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Cited by 224Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsinterferon and immune responses

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Tau activates microglia via the PQBP1-cGAS-STING pathway to promote brain inflammation

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Jin, Meihua, Shiwaku, Hiroki, Tanaka, Hikari, Obita, Takayuki, Ohuchi, Sakurako, Yoshioka, Yuki, Jin, Xiaocen, Kondo, Kanoh, Fujita, Kyota, Homma, Hidenori, Nakajima, Kazuyuki, Mizuguchi, Mineyuki, Okazawa, Hitoshi·2021·Nature Communications
DOI 10.1038/s41467-021-26851-2PMC PMC8592984
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Abstract

Brain inflammation generally accompanies and accelerates neurodegeneration. Here we report a microglial mechanism in which polyglutamine binding protein 1 (PQBP1) senses extrinsic tau 3R/4R proteins by direct interaction and triggers an innate immune response by activating a cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway. Tamoxifen-inducible and microglia-specific depletion of PQBP1 in primary culture in vitro and mouse brain in vivo shows that PQBP1 is essential for sensing-tau to induce nuclear translocation of nuclear factor κB (NFκB), NFκB-dependent transcription of inflammation genes, brain inflammation in vivo, and eventually mouse cognitive impairment. Collectively, PQBP1 is an intracellular receptor in the cGAS-STING pathway not only for cDNA of human immunodeficiency virus (HIV) but also for the transmissible neurodegenerative disease protein tau. This study characterises a mechanism of brain inflammation that is common to virus infection and neurodegenerative disorders.

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Figures

Fig. 1 PQBP1 interacts with extrinsic Tau in microglia. a Schematic presentation of deletion mutants of tau and PQBP1 used for SPR and NMR analyses. N1/2 N-terminal domain, R1/2/3/4 C-terminal microtubule-binding domain, PRR proline-rich region, WWD WW domain, PRD polar amino acid-rich domain, IDP/IDR intrinsically disordered region. b Equilibrium binding curves of PQBP1 to Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, and Tau 410 P179A/P216A monitored by SPR. Tau proteins were immobilized on sensor chip surfaces. The binding response at equilibrium was plotted against the concentration of PQBP1. Black, full-length PQBP1; red, PQBP1(1–94); blue, PQBP1 (94–176); green, PQBP1 (193–265). RU resonance unit. c 1H-15N HSQC spectra of Tau 1–150, 151–197, 198–243, 244–341, and 342–410 in the absence (black) and presence (red) of PQBP1(1–94). Amino acid residue numbers of tau are based on Tau 410. The molar ratio of Tau:PQBP1 (1–94) is 1:5. Residues showing significant chemical shift perturbation and line broadening of resonances are labeled in the HSQC spectra of Tau 151–197 and 198–243. Ctrl control, ppm parts per million. d Tau 410 amino acid sequence showing consensus motifs for interaction with PQBP1-WWD (red letter) and mutated prolines (underlined). e A representative western blot showing co-precipitation of tau with PQBP1 from a lysate of C57BL/6 microglia cultured for 3 days with 25 nM non-aggregated Tau-TAMRA (upper panels). Quantitative immunoprecipitation was performed with C57BL/6 microglia cultured for 3 days with 25 nM non-aggregated Tau-TAMRA. Input was the intensity of TAMRA-labeled Tau 410 in the C57BL/6 microglia lysate samples used for immunoprecipitation. Output was the intensity of TAMRA-labeled Tau 410 in samples after immunoprecipitation with anti-tau, anti-PQBP1, and human IgG. The fluorescence intensity of TAMRA-labeled Tau 410 was measured on a FLUOstar OPTIMA-6 microplate reader. n.s. not significant, AU arbitrary unit. N = 4 per group. P = 0.9963 (ctrl-IgG vs PQBP1), 0.8874 (ctrl-IgG vs tau), 0.9215 (PQBP1 vs tau), 9.63e−6 (PQBP1 vs ctrl-IgG), 3.69e−7 (tau vs ctrl-IgG). ##P < 0.01, n.s.P > 0.05 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 1 PQBP1 interacts with