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Cited by 127Alzheimer's disease research and treatmentsNeuroinflammation and Neurodegeneration MechanismsExtracellular vesicles in disease

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Trem2 deletion enhances tau dispersion and pathology through microglia exosomes

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Zhu, Bing, Liu, Yan, Hwang, Spring, Archuleta, Kailey, Huang, Huijie, Campos, Alex, Murad, Rabi, Piña-Crespo, Juan, Xu, Huaxi, Huang, Timothy Y.·2022·Molecular Neurodegeneration
DOI 10.1186/s13024-022-00562-8PMC PMC9438095
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Abstract

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder that manifests sequential Aβ and tau brain pathology with age-dependent onset. Variants in the microglial immune receptor TREM2 are associated with enhanced risk of onset in sporadic Alzheimer’s disease (AD). While recent studies suggest TREM2 dysfunction can aggravate tau pathology, mechanisms underlying TREM2-dependent modulation of tau pathology remains elusive. Methods: Here, we characterized differences in progressive tau spreading from the medial entorhinal cortex (MEC) to the hippocampus in wildtype (WT) and Trem2 knockout (KO) mice by injection of AAV-P301L tau into the MEC, and correlated changes in hippocampal tau histopathology with spatial and fear memory. We also compared effects of intraneuronal dispersion between cultured microglia and neurons using a microfluidic dispersion assay, analyzed differences in microglial tau trafficking following uptake, and quantified exosomal tau secretion and pathogenicity from purified WT and Trem2 KO exosomes. Results: Trem2 deletion in mice (Trem2 KO) can enhance tau spreading from the medial entorhinal cortex (MEC) to the hippocampus, which coincides with impaired synaptic function and memory behavior. Trem2 deletion in microglia enhances intraneuronal dispersion of tau in vitro between neuronal layers cultured in a microfluidic chamber, and the presence of exosome inhibitors can significantly reduce tau in exosomes and extracellular media from tau-loaded microglia. Although microglial Trem2 deletion has no effect on tau uptake, Trem2 deletion enhances distribution to endosomal and cellular pre-exosomal compartments following internalization. Trem2 deletion has little effect on exosome size, however, proteomic analysis indicates that Trem2 deletion can modulate changes in the microglial proteomic landscape with tau and LPS/ATP treatment conditions associated with exosome induction. Furthermore, exosomes from Trem2 KO microglia show elevated tau levels, and feature enhanced tau-seeding capacity in a tau FRET reporter line compared to exosomes from WT microglia. Conclusion: Together, our results reveal a role for Trem2 in suppressing exosomal tau pathogenicity, and demonstrates that Trem2 deletion can enhance tau trafficking, distribution and seeding through microglial exosomes. Graphical abstract:

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Figures

Fig. 1 Trem2 deletion aggravates pathogenic dispersion of tau pathology from the entorhinal cortex to the hippocampus. A Schematic depicting AAV-P301L tau (AAV-tau) injection and analysis. B AAV particles expressing human tau (AAV-P301L tau, “AAV-tau”) were stereotactically injected into the medial entorhinal cortex (MEC) of wildtype (WT) or Trem2 KO mice at 4 months of age, and histological images from the MEC (upper panels) or hippocampal dentate gyrus (DG) (lower panels) were obtained by confocal microscopy 5 weeks post-injection. Representative images from the MEC were stained with AT8 (pS202, T205 tau) and AT180 (pT231 tau) antibodies (green), in addition to Iba1 (purple), and DAPI (blue) as indicated. Bar = 100 μm. C Quantification of AT8 and AT180-positive cells/mm2 in the MEC region or DG from WT and Trem2 KO brain (n = 8 animals/genotype); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (****, p < 0.0001). D Representative histological images in MEC and DG brain regions stained for human tau (T13, green), Iba1 (purple) and nuclei (DAPI, blue) in WT and Trem2 KO mouse brain 5 weeks following AAV-tau injection. Bar = 50 μm. E Quantification of htau (T13)-positive cells/mm2 and number of Iba1-positive microglia in the MEC region or DG from WT and Trem2 KO brain (n = 8 animal/genotype); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (****p < 0.0001)
