AF+ microglia selectively accumulate intracellular storage bodies with age.
(A) Representative TEM images of sorted microglia subsets from 3- to 18-month-old mice. Red arrowheads point to storage bodies. Scale bars = 2 µm. Insets depict higher magnification images of …
Figure 4 with 1 supplement
Microglia AF subsets exhibit differential population dynamics upon depletion and replenishment of the microglia niche.
(A) Representative flow cytometry scatterplots showing CD45 and CD11B surface levels in brain single cell suspensions from mice at indicated timepoints post treatment with vehicle or BLZ945 and (B) …
Figure 4—figure supplement 1
Repopulating microglia exhibit lower levels of AF.
Related to Figure 4C,D. (A) Representative flow cytometry histogram and (B) corresponding quantitation of microglia subset proportions 70 days after treatment with vehicle or BLZ945. Significance …
Figure 5
Proteomic analysis of isolated AF+ and AF− microglia subsets reveals molecular differences in endolysosomal, autophagic and metabolic pathways.
(A) Volcano plot comparing protein abundance in AF+ and AF− microglia (x-axis: Log2 abundance difference, y-axis: negative log Benjamini-Hochberg adjusted p value from two-tailed Student’s t-test) …
-
Figure 5—source data 1
AF+ versus AF- subset associated proteins.
Sheet 1: Description of the column headers. Sheet 2: Proteins which were differentially regulated (adj. p value < 0.05) between subsets.
- https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data1-v2.xlsx
-
Figure 5—source data 2
Panther overrepresentation test of AF+ and AF- associated proteins.
Sheet 1: Description of the column headers. Sheet 2: Pathway overrepresentation testing for proteins upregulated in AF+ microglia. Sheet 3: Pathway overrepresentation testing for proteins upregulated in AF- microglia.
- https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data2-v2.xlsx
-
Figure 5—source data 3
Ingenuity pathway analysis of differentially regulated proteins in AF subsets.
Sheet 1: Description of the column headers. Sheet 2: Overlap of differentially regulated proteins with IPA canonical pathways based on input list of 351 proteins with adj. p val <0.01 and fold change > |1.3|. Sheet 3: Upstream regulators identified by IPA based on input list of 351 differentially regulated proteins with adj. p val <0.01 and fold change > |1.3|.
- https://cdn.elifesciences.org/articles/57495/elife-57495-fig5-data3-v2.xlsx
Figure 6 with 2 supplements
The accumulation of AF storage bodies in microglia is genetically-controlled.
(A, C, E, G, H, J) Representative flow cytometry histograms and quantitation of AF subset frequency and/or AF signal intensity in AF+ microglia isolated from mice of indicated genotypes. …
Figure 6—figure supplement 1
Characterization of microglia isolated from Mbp-deficient mice.
Related to Figure 6A,B. (A) Representative flow cytometry scatterplots depicting CLEC7A levels and AF intensity in microglia from mice of indicated genotypes and (B) corresponding quantitation of …
Figure 6—figure supplement 2
Characterization of microglia isolated from Atg5-deficient mice.
Related to Figure 6G,H,I. (A) Relative transcript expression of Atg5 quantified by RT-qPCR of FACS-isolated microglia from mice of indicated genotypes 1 or 8.5 months after tamoxifen-induced …
Figure 7 with 1 supplement
Advanced aging and lysosomal dysfunction lead to the preferential cellular loss of the AFhi microglia population.
(A) Representative flow cytometry histograms and (B, C) corresponding quantitation of AF subset frequencies (B) and absolute cell numbers (C) in 12- and 24-month-old naïve mice. Significance …
Figure 7—figure supplement 1
Lysosomal dysfunction leads to the preferential cellular loss of the AFhi microglia population.
