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Cited by 245Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsExtracellular vesicles in disease

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Plaque associated microglia hyper-secrete extracellular vesicles and accelerate tau propagation in a humanized APP mouse model

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Clayton, Kevin, Delpech, Jean Christophe, Herron, Shawn, Iwahara, Naotoshi, Ericsson, Maria, Saito, Takashi, Saido, Takaomi C., Ikezu, Seiko, Ikezu, Tsuneya·2021·Molecular Neurodegeneration
DOI 10.1186/s13024-021-00440-9PMC PMC7986521

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Abstract

Abstract Background Recent studies suggest that microglia contribute to tau pathology progression in Alzheimer’s disease. Amyloid plaque accumulation transforms microglia, the primary innate immune cells in the brain, into neurodegenerative microglia (MGnD), which exhibit enhanced phagocytosis of plaques, apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p-tau). It remains unclear how microglia promote disease progression while actively phagocytosing pathological proteins, therefore ameliorating pathology. Methods Adeno-associated virus expressing P301L tau mutant (AAV-P301L-tau) was stereotaxically injected into the medial entorhinal cortex (MEC) in C57BL/6 (WT) and humanized APP mutant knock-in homozygote ( App NL-G-F ) mice at 5 months of age. Mice were fed either chow containing a colony stimulating factor-1 receptor inhibitor (PLX5622) or control chow from 4 to 6 months of age to test the effect of microglia depletion. Animals were tested at 6 months of age for immunofluorescence, biochemistry, and FACS of microglia. In order to monitor microglial extracellular vesicle secretion in vivo, a novel lentiviral EV reporter system was engineered to express mEmerald-CD9 (mE-CD9) specifically in microglia, which was injected into the same region of MEC. Results Expressing P301L tau mutant in the MEC induced tau propagation to the granule cell layer of the hippocampal dentate gyrus, which was significantly exacerbated in App NL-G-F mice compared to WT control mice. Administration of PLX5622 depleted nearly all microglia in mouse brains and dramatically reduced propagation of p-tau in WT and to a greater extent in App NL-G-F mice, although it increased plaque burden and plaque-associated p-tau + dystrophic neurites. Plaque-associated MGnD microglia strongly expressed an EV marker, tumor susceptibility gene 101, indicative of heightened synthesis of EVs. Intracortical injection of mE-CD9 lentivirus successfully induced microglia-specific expression of mE-CD9 + EV particles, which were significantly enhanced in Mac2 + MGnD microglia compared to Mac2 − homeostatic microglia. Finally, consecutive intracortical injection of mE-CD9 lentivirus and AAV-P301L-tau into App NL-G-F mice revealed encapsulation of p-tau in microglia-specific mE-CD9 + EVs as determined by super-resolution microscopy and immuno-electron microscopy. Discussion Our findings suggest that MGnD microglia hyper-secrete p-tau + EVs while compacting Aβ plaques and clearing NP tau, which we propose as a novel mechanistic link between amyloid plaque deposition and exacerbation of tau propagation in App NL-G-F mice.