extrinsic Tau in microglia. a Schematic presentation of deletion mutants of tau and PQBP1 used for SPR and NMR analyses. N1/2 N-terminal domain, R1/2/3/4 C-terminal microtubule-binding domain, PRR proline-rich region, WWD WW domain, PRD polar amino acid-rich domain, IDP/IDR intrinsically disordered region. b Equilibrium binding curves of PQBP1 to Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, and Tau 410 P179A/P216A monitored by SPR. Tau proteins were immobilized on sensor chip surfaces. The binding response at equilibrium was plotted against the concentration of PQBP1. Black, full-length PQBP1; red, PQBP1(1–94); blue, PQBP1 (94–176); green, PQBP1 (193–265). RU resonance unit. c 1H-15N HSQC spectra of Tau 1–150, 151–197, 198–243, 244–341, and 342–410 in the absence (black) and presence (red) of PQBP1(1–94). Amino acid residue numbers of tau are based on Tau 410. The molar ratio of Tau:PQBP1 (1–94) is 1:5. Residues showing significant chemical shift perturbation and line broadening of resonances are labeled in the HSQC spectra of Tau 151–197 and 198–243. Ctrl control, ppm parts per million. d Tau 410 amino acid sequence showing consensus motifs for interaction with PQBP1-WWD (red letter) and mutated prolines (underlined). e A representative western blot showing co-precipitation of tau with PQBP1 from a lysate of C57BL/6 microglia cultured for 3 days with 25 nM non-aggregated Tau-TAMRA (upper panels). Quantitative immunoprecipitation was performed with C57BL/6 microglia cultured for 3 days with 25 nM non-aggregated Tau-TAMRA. Input was the intensity of TAMRA-labeled Tau 410 in the C57BL/6 microglia lysate samples used for immunoprecipitation. Output was the intensity of TAMRA-labeled Tau 410 in samples after immunoprecipitation with anti-tau, anti-PQBP1, and human IgG. The fluorescence intensity of TAMRA-labeled Tau 410 was measured on a FLUOstar OPTIMA-6 microplate reader. n.s. not significant, AU arbitrary unit. N = 4 per group. P = 0.9963 (ctrl-IgG vs PQBP1), 0.8874 (ctrl-IgG vs tau), 0.9215 (PQBP1 vs tau), 9.63e−6 (PQBP1 vs ctrl-IgG), 3.69e−7 (tau vs ctrl-IgG). ##P < 0.01, n.s.P > 0.05 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 2 Colocalization of PQBP1 and incorporated Tau in microglia. a Immunocytochemistry of primary microglia prepared from C57BL/6 mice cultured with Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A proteins in the medium. Colocalization of PQBP1 and extrinsic tau was detected frequently. The experiments were repeated four times independently with similar results. b In vitro polymerization of Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, and Tau 410 P179A/P216A full-length proteins. Only Tau 441 and Tau 410 P216A partially formed fibrils after 10 days of incubation, while the other full-length protein did not. The experiments were repeated four times independently with similar results. c Images of normal microglia before and after addition of polymerized or non-polymerized Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A, among which three species are shown in Supplementary Movies 1–3. h hour.
Fig. 2 Colocalization of PQBP1 and incorporated Tau in microglia. a Immunocytochemistry of primary microglia prepared from C57BL/6 mice cultured with Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A proteins in the medium. Colocalization of PQBP1 and extrinsic tau was detected frequently. The experiments were repeated four times independently with similar results. b In vitro polymerization of Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, and Tau 410 P179A/P216A full-length proteins. Only Tau 441 and Tau 410 P216A partially formed fibrils after 10 days of incubation, while the other full-length protein did not. The experiments were repeated four times independently with similar results. c Images of normal microglia before and after addition of polymerized or non-polymerized Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A, among which three species are shown in Supplementary Movies 1–3. h hour.CC BY · PubMed Central