Fig. 1 Trem2 deletion aggravates pathogenic dispersion of tau pathology from the entorhinal cortex to the hippocampus. A Schematic depicting AAV-P301L tau (AAV-tau) injection and analysis. B AAV particles expressing human tau (AAV-P301L tau, “AAV-tau”) were stereotactically injected into the medial entorhinal cortex (MEC) of wildtype (WT) or Trem2 KO mice at 4 months of age, and histological images from the MEC (upper panels) or hippocampal dentate gyrus (DG) (lower panels) were obtained by confocal microscopy 5 weeks post-injection. Representative images from the MEC were stained with AT8 (pS202, T205 tau) and AT180 (pT231 tau) antibodies (green), in addition to Iba1 (purple), and DAPI (blue) as indicated. Bar = 100 μm. C Quantification of AT8 and AT180-positive cells/mm2 in the MEC region or DG from WT and Trem2 KO brain (n = 8 animals/genotype); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (****, p < 0.0001). D Representative histological images in MEC and DG brain regions stained for human tau (T13, green), Iba1 (purple) and nuclei (DAPI, blue) in WT and Trem2 KO mouse brain 5 weeks following AAV-tau injection. Bar = 50 μm. E Quantification of htau (T13)-positive cells/mm2 and number of Iba1-positive microglia in the MEC region or DG from WT and Trem2 KO brain (n = 8 animal/genotype); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (****p < 0.0001)CC BY · PubMed Central
Fig. 2 Characterizing microglial activation with MEC AAV-tau transduction. A MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Cd68 (originally purple, shown in green), P2y12 (red) and nuclei (DAPI, blue), magnified regions are shown for Cd68/P2y12 colocalization. Bar = 50 μm. B Quantification of percentage P2y12 (top graphs), Cd68 (middle graphs) or P2y12/Cd68 double-positive microglia (bottom graphs) in the MEC region (left graphs) or DG (right graphs) from WT and Trem2 KO brain (n = 8 animal/genotype). C MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Iba1 (originally purple, shown in green), CD9 (red) and nuclei (DAPI, blue), magnified regions are shown for Iba1/CD9 colocalization. Bar = 50 μm. D Quantification of relative CD9 intensity (compared to WT, set to 1.0), or % CD9/Iba1 double-positive microglia in MEC or DG regions as indicated from WT and Trem2 KO brain (n = 8 animal/genotype). E MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Clec7a (originally purple, shown in green), Tmem119 (red) and nuclei (DAPI, blue), magnified regions are shown for Clec7a and Tmem119 colocalization. Bar = 50 μm. F Quantification of % Tmem119 positive (top graphs), Clec7a-positive (middle graphs), or Tmem119/Clec7a double positive (bottom graphs) microglia in MEC or DG regions as indicated from WT and Trem2 KO brain (n = 8 animal/genotype). In (B, D and F), graphs represent mean ± SE from WT and Trem2 KO brain (n = 8 animal/genotype). Statistical significance was determined using unpaired Student’s t-tests (**p < 0.01, ***p < 0.001, **** p < 0.0001)
Fig. 2 Characterizing microglial activation with MEC AAV-tau transduction. A MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Cd68 (originally purple, shown in green), P2y12 (red) and nuclei (DAPI, blue), magnified regions are shown for Cd68/P2y12 colocalization. Bar = 50 μm. B Quantification of percentage P2y12 (top graphs), Cd68 (middle graphs) or P2y12/Cd68 double-positive microglia (bottom graphs) in the MEC region (left graphs) or DG (right graphs) from WT and Trem2 KO brain (n = 8 animal/genotype). C MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Iba1 (originally purple, shown in green), CD9 (red) and nuclei (DAPI, blue), magnified regions are shown for Iba1/CD9 colocalization. Bar = 50 μm. D Quantification of relative CD9 intensity (compared to WT, set to 1.0), or % CD9/Iba1 double-positive microglia in MEC or DG regions