Related to Figure 7E,H,I,J. (A) Quantitation of the frequency of AnnexinV+, DAPI− microglia at 12- and 24 months of age in C57BL/6 mice. Significance established with 2-way repeated measures ANOVA …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | BLZ945; CSF1R inhibitor | PMID:24056773 | CAS No. 953769-46-5 | |
| Chemical compound, drug | Tamoxifen | Millipore-Sigma | Cat# T5648 | |
| Biological sample (C. macaque) | NHP brain samples | Charles River Labs | freshly isolated same day | |
| Other | DAPI | ThermoFisher | Cat# 62248 | Cell impermeant blue-fluorescent DNA stain for viability used at a concentration of 0.1 µg/mL |
| Antibody | anti-mouse CD11b BV510 (rat monoclonal) | Biolegend | RRID:AB_2561390 | FlowCyt (1:200) |
| Antibody | anti-mouse CD45 BV785 (rat monoclonal) | Biolegend | RRID:AB_2564590 | FlowCyt (1:200) |
| Antibody | anti-mouse CX3CR1 BV421 (rat monoclonal) | Biolegend | RRID:AB_2565706 | FlowCyt (1:200) |
| Antibody | anti-mouse P2RY12 PE (rat monoclonal) | Biolegend | RRID:AB_2721644 | FlowCyt (1:200) |
| Antibody | anti-mouse TMEM119 (rabbit monoclonal) | AbCam | RRID:AB_2744673 | FlowCyt (1:100) |
| Antibody | anti-rabbit AlexaFluor488 (rat monoclonal) | Biolegend | RRID:AB_2563203 | FlowCyt (1:400) |
| Other | AnnexinV AlexaFluor647 | ThermoFisher | Cat# A23204 | Apoptotic cell labeling reagent |
| Antibody | anti-mouse CD68 FITC (rat monoclonal) | Biolegend | RRID:AB_10575475 | FlowCyt (1:300) |
| Antibody | anti-mouse LAMP1 AlexaFluor647 (rat monoclonal) | Biolegend | RRID:AB_571990 | FlowCyt (1:200) |
| Antibody | anti-mouse KI-67 BV421 (rat monoclonal) | Biolegend | RRID:AB_2629748 | FlowCyt (1:100) |
| Antibody | anti-mouse Ly6G PE (rat monoclonal) | BD Biosciences | RRID:AB_394208 | FlowCyt (1:100) |
| Antibody | anti-mouse Ly6C PE (rat monoclonal) | Biolegend | RRID:AB_1186133 | FlowCyt (1:100) |
| Antibody | anti-mouse CD3 PE (rat monoclonal) | BD Biosciences | RRID:AB_395699 | FlowCyt (1:100) |
| Antibody | anti-mouse CD19 PE (rat monoclonal) | BD Biosciences | RRID:AB_395050 | FlowCyt (1:100) |
| Antibody | anti-mouse NK1.1 PE (rat monoclonal) | BD Biosciences | RRID:AB_396674 | FlowCyt (1:100) |
| Antibody | anti-mouse CD45 AF488 (rat monoclonal) | Biolegend | RRID:AB_493531 | FlowCyt (1:100) |
| Antibody | anti-mouse CX3CR1 BV785 (rat monoclonal) | Biolegend | RRID:AB_2565938 | FlowCyt (1:200) |
| Antibody | anti-mouse CD11b APC (rat monoclonal) | Biolegend | RRID:AB_312794 | FlowCyt (1:200) |
| Antibody | anti-NHP CD45 BV786 (mouse monoclonal) | BD Biosciences | RRID:AB_2738454 | FlowCyt (5 µL/test) |
| Antibody | anti-NHP CD11b BV510 (mouse monoclonal) | BD Biosciences | RRID:AB_2737996 | FlowCyt (5 µL/test) |
| Genetic reagent (M. musculus) | Cx3cr1tm2.1(cre/ERT2)Jung (Cx3cr1–CreERT2) | PMID:23273845 | RRID:IMSR_JAX:020940 | Dr. Steffen Jung (Weizmann Institute of Science, Israel) |
| Genetic reagent (M. musculus) | Atg5flox | PMID:16625204 | Dr. Noboru Mizushima, (University of Tokyo) | |
| Genetic reagent (M. musculus) | Fcer1g-/- | Taconic Biosciences | Model 583 | PMID:8313472 |
| Genetic reagent (M. musculus) | Trem2-/- | KOMP repository | RRID:MMRRC_050209-UCD | |
| Genetic reagent (M. musculus) | Mbpshi | Jackson Laboratory | RRID:IMSR_JAX:001428 | PMID:6168677 |
| Genetic reagent (M. musculus) | Cln3e × 7/ex8 (Cln3 KI) | PMID:12374761 | Dr. Susan Cotman (Harvard Medical School) | |
| Software, algorithm | FlowJo V10 | TreeStar | RRID:SCR_008520 | |
| Software, algorithm | Prism 8.0 | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | FIJI | FIJI | RRID:SCR_002285 | |
| Software, algorithm | MaxQuant Version 1.6.0.16 | Max Planck | RRID:SCR_014485 | |
| Software, algorithm | Perseus | Max Planck | RRID:SCR_015753 | |
| Software, algorithm | Ingenuity Pathway Analysis | Qiagen | RRID:SCR_008653 | |
| Other | Percoll | GE Healthcare | Cat# 17-0891-01 | Medium for density gradient centrifugation |
| Other | Mitotracker DeepRedFM | ThermoFisher | Cat# M22426 | Mitochondrial fluorescent dye, used at 50 nM |
| Other | CellROX DeepRed | ThermoFisher | Cat# C10491 | ROS fluorescent detection agent, used at 500 nM |
| Sequence based reagent | Atg5 Taqman primers | ThermoFisher | AssayID: Mm01187301_m1, Cat# 4351372 | Spanning exons 3–4 |
| Commercial assay or kit | RNEasy Plus Micro | Qiagen | Cat# 74034 | |
| Commercial assay or kit | SuperScript IV VILO kit | ThermoFisher | Cat# 11754050 | |
| Sequence based reagent | Gapdh endogenous control primers, M. musculus | ThermoFisher | Cat# 4352339E | |
| Commercial assay or kit | QuantiTect Multiplex PCR | Qiagen | Cat# 204541 | |
| Software, algorithm | PANTHER | PANTHER | RRID:SCR_004869 | |
| Peptide, recombinant protein | Lysyl Endopeptidase, Mass Spectrometry Grade (Lys-C) | Wako | Cat# 121–05063 | |
| Peptide, recombinant protein | Sequencing Grade Modified Trypsin | Promega | Cat# V5113 |