Fig. 3 Extrinsic Tau proteins induce nuclear translocation of NFκB and expression of pro-inflammatory genes in microglia. a Immunocytochemistry of primary microglia prepared from newborn C57BL/6 mice reveals nuclear translocation of NFκB at 48 h after addition of recombinant full-length Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A protein in the non-polymerized or polymerized state to culture medium. Images were obtained by confocal microscopy. Red or white arrows indicate microglia with or without nuclear translocation of NFκB. Lower panels show high magnification of representative cells. In the presence of tau proteins in culture medium, nuclear translocation of NFκB was induced in most microglia, while in the absence of tau proteins NFκB remained in the cytoplasm. b The effects of various tau species in the polymerized and non-polymerized state on nuclear translocation of NFκB were compared. Aggregation of Tau 410/441 reduces the frequency of NFκB nuclear translocation. PQBP1-binding proline single mutants (Tau 410 P179A, Tau 410 P216A) partially, while double proline mutant (Tau 410 P179A/P216A) substantially, lost the ability to induce NFκB nuclear translocation. N = 5, n = 20 (cells). ##P < 0.01 in Tukey’s HSD test. c Isoforms, mutations, and aggregation states of tau affect induction of Tnf and Isg54 gene expression. Consistent with immunocytochemistry of NFκB, non-polymerized Tau 410 induced pro-inflammatory genes most efficiently, while Tau 441 possesses 60–80% of the induction ability. PQBP1-binding proline single mutants (Tau 410 P179A, Tau 410 P216A) partially, while double proline mutant (Tau 410 P179A/P216A) substantially, lost transactivation of Tnf and Isg54 gene expression. RT-qPCR reverse transcriptase quantitative polymerase chain reaction. N = 3. ##P < 0.01 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 3 Extrinsic Tau proteins induce nuclear translocation of NFκB and expression of pro-inflammatory genes in microglia. a Immunocytochemistry of primary microglia prepared from newborn C57BL/6 mice reveals nuclear translocation of NFκB at 48 h after addition of recombinant full-length Tau 410, Tau 441, Tau 410 P179A, Tau 410 P216A, or Tau 410 P179A/P216A protein in the non-polymerized or polymerized state to culture medium. Images were obtained by confocal microscopy. Red or white arrows indicate microglia with or without nuclear translocation of NFκB. Lower panels show high magnification of representative cells. In the presence of tau proteins in culture medium, nuclear translocation of NFκB was induced in most microglia, while in the absence of tau proteins NFκB remained in the cytoplasm. b The effects of various tau species in the polymerized and non-polymerized state on nuclear translocation of NFκB were compared. Aggregation of Tau 410/441 reduces the frequency of NFκB nuclear translocation. PQBP1-binding proline single mutants (Tau 410 P179A, Tau 410 P216A) partially, while double proline mutant (Tau 410 P179A/P216A) substantially, lost the ability to induce NFκB nuclear translocation. N = 5, n = 20 (cells). ##P < 0.01 in Tukey’s HSD test. c Isoforms, mutations, and aggregation states of tau affect induction of Tnf and Isg54 gene expression. Consistent with immunocytochemistry of NFκB, non-polymerized Tau 410 induced pro-inflammatory genes most efficiently, while Tau 441 possesses 60–80% of the induction ability. PQBP1-binding proline single mutants (Tau 410 P179A, Tau 410 P216A) partially, while double proline mutant (Tau 410 P179A/P216A) substantially, lost transactivation of Tnf and Isg54 gene expression. RT-qPCR reverse transcriptase quantitative polymerase chain reaction. N = 3. ##P < 0.01 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 4 PQBP1–cGAS–STING pathway mediates Tau-induced NFκB activation in microglia. a Protocol of tamoxifen-induced Pqbp1 knockout in primary microglia prepared from newborn Pqbp1-cKO mice. WB western blot. b