as indicated from WT and Trem2 KO brain (n = 8 animal/genotype). E MEC and DG regions from WT or Trem2 KO mouse brain 5 weeks following AAV-tau injection. Sections were stained for AT8 (originally green, shown in purple), Clec7a (originally purple, shown in green), Tmem119 (red) and nuclei (DAPI, blue), magnified regions are shown for Clec7a and Tmem119 colocalization. Bar = 50 μm. F Quantification of % Tmem119 positive (top graphs), Clec7a-positive (middle graphs), or Tmem119/Clec7a double positive (bottom graphs) microglia in MEC or DG regions as indicated from WT and Trem2 KO brain (n = 8 animal/genotype). In (B, D and F), graphs represent mean ± SE from WT and Trem2 KO brain (n = 8 animal/genotype). Statistical significance was determined using unpaired Student’s t-tests (**p < 0.01, ***p < 0.001, **** p < 0.0001)CC BY · PubMed Central
Fig. 3 Trem2 deletion aggravates memory and synaptic impairment associated with AAV-tau expression. A-B Trem2 deletion aggravates tau-mediated spatial memory impairment. WT or Trem2 KO animals were stereotactically injected with AAV control (AAV9-synapsin GFP) or AAV-tau constructs into the MEC region at 4 months of age, and subjected to 5 days of navigational training using the Barnes land maze (A); training of a cohort 5-weeks following AAV injection is shown. B 2 days following last training, spatial memory was evaluated by probe test in cohorts 2 weeks (left graph, n = 6 animals per genotype/treatment) or 5 weeks (right graph, WT/AAV con – n = 10; Trem2 KO/con – n = 10; WT/AAV tau – n = 9; Trem2 KO/AAV-tau – n = 8 animals) after AAV injection. Representative traces of motion tracks for WT and Trem2 KO animals 5 weeks after AAV injection are shown; red circle indicates the target hole (traces, right panel). C-D Trem2 deletion aggravates tau-mediated contextual fear memory impairment. WT or Trem2 KO animals were stereotactically injected with AAV control or AAV-tau constructs as in (A), and characterized for freezing time during pre-conditioning 5 weeks after AAV injection (C), and in fear conditioning probe tests 2 weeks (top, n = 6 animals per genotype/treatment) or 5 weeks (bottom, WT/AAV con – n = 10; Trem2 KO/con – n = 10; WT/AAV tau – n = 9; Trem2 KO/tau – n = 8 animals) after AAV injection (D). E Population spike amplitude recorded in the DG region from acute hippocampal slices in WT and Trem2 KO animals 5 weeks following AAV injection; population spike amplitudes are shown (WT control, 12 slices from n = 4 animals; WT AAV-tau, 13 slices from n = 4 animals; Trem2 KO control, 15 slices from n = 5 animals; Trem2 KO AAV-tau, 15 slices from n = 5 animals). Representative traces on the left depict depolarizing spikes following stimulation in WT and Trem2 KO hippocampus as indicated. Graphs depict mean ± SE (error bars in (E) represent SE from total slice recordings from each experimental group), statistical significance was determined using One-way ANOVA with Dunn’s multiple comparison (D) and Two-way ANOVA (B, E) (*p < 0.05, **p < 0.01). F Representative histological images in MEC and DG brain regions stained for human tau (T13, green), PSD95 (red), Iba1 (purple) and nuclei (DAPI, blue) in WT and Trem2 KO mouse brain 5 weeks following AAV-tau injection. Bar = 50 μm. G Quantification of PSD95 particles in DG from WT and Trem2 KO brain (n = 8 animal/genotype) in (F); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (***p < 0.001)
Fig. 3 Trem2 deletion aggravates memory and synaptic impairment associated with AAV-tau expression. A-B Trem2 deletion aggravates tau-mediated spatial memory impairment. WT or Trem2 KO animals were stereotactically injected with AAV control (AAV9-synapsin GFP) or AAV-tau constructs into the MEC region at 4 months of age, and subjected to 5 days of navigational training using the Barnes land maze (A); training of a cohort 5-weeks following AAV injection is shown. B 2 days following last training, spatial memory was evaluated by probe test in cohorts 2 weeks (left graph, n = 6 animals per genotype/treatment) or 5 weeks (right graph, WT/AAV con – n = 10; Trem2 KO/con – n = 10; WT/AAV tau – n = 9; Trem2 KO/AAV-tau – n = 8 animals) after AAV injection. Representative traces of motion tracks for WT and Trem2 KO animals 5 weeks after AAV injection are shown; red circle indicates the target hole (traces, right panel). C-D Trem2 deletion aggravates tau-mediated contextual fear memory impairment. WT or Trem2 KO animals were stereotactically injected with AAV control or AAV-tau constructs as in (A), and characterized for freezing time during pre-conditioning 5 weeks after AAV injection (C), and in fear conditioning probe tests 2 weeks (top, n = 6 animals per genotype/treatment) or 5 weeks (bottom, WT/AAV con – n = 10; Trem2 KO/con – n = 10; WT/AAV tau – n = 9; Trem2 KO/tau – n = 8 animals) after AAV injection (D). E Population spike amplitude recorded in the DG region from acute hippocampal slices in WT and Trem2 KO animals 5 weeks following AAV injection; population spike amplitudes are shown (WT control, 12 slices from n = 4 animals; WT AAV-tau, 13 slices from n = 4 animals; Trem2 KO control, 15 slices from n = 5 animals; Trem2 KO AAV-tau, 15 slices from n = 5 animals). Representative traces on the left depict depolarizing spikes following stimulation in WT and Trem2 KO hippocampus as indicated. Graphs depict mean ± SE (error bars in (E) represent SE from total slice recordings from each experimental group), statistical significance was determined using One-way ANOVA with Dunn’s multiple comparison (D) and Two-way ANOVA (B, E) (*p < 0.05, **p < 0.01). F Representative histological images in MEC and DG brain regions stained for human tau (T13, green), PSD95 (red), Iba1 (purple) and nuclei (DAPI, blue) in WT and Trem2 KO mouse brain 5 weeks following AAV-tau injection. Bar = 50 μm. G Quantification of PSD95 particles in DG from WT and Trem2 KO brain (n = 8 animal/genotype) in (F); graphs represent mean ± SE. Statistical significance was determined using unpaired Student’s t-tests (***p < 0.001)CC BY · PubMed Central
Fig. 4 Trem2 deletion enhances interneuronal tau transduction. A Schematic representation of a microglia-mediated interneuronal tau transduction system using a microfluidic 3-chamber culture system (left); a representative 3-chamber culture setup is shown (right). WT murine neurons are cultured in first and 3rd layers (Layers 1 and 3) (tubulin, red), and WT or Trem2 KO microglia (Iba1, purple) are cultured in the intermediary chamber (Layer 2). AAV-P301L tau is transduced in the first layer, and effects of microglia in intermediary Layer 2 are determined on transmission of tau into neuronal Layer 3 by staining for human tau. B Neurons are exposed to 1 × 1010 VP/ml AAV-P301L tau in Layer 1, and transduction of P301L tau from Layer 1 into Layers 2 (microglia) and 3 (neurons) is quantified following transduction in the presence of WT or Trem2 KO microglia, or without microglia in intermediary Layer 2 as indicated. Fluorescence imaging was used to characterize neuronal processes (tubulin, red), recombinant tau (h-tau, green), and microglia (Iba1). C Fluorescence intensity of human tau, tubulin, and neuronal numbers quantified in Layers 1 and 3, and quantification of microglia (Iba1) and Iba1/tau overlap in Layer 2. Graph depicts mean ± SE of 3 independent experiments (individual plots), averaged from 3 replicates per experiment. Statistical significance was determined by One-way ANOVA, with Tukey’s multiple comparison (*p < 0.05, **p < 0.01, ***p < 0.001)
Fig. 4 Trem2 deletion enhances interneuronal tau transduction. A Schematic representation of a microglia-mediated interneuronal tau transduction system using a microfluidic 3-chamber culture system (left); a representative 3-chamber culture setup is shown (right). WT murine neurons are cultured in first and 3rd layers (Layers 1 and 3) (tubulin, red), and WT or Trem2 KO microglia (Iba1, purple) are cultured in the intermediary chamber (Layer 2). AAV-P301L tau is transduced in the first layer, and effects of microglia in intermediary Layer 2 are determined on transmission of tau into neuronal Layer 3 by staining for human tau. B Neurons are exposed to 1 × 1010 VP/ml AAV-P301L tau in Layer 1, and transduction of P301L tau from