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Fig. 1 PLX5622 treatment ablates microglia and increases amyloid plaque burden. a. Schematic of study design with PLX5622 administration. The table displays the number of mice used per experimental group. b. Representative images of IBA1 staining in the cortical region from WT and AppNL-G-F mice administered with control (CTRL) or PLX5622 chow. c. Unbiased quantification of percentage IBA1+ area in the cortex. d. Representative images of Thioflavin-S staining in the cortex. e. Unbiased quantification of plaque characteristics in Thioflavin-S stained slices in the cortex. f. Representative images of 4G8 staining in the cortex. g. Unbiased quantification of plaque characteristics in 4G8 stained slices in the cortex. h. Representative images of 82E1 staining in the cortex. i. Unbiased quantification of the plaque characteristics in 82E1 stained slices in the cortex. Representative images displayed in a-i are a mix of male and female mice. All values displayed in a-i represent the mean ± standard error (SEM) for a minimum of 6 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way analysis of variance (ANOVA) with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via Unpaired t-test. *p < 0.05, ** p < 0.01, *** p < 0.001, between indicated groups. #### p < 0.0001 for the PLX5622 factor
Fig. 1 PLX5622 treatment ablates microglia and increases amyloid plaque burden. a. Schematic of study design with PLX5622 administration. The table displays the number of mice used per experimental group. b. Representative images of IBA1 staining in the cortical region from WT and AppNL-G-F mice administered with control (CTRL) or PLX5622 chow. c. Unbiased quantification of percentage IBA1+ area in the cortex. d. Representative images of Thioflavin-S staining in the cortex. e. Unbiased quantification of plaque characteristics in Thioflavin-S stained slices in the cortex. f. Representative images of 4G8 staining in the cortex. g. Unbiased quantification of plaque characteristics in 4G8 stained slices in the cortex. h. Representative images of 82E1 staining in the cortex. i. Unbiased quantification of the plaque characteristics in 82E1 stained slices in the cortex. Representative images displayed in a-i are a mix of male and female mice. All values displayed in a-i represent the mean ± standard error (SEM) for a minimum of 6 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way analysis of variance (ANOVA) with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via Unpaired t-test. *p < 0.05, ** p < 0.01, *** p < 0.001, between indicated groups. #### p < 0.0001 for the PLX5622 factorCC BY · PubMed Central
Fig. 2 Aβ deposition accelerates tau propagation in a microglia-dependent manner, while microglial depletion enhances NP tau formation. a. Schematic of injection coordinate and tri-synaptic pathway. b. Representative images of AT8 staining in the MEC and the GCL region of the DG. c. Unbiased quantification of AT8+ cell propagation from the MEC to the GCL. Tau propagation values across all 4 treatment groups are displayed. d. Representative image of plaques in the MEC of non-injected AppNL-G-F mice, injected mice receiving control chow, and injected mice receiving PLX5622. e. Unbiased quantification of percentage AT8+ plaque area within the MEC. Representative images displayed in a-e are a mix of male and female mice. All values displayed in a-e represent the mean ± SEM. Graphs comparing values across 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing values across 3 groups were analyzed via 1-way ANOVA with Fisher’s LSD post-hoc analysis for individual comparisons. * p < 0.05, ** p < 0.01 between indicated groups
Fig. 2 Aβ deposition accelerates tau propagation in a microglia-dependent manner, while microglial depletion enhances NP tau formation. a. Schematic of injection coordinate and tri-synaptic pathway. b. Representative images of AT8 staining in the MEC and the GCL region of the DG. c. Unbiased quantification of AT8+ cell propagation from the MEC to the GCL. Tau propagation values across all 4 treatment groups are displayed. d. Representative image of plaques in the MEC of non-injected AppNL-G-F mice, injected mice receiving control chow, and injected mice receiving PLX5622. e. Unbiased quantification of percentage AT8+ plaque area within the MEC. Representative images displayed in a-e are a mix of male and female mice. All values displayed in a-e represent the mean ± SEM. Graphs comparing values across 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing values across 3 groups were analyzed via 1-way ANOVA with Fisher’s LSD post-hoc analysis for individual comparisons. * p < 0.05, ** p < 0.01 between indicated groupsCC BY · PubMed Central
Fig. 3 Increased synaptic tagging and EV release signatures in the amyloid bearing brain. a. Representative images of C1Q, 4G8, and DAPI staining in the hippocampal region of WT and AppNL-G-F mice administered with control (CTRL) or PLX5622 chow as well as C1Q KO mice. b. Unbiased quantification of C1Q intensity in the OML of the hippocampus. c. Representative stacked confocal images of 4G8, Clec7A, and Tsg101 staining of plaques in the OML. See also Supplemental Fig. S3A and Video S1. d. Unbiased quantification of percentage Clec7A+ area across the OML for all groups and of the plaque-specific regions. e. Unbiased quantification of Tsg101 intensity of plaque-positive regions in the OML compared to background signal. f. qPCR data displaying fold-increases in MGnD and EV-associated markers ApoE, C1Qa, CD9, CD81, CD63, Tsg101, and P2RY12 expressed in Clec7A+ over Clec7A− microglia. Representative images displayed in a-f are a mix of male and female mice. All values displayed in a-f represent the mean ± SEM for a minimum of 3 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via Unpaired t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between indicated groups. ## p < 0.01 for the PLX5622 factor
Fig. 3 Increased synaptic tagging and EV release signatures in the amyloid bearing brain. a. Representative images of C1Q, 4G8, and DAPI staining in the hippocampal region of WT and AppNL-G-F mice administered with control (CTRL) or PLX5622 chow as well as C1Q KO mice. b. Unbiased quantification of C1Q intensity in the OML of the hippocampus. c. Representative stacked confocal images of 4G8, Clec7A, and Tsg101 staining of plaques in the OML. See also Supplemental Fig. S3A and Video S1. d. Unbiased quantification of percentage Clec7A+ area across the OML for all groups and of the plaque-specific regions. e. Unbiased quantification of Tsg101 intensity of plaque-positive regions in the OML compared to background signal. f. qPCR data displaying fold-increases in MGnD and EV-associated markers ApoE, C1Qa, CD9, CD81, CD63, Tsg101, and P2RY12 expressed in Clec7A+ over Clec7A− microglia. Representative images displayed in a-f are a mix of male and female mice. All values displayed in a-f represent the mean ± SEM for a minimum of 3 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via Unpaired t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 between indicated groups. ## p < 0.01 for the PLX5622 factorCC BY · PubMed Central