Effect of PQBP1 deficiency, cGAS knockdown, and STING knockdown on Tau 410/441-induced nuclear translocation of NFκB in microglia. Activated microglia with nuclear NFκB are indicated with red arrows. Non-activated microglia are indicated with white arrows. Images were obtained by confocal microscopy. c Quantitative analyses of % nuclear NFκB-positive microglia in six visual fields in normal condition, PQBP1 deficiency, cGAS knockdown, and STING knockdown. N = 6 (visual fields). ##P < 0.01 in Tukey’s HSD test (vs non-induced Pqbp1-cKO). d Western blots reveal activation of NFκB (pSer536-p65) and IRF3 (pSer396-IRF3) after culture with non-polymerized Tau 410/441 in normal microglia, but not in tamoxifen-induced Pqbp1-cKO, siRNA-mediated cGAS-KD, or siRNA-mediated STING-KD microglia. This experiment was repeated independently, three times, with similar results. e Effect of PQBP1 depletion on microglia migration. FBS fetal bovine serum. N = 4 wells. Statistical test: Student’s t-test (two-sided). f Effect of PQBP1 depletion on microglia phagocytosis by using FITC-beads. AU arbitrary unit. N = 4 wells. For statistical analysis, a Student’s t-test (two-sided) was performed. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 4 PQBP1–cGAS–STING pathway mediates Tau-induced NFκB activation in microglia. a Protocol of tamoxifen-induced Pqbp1 knockout in primary microglia prepared from newborn Pqbp1-cKO mice. WB western blot. b Effect of PQBP1 deficiency, cGAS knockdown, and STING knockdown on Tau 410/441-induced nuclear translocation of NFκB in microglia. Activated microglia with nuclear NFκB are indicated with red arrows. Non-activated microglia are indicated with white arrows. Images were obtained by confocal microscopy. c Quantitative analyses of % nuclear NFκB-positive microglia in six visual fields in normal condition, PQBP1 deficiency, cGAS knockdown, and STING knockdown. N = 6 (visual fields). ##P < 0.01 in Tukey’s HSD test (vs non-induced Pqbp1-cKO). d Western blots reveal activation of NFκB (pSer536-p65) and IRF3 (pSer396-IRF3) after culture with non-polymerized Tau 410/441 in normal microglia, but not in tamoxifen-induced Pqbp1-cKO, siRNA-mediated cGAS-KD, or siRNA-mediated STING-KD microglia. This experiment was repeated independently, three times, with similar results. e Effect of PQBP1 depletion on microglia migration. FBS fetal bovine serum. N = 4 wells. Statistical test: Student’s t-test (two-sided). f Effect of PQBP1 depletion on microglia phagocytosis by using FITC-beads. AU arbitrary unit. N = 4 wells. For statistical analysis, a Student’s t-test (two-sided) was performed. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 5 PQBP1 is essential for recruiting cGAS to Tau. Confocal microscopy images of primary Pqbp1-cKO microglia stained with anti-cGAS and -tau antibodies. a Cytoplasmic tau foci after incorporation recruit cGAS in the presence of PQBP1 (left panels), while in the absence of PQBP1, cGAS is mislocalized and not merged with cytoplasmic foci of tau, which is phosphorylated after endocytosis. b Quantification of cGAS-tau colocalized cells in four wells of non-induced and tamoxifen-induced Pqbp1-cKO microglia after tau addition to primary culture medium. N = 4. ##P < 0.01 in Tukey’s HSD test. c Schemes showing the concepts of the luciferase assay used to monitor NFκB activity under tau stimulation and PQBP1–cGAS–STING pathway perturbation (d) and of RT-qPCR used to monitor expression levels of NFκB-target genes (e). d Luciferase assay to monitor NFκB activity under Tau 410/441 stimulation together with PQBP1–cGAS–STING pathway perturbation by tamoxifen-induced Pqbp1-cKO, siRNA against cGAS and siRNA against STING. N = 3. ##P < 0.01 in Tukey’s HSD test. e RT-qPCR to monitor expression levels of NFκB- or IRF3-target genes (Tnf, Isg54, Cxcl10, and Ifnβ) under Tau 410/441 stimulation together with PQBP1–cGAS–STING pathway perturbation by tamoxifen-induced Pqbp1-cKO, siRNA against cGAS, and siRNA against STING. N = 3. ##P < 0.01 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 5 PQBP1 is essential for recruiting cGAS to Tau. Confocal microscopy images of primary Pqbp1-cKO microglia stained with anti-cGAS and -tau antibodies. a Cytoplasmic tau foci after incorporation recruit cGAS in the presence of PQBP1 (left panels), while in the absence of PQBP1, cGAS is mislocalized and not merged with cytoplasmic foci of tau, which is phosphorylated after endocytosis. b Quantification of cGAS-tau colocalized cells in four wells of non-induced and tamoxifen-induced Pqbp1-cKO microglia after tau addition to primary culture medium. N = 4. ##P < 0.01 in Tukey’s HSD test. c Schemes showing the concepts of the luciferase assay used to monitor NFκB activity under tau stimulation and PQBP1–cGAS–STING pathway perturbation (d) and of RT-qPCR used to monitor expression levels of NFκB-target genes (e). d Luciferase assay to monitor NFκB activity under Tau 410/441 stimulation together with PQBP1–cGAS–STING pathway perturbation by tamoxifen-induced Pqbp1-cKO, siRNA against cGAS and siRNA against STING. N = 3. ##P < 0.01 in Tukey’s HSD test. e RT-qPCR to monitor expression levels of NFκB- or IRF3-target genes (Tnf, Isg54, Cxcl10, and Ifnβ) under Tau 410/441 stimulation together with PQBP1–cGAS–STING pathway perturbation by tamoxifen-induced Pqbp1-cKO, siRNA against cGAS, and siRNA against STING. N = 3. ##P < 0.01 in Tukey’s HSD test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 6 PQBP1 is essential for in vivo activation of microglia by extrinsic Tau. a Protocol of Tau 410 monomer (2.2 μg/μl, 1 μl/injection, 4.4 μg/mouse) or equivalent PBS injection to bilateral entorhinal cortices of non-induced or tamoxifen-induced Pqbp1-cKO mice. Four groups were made for analyses. i.p. intra-peritoneal injection, qPCR quantitative RT-PCR, IHC immunohistochemistry. b Immunohistochemistry of Iba1-positive microglia with PQBP1-Tau colocalization, cGAS-tau colocalization, and NFκB nuclear translocation in injected areas of non-induced Pqbp1-cKO and tamoxifen-induced Pqbp1-cKO. (Right) Quantitative analyses of PQBP1-Tau colocalized microglia, cGAS-tau colocalized microglia, and activated microglia (nuclear NFκB-positive) among Tau-incorporating microglia. N = 5. P value: [Upper] 3.55e−9 (non-induced), 5.71e−9 (Tau410). [Middle] 1.00e−7 (non-induced), 1.00e−7 (Tau410). [Lower] 2.29e−14 (non-induced), 6.98e−12 (Tau410). c RT-qPCR of induction of Tnf and Isg54 by Tau 410 in entorhinal cortex from non-induced or tamoxifen-induced Pqbp1-cKO mice. N = (4, 6, 3, 6). P value: [Upper] 0.0011 (non-induced), 0.0005 (Tau410). [Lower] 0.0031 (non-induced), 0.0080 (Tau410). d Y-maze test at 7, 10, and 14 days and Morris water maze test at 14 days after tau injection into bilateral entorhinal. Data points (Morris water maze test): mean ± SEM. Y-maze: N = (8, 8, 7, 8) for 7 days, (7, 8, 7, 8) for 10 days and (8, 8, 7, 8) for 14 days. Morris water maze: N = (7, 8, 6, 7). P value: [Y-maze; 10 days] 0.0439 (non-induced), and 0.0055 (Tau410). [14 days] 0.0014 (non-induced), and 0.0018 (Tau410). [Morris water maze, Day4] 0.0043 (non-induced), and 0.0048 (Tau410). e In vivo live imaging of microglia by two-photon microscopy with B6.129P2(Cg)-Cx3cr1tm1Litt/J (Cx3cr-1GFP) mice in which microglia were labeled by the endogenous Cx3cr1 enhancer/promoter-driven EGFP protein. f Immunohistochemistry confirmed PQBP1-KD by siRNA-PQBP1 injection. (Graph) Quantification of PQBP1 signal intensity/cell. Data points: mean ± SEM. n = 100 microglia. P = 1.71e−102, **P < 0.01 in two-sided Student’s t-test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range. Tuple of N numbers indicates PBS/non-induced, Tau410/non-induced, PBS/induced Pqbp1-cKO, and Tau410/induced Pqbp1-cKO), respectively. For multiple group comparisons, Tukey’s HSD test was performed (##P < 0.01, #P < 0.05).