Layer 1 into Layers 2 (microglia) and 3 (neurons) is quantified following transduction in the presence of WT or Trem2 KO microglia, or without microglia in intermediary Layer 2 as indicated. Fluorescence imaging was used to characterize neuronal processes (tubulin, red), recombinant tau (h-tau, green), and microglia (Iba1). C Fluorescence intensity of human tau, tubulin, and neuronal numbers quantified in Layers 1 and 3, and quantification of microglia (Iba1) and Iba1/tau overlap in Layer 2. Graph depicts mean ± SE of 3 independent experiments (individual plots), averaged from 3 replicates per experiment. Statistical significance was determined by One-way ANOVA, with Tukey’s multiple comparison (*p < 0.05, **p < 0.01, ***p < 0.001)CC BY · PubMed Central
Fig. 5 Trem2 deletion promotes tau distribution to endosomal and pre-exosomal compartments, and promotes exosomal tau release. A Trem2 deletion enhances endosomal tau sorting to pre-exosomal compartments. WT or Trem2 KO microglia were incubated with 2.5 μg/ml tau oligomers for 24 h, and stained for endosomal (Rab5, Rab7), lysosomal (LAMP1), and markers for pre-exosomal vesicles (CD63, Tsg101) (red), and co-localization with human tau (green) was visualized by confocal microscopy. Region of overlap between intracellular markers (red) and tau (green) were determined as a proportion of total internalized tau in microglia the graphs shown (mean ± SE, where individual plots represent one cell from 3 independent experiments). Bar = 5 μm. Statistical significance was determined by unpaired Student’s t-test (***p < 0.001). B Experimental schematic: WT or Trem2 KO microglia were loaded with 2.5 μg/ml tau oligomers for 24 h, washed with PBS and treated with 10 μM GW4869 for 3 h. Microglia were then treated with LPS/ATP and conditioned media was collected and subjected to tau quantification (ELISA) or neuronal tau uptake. C Exosomes were purified from conditioned media from tau-loaded WT or Trem2 KO microglia in the presence (+inhib) or absence (con) of GW4869, and subjected to ELISA analysis to quantify exosomal tau released with LPS/ATP in conditioned media. D-F Quantifying tau uptake from microglia conditioned media in primary neurons. Primary WT mouse neurons were incubated for 48 h with conditioned media from tau-loaded WT or Trem2 KO microglia untreated (D) or treated with GW4869 (E), and tau uptake was observed by confocal microscopy in cells stained for tau (T13, green) and tubulin (red); nuclei were visualized by DAPI staining (blue). Bar = 20 μm. F Quantification of tau puncta in neurons (left graph, tau puncta/neuron) or tau staining intensity (right graph, arbitrary units). Graphs represent mean ± SE, statistical significance was determined by One-way ANOVA with Tukey’s multiple comparison (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). G Electron micrograph of exosomes prepared from WT or Trem2 KO microglia. Bar = 100 nm. H, I Trem2 deletion does not affect exosomal size. Exosomes purified from WT or Trem2 KO microglia were plotted for exosomal vesicle size (H), as well as vesicle size distribution (I). Mean and quartiles are shown in violin plots in (H), graph in (I) represents mean ± SE
Fig. 5 Trem2 deletion promotes tau distribution to endosomal and pre-exosomal compartments, and promotes exosomal tau release. A Trem2 deletion enhances endosomal tau sorting to pre-exosomal compartments. WT or Trem2 KO microglia were incubated with 2.5 μg/ml tau oligomers for 24 h, and stained for endosomal (Rab5, Rab7), lysosomal (LAMP1), and markers for pre-exosomal vesicles (CD63, Tsg101) (red), and co-localization with human tau (green) was visualized by confocal microscopy. Region of overlap between intracellular markers (red) and tau (green) were determined as a proportion of total internalized tau in microglia the graphs shown (mean ± SE, where individual plots represent one cell from 3 independent experiments). Bar = 5 μm. Statistical significance was determined by unpaired Student’s t-test (***p < 0.001). B Experimental schematic: WT or Trem2 KO microglia were loaded with 2.5 μg/ml tau oligomers for 24 h, washed with PBS