Fig. 4 In vivo imaging of EV secretion from microglia after lentiviral microglia-specific expression of mE-CD9. a. Schematic of mE-CD9 lentiviral construct. b. Representative images of mE-CD9 lentivirus transduction in WT and AppNL-G-F microglia following injection and 10-day incubation. c. Representative images of individual microglia and released mE-CD9+ particles showing FSB, Mac2, and mE-CD9. d. Unbiased quantification of mE-CD9+ voxels surrounding individual microglia (n = 12 microglia per group from 3 mice per group). e. Regression plot of Mac2 staining intensity versus the number of mE-CD9 particles released by individual microglia. Representative images displayed in a-e are a mix of male and female mice. All values displayed in a-e represent the mean ± SEM for a minimum of 3 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via unpaired t-test. ** p < 0.01, *** p < 0.001, between indicated groups
Fig. 4 In vivo imaging of EV secretion from microglia after lentiviral microglia-specific expression of mE-CD9. a. Schematic of mE-CD9 lentiviral construct. b. Representative images of mE-CD9 lentivirus transduction in WT and AppNL-G-F microglia following injection and 10-day incubation. c. Representative images of individual microglia and released mE-CD9+ particles showing FSB, Mac2, and mE-CD9. d. Unbiased quantification of mE-CD9+ voxels surrounding individual microglia (n = 12 microglia per group from 3 mice per group). e. Regression plot of Mac2 staining intensity versus the number of mE-CD9 particles released by individual microglia. Representative images displayed in a-e are a mix of male and female mice. All values displayed in a-e represent the mean ± SEM for a minimum of 3 animals per group. Graphs comparing values across all 4 groups were analyzed via 2-way ANOVA with Tukey post-hoc analysis for individual comparisons. Graphs comparing two groups were analyzed via unpaired t-test. ** p < 0.01, *** p < 0.001, between indicated groupsCC BY · PubMed Central
Fig. 5 Pathologic tau is secreted through microglia-derived EVs. a: The timeline of injection of mE-CD9 lentivirus and AAV-P301L-tau. b: Representative low-magnification images of the injection site in the MEC showing FSB (blue: amyloid plaque), mE-CD9 (green), Mac2 (red), and AT8 (magenta). c: Left: Representative images of homeostatic microglia (Mac2−) and MGnD (Mac2+) with phagocytosed AT8+ p-tau and secreted mE-CD9+ EVs (white) surrounding microglia. Right: Surface rendering images by IMARIS software showing internalized p-tau within microglia or EVs. d: Representative images of microglia-derived mE-CD9+ EVs (white) containing p-tau (magenta). See also Supplementary Video S3. e: Immuno-gold electron microscopy images of microglia-derived EV containing mE-CD9 fusion protein (GFP+ 10 nm immuno-gold dots) and p-tau (AT8+ 5 nm immuno-gold dots). f: Quantification of mE-CD9+ EVs released from homeostatic microglia (Mac2−) and MGnD (Mac2+) in the injected region. g: Volume of phagocytosed p-tau per microglia h: Quantification of total p-tau released through mE-CD9+ EVs per microglia. Representative images displayed in a-h are a mix of male and female mice. f-h: n = 8 microglia per group from 5 WT and 5 AppNL-G-F animals. Graphs comparing values across all 4 groups were analyzed via 2-way analysis of variance (ANOVA) with Tukey post-hoc analysis for individual comparisons. * p < 0.05 between indicated groups, # p < 0.05, ## p < 0.01 for the Mac2 factor
Fig. 5 Pathologic tau is secreted through microglia-derived EVs. a: The timeline of injection of mE-CD9 lentivirus and AAV-P301L-tau. b: Representative low-magnification images of the injection site in the MEC showing FSB (blue: amyloid plaque), mE-CD9 (green), Mac2 (red), and AT8 (magenta). c: Left: Representative images of homeostatic microglia (Mac2−) and MGnD (Mac2+) with phagocytosed AT8+ p-tau and secreted mE-CD9+ EVs (white) surrounding microglia. Right: Surface rendering images by IMARIS software showing internalized p-tau within microglia or EVs. d: Representative images of microglia-derived mE-CD9+ EVs (white) containing p-tau (magenta). See also Supplementary Video S3. e: Immuno-gold electron microscopy images of microglia-derived EV containing mE-CD9 fusion protein (GFP+ 10 nm immuno-gold dots) and p-tau (AT8+ 5 nm immuno-gold dots). f: Quantification of mE-CD9+ EVs released from homeostatic microglia (Mac2−) and MGnD (Mac2+) in the injected region. g: Volume of phagocytosed p-tau per microglia h: Quantification of total p-tau released through mE-CD9+ EVs per microglia. Representative images displayed in a-h are a mix of male and female mice. f-h: n = 8 microglia per group from 5 WT and 5 AppNL-G-F animals. Graphs comparing values across all 4 groups were analyzed via 2-way analysis of variance (ANOVA) with Tukey post-hoc analysis for individual comparisons. * p < 0.05 between indicated groups, # p < 0.05, ## p < 0.01 for the Mac2 factorCC BY · PubMed Central
Fig. 6 Schematic diagram of Aβ plaque deposition and microglia-mediated tau propagation. In the WT mouse brain, tau propagation from the MEC to GCL neurons is sensitive to microglia. In AppNL-G-F mouse brains, Aβ plaque associated microglia are more phagocytic and hyper-secrete tau-seeding EVs compared to homeostatic microglia, resulting in enhanced tau propagation from MEC to GCL regions. Both Aβ plaque and NP tau pathology are increased following microglia depletion, suggesting their active roles on compaction and clearance of Aβ plaques and NP tau
Fig. 6 Schematic diagram of Aβ plaque deposition and microglia-mediated tau propagation. In the WT mouse brain, tau propagation from the MEC to GCL neurons is sensitive to microglia. In AppNL-G-F mouse brains, Aβ plaque associated microglia are more phagocytic and hyper-secrete tau-seeding EVs compared to homeostatic microglia, resulting in enhanced tau propagation from MEC to GCL regions. Both Aβ plaque and NP tau pathology are increased following microglia depletion, suggesting their active roles on compaction and clearance of Aβ plaques and NP tauCC BY · PubMed Central