Fig. 6 PQBP1 is essential for in vivo activation of microglia by extrinsic Tau. a Protocol of Tau 410 monomer (2.2 μg/μl, 1 μl/injection, 4.4 μg/mouse) or equivalent PBS injection to bilateral entorhinal cortices of non-induced or tamoxifen-induced Pqbp1-cKO mice. Four groups were made for analyses. i.p. intra-peritoneal injection, qPCR quantitative RT-PCR, IHC immunohistochemistry. b Immunohistochemistry of Iba1-positive microglia with PQBP1-Tau colocalization, cGAS-tau colocalization, and NFκB nuclear translocation in injected areas of non-induced Pqbp1-cKO and tamoxifen-induced Pqbp1-cKO. (Right) Quantitative analyses of PQBP1-Tau colocalized microglia, cGAS-tau colocalized microglia, and activated microglia (nuclear NFκB-positive) among Tau-incorporating microglia. N = 5. P value: [Upper] 3.55e−9 (non-induced), 5.71e−9 (Tau410). [Middle] 1.00e−7 (non-induced), 1.00e−7 (Tau410). [Lower] 2.29e−14 (non-induced), 6.98e−12 (Tau410). c RT-qPCR of induction of Tnf and Isg54 by Tau 410 in entorhinal cortex from non-induced or tamoxifen-induced Pqbp1-cKO mice. N = (4, 6, 3, 6). P value: [Upper] 0.0011 (non-induced), 0.0005 (Tau410). [Lower] 0.0031 (non-induced), 0.0080 (Tau410). d Y-maze test at 7, 10, and 14 days and Morris water maze test at 14 days after tau injection into bilateral entorhinal. Data points (Morris water maze test): mean ± SEM. Y-maze: N = (8, 8, 7, 8) for 7 days, (7, 8, 7, 8) for 10 days and (8, 8, 7, 8) for 14 days. Morris water maze: N = (7, 8, 6, 7). P value: [Y-maze; 10 days] 0.0439 (non-induced), and 0.0055 (Tau410). [14 days] 0.0014 (non-induced), and 0.0018 (Tau410). [Morris water maze, Day4] 0.0043 (non-induced), and 0.0048 (Tau410). e In vivo live imaging of microglia by two-photon microscopy with B6.129P2(Cg)-Cx3cr1tm1Litt/J (Cx3cr-1GFP) mice in which microglia were labeled by the endogenous Cx3cr1 enhancer/promoter-driven EGFP protein. f Immunohistochemistry confirmed PQBP1-KD by siRNA-PQBP1 injection. (Graph) Quantification of PQBP1 signal intensity/cell. Data points: mean ± SEM. n = 100 microglia. P = 1.71e−102, **P < 0.01 in two-sided Student’s t-test. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range. Tuple of N numbers indicates PBS/non-induced, Tau410/non-induced, PBS/induced Pqbp1-cKO, and Tau410/induced Pqbp1-cKO), respectively. For multiple group comparisons, Tukey’s HSD test was performed (##P < 0.01, #P < 0.05).CC BY · PubMed Central
Fig. 7 PQBP1-Tau interaction is essential for in vivo activation of microglia. a Protocol of injection of Tau 410 or Tau 410 PQBP1-binding mutant monomers (0.5 μg/μl, 4.0 μl/injection, 4.0 μg/mouse) or equivalent PBS to bilateral entorhinal cortices of non-induced or tamoxifen-induced Pqbp1-cKO mice. Five groups were used for analyses. b, c Immunohistochemistry of Iba1-positive microglia with PQBP1 + tau, cGAS+tau, NFκB-p65+tau colocalization in injected area of non-induced Pqbp1-cKO, and tamoxifen-induced Pqbp1-cKO. Quantitative analyses of PQBP1-Tau colocalized microglia, cGAS-tau colocalized microglia, and activated microglia (nuclear NFκB-positive) among Tau-incorporating microglia are shown in the graphs on the right. N = 10 (visual fields, from 3 mice). ##P < 0.01 in Tukey’s HSD test. d, e RT-qPCR of Tnf and Isg54 induction by Tau 410 and the PQBP1-binding mutants with entorhinal cortex tissues prepared from non-induced or tamoxifen-induced Pqbp1-cKO mice. N = 3. ##P < 0.01, n.s.P > 0.05 in Tukey’s HSD test (two-sided, multiple comparison). Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 7 PQBP1-Tau interaction is essential for in vivo activation of microglia. a Protocol of injection of Tau 410 or Tau 410 PQBP1-binding mutant monomers (0.5 μg/μl, 4.0 μl/injection, 4.0 μg/mouse) or equivalent PBS to bilateral entorhinal cortices of non-induced or tamoxifen-induced Pqbp1-cKO mice. Five groups were used for analyses. b, c Immunohistochemistry of Iba1-positive microglia with PQBP1 + tau, cGAS+tau, NFκB-p65+tau colocalization in injected area of non-induced Pqbp1-cKO, and tamoxifen-induced Pqbp1-cKO. Quantitative analyses of PQBP1-Tau colocalized microglia, cGAS-tau colocalized microglia, and activated microglia (nuclear NFκB-positive) among Tau-incorporating microglia are shown in the graphs on the right. N = 10 (visual fields, from 3 mice). ##P < 0.01 in Tukey’s HSD test. d, e RT-qPCR of Tnf and Isg54 induction by Tau 410 and the PQBP1-binding mutants with entorhinal cortex tissues prepared from non-induced or tamoxifen-induced Pqbp1-cKO mice. N = 3. ##P < 0.01, n.s.P > 0.05 in Tukey’s HSD test (two-sided, multiple comparison). Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 8 Genetic interaction of PQBP1 with LRP1 or TREM2 in microglia. a Immunocytochemistry revealed high efficiency of tamoxifen-induced Pqbp1-cKO and D10A-mutant Cas9 nuclease/sgRNA-mediated KO of Lrp1/Trem2. Right graphs show quantitative analyses of signal intensities of target genes. Non-TF non-transfected. N = 5 wells. In the analysis of CRIPER-GFP signals: P = 7.35e−12 (Ctrl vs Non-TF), 2.22e−12 (Lrp1-KO vs Non-TF), 3.31e−13 (Trem2-KO vs Non-TF); in the analysis of LRP1 signal: P = 1.09e−11 (Lrp1-KO vs Non-TF); in the analysis of PQBP1 signals: P = 1.92e−14 (Pqbp1-cKO vs Non-TF); in the analysis of TREM2 signals: P = 6.76e−14 (Trem2-KO vs Non-TF); ##P < 0.01 in Tukey’s HSD test (vs Non-TF). b Protocol for double KO of Pqbp1-Lrp1 or Pqbp1-Trem2 genes in primary microglia and their activation of NFκB by Tau 410. Tamox tamoxifen. c RFP signal assay to monitor activation of NFκB by adding Tau 410 in single KO microglia of Lrp1 or Trem2 or double KO microglia of Pqbp1-Lrp1 or Pqbp1-Trem2. N = 5 wells. In tau signal assay of non-induced Pqbp1-cKO group: P = 1.002e−13 [Non-TF (Tau+) vs (Tau−)], 1.005e−13 [Ctrl (Tau+) vs (Tau−)], 5.65e−11 [Lrp1-KO (Tau+) vs (Tau−)], 4.597e−12 [Trem2-KO (Tau+) vs (Tau−)]; in tau signal assay of induced Pqbp1-cKO group: P = 1.005e−13 [Non-TF (Tau+) vs (Tau−)], 1.006e−13 [Ctrl (Tau+) vs (Tau−)], 1.73e−11 [Lrp1-KO (Tau+) vs (Tau−)], 1.98e−11 [Trem2-KO (Tau+) vs (Tau−)]; in RFP signal assay of non-induced Pqbp1-cKO group: P = 4.60e−13 [Non-TF (Tau + ) vs (Tau−)], 1.23e−13 [Ctrl (Tau+) vs (Tau−)], 4.93e−7 [Lrp1-KO (Tau+) vs (Tau−)], 1.15e−12 [Trem2-KO (Tau+) vs (Tau−)]; in RFP signal assay of induced Pqbp1-cKO group: P = 1.005e−13 [Non-TF (Tau+) vs (Tau−)], 1.006e−13 [Ctrl (Tau+) vs (Tau−)], 1.73e−11 [Lrp1-KO (Tau+) vs (Tau−)], 0.9998 [Trem2-KO (Tau+) vs (Tau−)]; #P < 0.05, ##P < 0.01 in Tukey’s HSD test. d Luciferase assay to monitor activation of NFκB by adding Tau 410 to single KO microglia of Lrp1 or Trem2 or double KO microglia of Pqbp1-Lrp1 or Pqbp1-Trem2. N = 4 wells. In non-induced Pqbp1-cKO group: P = 2.20e−11 [Non-TF (Tau+) vs (Tau−)], 3.31e−11 [Ctrl (Tau+) vs (Tau−)], 7.40e−5 [Lrp1-KO (Tau+) vs (Tau−)], 5.76e−15 [Trem2-KO (Tau+) vs (Tau−)]; in induced Pqbp1-cKO group: P = 8.13e−5 [Non-TF (Tau+) vs (Tau−)], 0.0013 [Ctrl (Tau+) vs (Tau−)], 0.0028 [Lrp1-KO (Tau+) vs (Tau−)], 0.9498 [Trem2-KO (Tau+) vs (Tau−)]; #P < 0.05, ##P < 0.01 in Tukey’s HSD test. e A hypothetical scheme of two pathways from extrinsic tau to activation of nuclear NFκB in microglia. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 8 Genetic interaction of PQBP1 with LRP1 or TREM2 in microglia. a Immunocytochemistry revealed high efficiency of tamoxifen-induced Pqbp1-cKO and D10A-mutant Cas9 nuclease/sgRNA-mediated KO of Lrp1/Trem2. Right graphs show quantitative analyses of signal intensities of target genes. Non-TF non-transfected. N = 5 wells. In the analysis of CRIPER-GFP signals: P = 7.35e−12 (Ctrl vs Non-TF), 2.22e−12 (Lrp1-KO vs Non-TF), 3.31e−13 (Trem2-KO vs Non-TF); in the analysis of LRP1 signal: P = 1.09e−11 (Lrp1-KO vs Non-TF); in the analysis of PQBP1 signals: P = 1.92e−14 (Pqbp1-cKO vs Non-TF); in the analysis of TREM2 signals: P = 6.76e−14 (Trem2-KO vs Non-TF); ##P < 0.01 in Tukey’s HSD test (vs Non-TF). b Protocol for double KO of Pqbp1-Lrp1 or Pqbp1-Trem2 genes in primary microglia and their activation of NFκB by Tau 410. Tamox tamoxifen. c RFP signal assay to monitor activation of NFκB by adding Tau 410 in