and treated with 10 μM GW4869 for 3 h. Microglia were then treated with LPS/ATP and conditioned media was collected and subjected to tau quantification (ELISA) or neuronal tau uptake. C Exosomes were purified from conditioned media from tau-loaded WT or Trem2 KO microglia in the presence (+inhib) or absence (con) of GW4869, and subjected to ELISA analysis to quantify exosomal tau released with LPS/ATP in conditioned media. D-F Quantifying tau uptake from microglia conditioned media in primary neurons. Primary WT mouse neurons were incubated for 48 h with conditioned media from tau-loaded WT or Trem2 KO microglia untreated (D) or treated with GW4869 (E), and tau uptake was observed by confocal microscopy in cells stained for tau (T13, green) and tubulin (red); nuclei were visualized by DAPI staining (blue). Bar = 20 μm. F Quantification of tau puncta in neurons (left graph, tau puncta/neuron) or tau staining intensity (right graph, arbitrary units). Graphs represent mean ± SE, statistical significance was determined by One-way ANOVA with Tukey’s multiple comparison (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). G Electron micrograph of exosomes prepared from WT or Trem2 KO microglia. Bar = 100 nm. H, I Trem2 deletion does not affect exosomal size. Exosomes purified from WT or Trem2 KO microglia were plotted for exosomal vesicle size (H), as well as vesicle size distribution (I). Mean and quartiles are shown in violin plots in (H), graph in (I) represents mean ± SECC BY · PubMed Central
Fig. 6 Characterization of proteomic profiles associated with exosome induction in Trem2 KO microglia. A Schematic of the proteomic analysis scheme used; WT or Trem2 KO microglia were left untreated or treated with tau oligomers (run 1) or LPS/ATP (run 2) and microglia lysates were subjected to proteomic analysis by label-free mass spectrometry. B, C PCA plots depicting distribution and clustering of proteomic profiles from WT and Trem2 KO (“KO”) microglia untreated or exposed to tau oligomers (B) or LPS/ATP (C). PCA distribution of 6 individual replicate samples for each treatment group are shown. D Total (left graph) and upregulated (black)/downregulated (gray) (right graph) differentially expressed proteins (DEPs) in microglial cell lysates in four comparative analyses indicated (run 1): Trem2 KO vs WT (both untreated), Trem2 KO vs WT (both +tau), WT – tau vs untreated, and Trem2 KO – tau vs untreated. E Total (left graph) and upregulated (black)/downregulated (gray) (right graph) DEPs in microglial cell lysates in four comparative analyses indicated (run 2): Trem2 KO vs WT (both untreated), Trem2 KO vs WT (both +LPS/ATP), WT – LPS/ATP vs untreated, and Trem2 KO – LPS/ATP vs untreated. F-I Heatmap distribution and scatterplot distribution of overlapping DEPs in WT (x-axis) and Trem2 KO (“KO”) (y-axis) microglia with tau (run 1) and LPS/ATP (run 2) treatment. F Heatmap showing Log2 fold-change and adj p-value of 2867 DEPs in WT and KO microglia with tau treatment; adjacent heatmap depicts z-score values of the genes listed on the left heatmap for individual samples (n = 6) from WT (untreated), Trem2 KO (untreated), WT (+tau) or Trem2 KO (+tau) microglia lysates as indicated; “NA” values derived from missing peptide values are indicated in gray. Log2 fold-change, z-score (red/blue) and p-value (red/yellow) scales are shown. G Scatterplot distribution of 927 overlapping DEPs identified in (F). H Log2 fold-change and adj p-value of 3481 DEPs in WT and KO microglia with LPS/ATP treatment; adjacent heatmap depicts z-score values of the genes listed on the left heatmap for individual samples (n = 6) from WT (untreated), Trem2 KO (untreated), WT (+LPS/ATP) or Trem2 KO (+LPS/ATP) microglia lysates as indicated; “NA” values derived from missing peptide values are indicated in gray. Log2 fold-change, z-score (red/blue) and p-value (red/yellow) scales are shown. I Scatterplot distribution of 1398 overlapping DEPs identified in (H). J Heatmap distribution, Log2 fold-change of GO CC “extracellular exosome” DEPs from combined runs 1 (tau) and 2 (LPS/ATP) from the 8 pairwise comparisons indicated. Z-scores of 6 replicates from each run are shown on the right, “NA” values derived from missing peptide values are indicated in gray