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Authors
Clayton, K., Delpech, J. C., Herron, S., Iwahara, N., Ericsson, M., Saito, T., Saido, T. C., Ikezu, S., & Ikezu, T.
Affiliations
  • Boston University (US)
  • Harvard University (US)
  • RIKEN Center for Brain Science (JP)
Corresponding author
Ikezu, Tsuneya
ORCID
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2021-03-22
Volume
16
Issue
1
Pages
18-18
ISSN
1750-1326 (ISSN-L)
DOI
10.1186/s13024-021-00440-9
PMC
PMC7986521
OpenAlex
W3137321433
Cited by
245
References
73
Citations by year
2026: 35 · 2025: 42 · 2024: 49 · 2023: 43 · 2022: 49 · 2021: 26
FWCI
26.24
Type
journal article
Language
en
Update notices
correction (2021-04-14)
Open access
Licence
Locations
  • Molecular Neurodegeneration (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • OpenBU (Boston University) (submittedVersion)
  • Oskar-Bordeaux (Universite de Bordeaux) (submittedVersion, cc-by)
Funders
  • Alzheimer's Association (DVT-14-320835)
  • BrightFocus Foundation (A2016551S)
  • Cure Alzheimer's Fund (N/A)
  • National Institute on Aging (R01 AG066429, RF1 AG054199, R56 AG057469, P30 AG0138423, F31 AG057170)
  • NIGMS (T32 GM008541)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
microglia, extracellular vesicles, tau propagation, Alzheimer's disease, amyloid plaques, PLX5622, CSF1R inhibition, microglial depletion, CD9, TSG101, medial entorhinal cortex, dentate gyrus, dystrophic neurites, tau phosphorylation, plaque-associated microglia, disease progression
MeSH terms
Extracellular Vesicles, Animals, Female, Genetic Vectors, Humans, Injections, Male, Mice, Inbred C57BL, Organic Chemicals, Recombinant Proteins, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor, Neurites, Amyloid beta-Protein Precursor, tau Proteins, Point Mutation, Microglia, Entorhinal Cortex, Dentate Gyrus, Mutation, Missense, Mice, Gene Knock-In Techniques, Plaque, Amyloid, Protein Aggregation, Pathological

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