Fig. 6 Characterization of proteomic profiles associated with exosome induction in Trem2 KO microglia. A Schematic of the proteomic analysis scheme used; WT or Trem2 KO microglia were left untreated or treated with tau oligomers (run 1) or LPS/ATP (run 2) and microglia lysates were subjected to proteomic analysis by label-free mass spectrometry. B, C PCA plots depicting distribution and clustering of proteomic profiles from WT and Trem2 KO (“KO”) microglia untreated or exposed to tau oligomers (B) or LPS/ATP (C). PCA distribution of 6 individual replicate samples for each treatment group are shown. D Total (left graph) and upregulated (black)/downregulated (gray) (right graph) differentially expressed proteins (DEPs) in microglial cell lysates in four comparative analyses indicated (run 1): Trem2 KO vs WT (both untreated), Trem2 KO vs WT (both +tau), WT – tau vs untreated, and Trem2 KO – tau vs untreated. E Total (left graph) and upregulated (black)/downregulated (gray) (right graph) DEPs in microglial cell lysates in four comparative analyses indicated (run 2): Trem2 KO vs WT (both untreated), Trem2 KO vs WT (both +LPS/ATP), WT – LPS/ATP vs untreated, and Trem2 KO – LPS/ATP vs untreated. F-I Heatmap distribution and scatterplot distribution of overlapping DEPs in WT (x-axis) and Trem2 KO (“KO”) (y-axis) microglia with tau (run 1) and LPS/ATP (run 2) treatment. F Heatmap showing Log2 fold-change and adj p-value of 2867 DEPs in WT and KO microglia with tau treatment; adjacent heatmap depicts z-score values of the genes listed on the left heatmap for individual samples (n = 6) from WT (untreated), Trem2 KO (untreated), WT (+tau) or Trem2 KO (+tau) microglia lysates as indicated; “NA” values derived from missing peptide values are indicated in gray. Log2 fold-change, z-score (red/blue) and p-value (red/yellow) scales are shown. G Scatterplot distribution of 927 overlapping DEPs identified in (F). H Log2 fold-change and adj p-value of 3481 DEPs in WT and KO microglia with LPS/ATP treatment; adjacent heatmap depicts z-score values of the genes listed on the left heatmap for individual samples (n = 6) from WT (untreated), Trem2 KO (untreated), WT (+LPS/ATP) or Trem2 KO (+LPS/ATP) microglia lysates as indicated; “NA” values derived from missing peptide values are indicated in gray. Log2 fold-change, z-score (red/blue) and p-value (red/yellow) scales are shown. I Scatterplot distribution of 1398 overlapping DEPs identified in (H). J Heatmap distribution, Log2 fold-change of GO CC “extracellular exosome” DEPs from combined runs 1 (tau) and 2 (LPS/ATP) from the 8 pairwise comparisons indicated. Z-scores of 6 replicates from each run are shown on the right, “NA” values derived from missing peptide values are indicated in grayCC BY · PubMed Central
Fig. 7 Trem2 KO exosomes exhibit enhanced tau seeding activity. A Western blot of tau and Alix in lysates and exosomal preparations from tau-loaded WT microglia with or without LPS/ATP stimulation. B Comparison of tau and Alix in lysates and exosomes from tau-loaded WT and Trem2 KO microglia following LPS/ATP stimulation by Western blot analysis. C ELISA measurement of tau levels in WT and Trem2 KO microglial exosomes from 3 independent cultures, 2 replicate ELISA runs per culture. D Experimental design schematic, WT and Trem2 KO microglia exposed to tau oligomers were treated (or untreated) with exosome inhibitor (GW4869), followed by exosomal induction with LPS/ATP. Purified exosomes were then assayed for tau seeding using the tau-RD FRET biosensor cell line, or injected into mouse brain for histopathological analysis. E Exosomes were purified from 1 × 107 WT or Trem2 KO microglia in the absence of tau (no tau), pre-loaded with tau oligomers (+ tau) and induced with LPS/ATP, or Trem2 KO microglia pre-loaded with tau and pre-treated with 10 μM GW4869 prior to LPS/ATP release (+ tau, + exosome inhib), and applied to a tau RD reporter cell line. Tau fret signals were then visualized by fluorescence microscopy. Bar = 10 μm. F Quantification of the area of FRET-positive signals detected (μm2) per cell from representative images in (E). G Characterizing pathological effects of exosomal tau preparations in vivo. Exosomal preparations from tau-loaded or control (“no tau”) WT or Trem2 KO microglia were stereotactically injected into 5-month old WT mouse hippocampus, and stained for AT8 pathology (green) or nuclei (DAPI, blue) 21 days after injection. Bar = 50 μm. H Number of AT8-positive cells (per 2.5 × 105 μm2 area) were scored from 6 independently injected animals per treatment/genotype, graph represent mean ± SE. Graphs in (C), (F) and (H) depict mean ± SE. Statistical significance was determined by Student’s t-test (C), One-way ANOVA with Tukey’s multiple comparison (F, H), **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 7 Trem2 KO exosomes exhibit enhanced tau seeding activity. A Western blot of tau and Alix in lysates and exosomal preparations from tau-loaded WT microglia with or without LPS/ATP stimulation. B Comparison of tau and Alix in lysates and exosomes from tau-loaded WT and Trem2 KO microglia following LPS/ATP stimulation by Western blot analysis. C ELISA measurement of tau levels in WT and Trem2 KO microglial exosomes from 3 independent cultures, 2 replicate ELISA runs per culture. D Experimental design schematic, WT and Trem2 KO microglia exposed to tau oligomers were treated (or untreated) with exosome inhibitor (GW4869), followed by exosomal induction with LPS/ATP. Purified exosomes were then assayed for tau seeding using the tau-RD FRET biosensor cell line, or injected into mouse brain for histopathological analysis. E Exosomes were purified from 1 × 107 WT or Trem2 KO microglia in the absence of tau (no tau), pre-loaded with tau oligomers (+ tau) and induced with LPS/ATP, or Trem2 KO microglia pre-loaded with tau and pre-treated with 10 μM GW4869 prior to LPS/ATP release (+ tau, + exosome inhib), and applied to a tau RD reporter cell line. Tau fret signals were then visualized by fluorescence microscopy. Bar = 10 μm. F Quantification of the area of FRET-positive signals detected (μm2) per cell from representative images in (E). G Characterizing pathological effects of exosomal tau preparations in vivo. Exosomal preparations from tau-loaded or control (“no tau”) WT or Trem2 KO microglia were stereotactically injected into 5-month old WT mouse hippocampus, and stained for AT8 pathology (green) or nuclei (DAPI, blue) 21 days after injection. Bar = 50 μm. H Number of AT8-positive cells (per 2.5 × 105 μm2 area) were scored from 6 independently injected animals per treatment/genotype, graph represent mean ± SE. Graphs in (C), (F) and (H) depict mean ± SE. Statistical significance was determined by Student’s t-test (C), One-way ANOVA with Tukey’s multiple comparison (F, H), **p < 0.01, ***p < 0.001, ****p < 0.0001CC BY · PubMed Central

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Metadata

Authors
Zhu, B., Liu, Y., Hwang, S., Archuleta, K., Huang, H., Campos, A., Murad, R., Piña-Crespo, J., Xu, H., & Huang, T. Y.
Affiliations
  • Sanford Burnham Prebys Medical Discovery Institute (US)
  • Chongqing Medical University (CN)
Corresponding author
  • Xu, Huaxi
  • Huang, Timothy Y.
ORCID
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2022-09-02
Volume
17
Issue
1
Pages
58-58
ISSN
1750-1326 (ISSN-L)
DOI
10.1186/s13024-022-00562-8
PMC
PMC9438095
OpenAlex
W4294169024
Cited by
127
References
69
Citations by year
2026: 28 · 2025: 35 · 2024: 35 · 2023: 26 · 2022: 3
FWCI
15.33
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Neurodegeneration (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institute on Aging (R21 AG070676, RF1 056130)
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
TREM2, tau pathology, Alzheimer's disease, microglia, exosomes, tau propagation, tau seeding, medial entorhinal cortex, hippocampus, memory impairment, TREM2 knockout mice
MeSH terms
Alzheimer Disease, Animals, Membrane Glycoproteins, Receptors, Immunologic, Microglia, Mice, Knockout, Proteomics, Mice, Exosomes

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