single KO microglia of Lrp1 or Trem2 or double KO microglia of Pqbp1-Lrp1 or Pqbp1-Trem2. N = 5 wells. In tau signal assay of non-induced Pqbp1-cKO group: P = 1.002e−13 [Non-TF (Tau+) vs (Tau−)], 1.005e−13 [Ctrl (Tau+) vs (Tau−)], 5.65e−11 [Lrp1-KO (Tau+) vs (Tau−)], 4.597e−12 [Trem2-KO (Tau+) vs (Tau−)]; in tau signal assay of induced Pqbp1-cKO group: P = 1.005e−13 [Non-TF (Tau+) vs (Tau−)], 1.006e−13 [Ctrl (Tau+) vs (Tau−)], 1.73e−11 [Lrp1-KO (Tau+) vs (Tau−)], 1.98e−11 [Trem2-KO (Tau+) vs (Tau−)]; in RFP signal assay of non-induced Pqbp1-cKO group: P = 4.60e−13 [Non-TF (Tau + ) vs (Tau−)], 1.23e−13 [Ctrl (Tau+) vs (Tau−)], 4.93e−7 [Lrp1-KO (Tau+) vs (Tau−)], 1.15e−12 [Trem2-KO (Tau+) vs (Tau−)]; in RFP signal assay of induced Pqbp1-cKO group: P = 1.005e−13 [Non-TF (Tau+) vs (Tau−)], 1.006e−13 [Ctrl (Tau+) vs (Tau−)], 1.73e−11 [Lrp1-KO (Tau+) vs (Tau−)], 0.9998 [Trem2-KO (Tau+) vs (Tau−)]; #P < 0.05, ##P < 0.01 in Tukey’s HSD test. d Luciferase assay to monitor activation of NFκB by adding Tau 410 to single KO microglia of Lrp1 or Trem2 or double KO microglia of Pqbp1-Lrp1 or Pqbp1-Trem2. N = 4 wells. In non-induced Pqbp1-cKO group: P = 2.20e−11 [Non-TF (Tau+) vs (Tau−)], 3.31e−11 [Ctrl (Tau+) vs (Tau−)], 7.40e−5 [Lrp1-KO (Tau+) vs (Tau−)], 5.76e−15 [Trem2-KO (Tau+) vs (Tau−)]; in induced Pqbp1-cKO group: P = 8.13e−5 [Non-TF (Tau+) vs (Tau−)], 0.0013 [Ctrl (Tau+) vs (Tau−)], 0.0028 [Lrp1-KO (Tau+) vs (Tau−)], 0.9498 [Trem2-KO (Tau+) vs (Tau−)]; #P < 0.05, ##P < 0.01 in Tukey’s HSD test. e A hypothetical scheme of two pathways from extrinsic tau to activation of nuclear NFκB in microglia. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 9 Direct and indirect pathways mediating neuronal toxicity of Tau. a Immunohistochemistry of Tau-injected mice revealed most of the non-microglia cells incorporating tau were MAP2-positive neurons but not GFAP-positive astrocytes. The ratios of Tau-incorporating cells were equivalent in non-induced and tamoxifen-induced Pqbp1-cKO mice, as the basis for analyses in (b). N = 20 images from three mice. Statistical test: Student’s t-test. b Frequencies of neuronal cell death (TUNEL-positive and NeuN-positive dying neurons) by addition of normal or PQBP1-binding mutant Tau 410 were changed in Tau-negative neurons in addition to that of Tau-positive neurons. Indirect effect of Tau 410 monomer on Tau-negative neurons was suppressed by PQBP1-binding mutation or by tamoxifen-induced Pqbp1-cKO, while the direct effect of Tau 410 polymers on Tau-positive neurons was not affected by PQBP1-binding mutation or by tamoxifen-induced Pqbp1-cKO. TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Values in each group are presented as mean ± SEM. N = 4 fields. #P < 0.05, ##P < 0.01 in Tukey’s HSD test. c Direct effect of monomer and polymerized Tau 410 on primary neurons. Non-polymerized Tau 410 was not toxic to neurons in the absence of microglia. N = 5 visual fields. n.s.P > 0.05 in Tukey’s HSD test. d Direct and indirect pathways from extracellular tau to neuronal damage. Tau polymer directly damages neurons, while tau monomer is incorporated into LPL-negative non-DAM microglia (Supplementary Fig. 8), and indirectly damages neurons via cytokines. Though not included in this study, DAM around Aβ plaques might play a different role to prevent tau seeding. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.
Fig. 9 Direct and indirect pathways mediating neuronal toxicity of Tau. a Immunohistochemistry of Tau-injected mice revealed most of the non-microglia cells incorporating tau were MAP2-positive neurons but not GFAP-positive astrocytes. The ratios of Tau-incorporating cells were equivalent in non-induced and tamoxifen-induced Pqbp1-cKO mice, as the basis for analyses in (b). N = 20 images from three mice. Statistical test: Student’s t-test. b Frequencies of neuronal cell death (TUNEL-positive and NeuN-positive dying neurons) by addition of normal or PQBP1-binding mutant Tau 410 were changed in Tau-negative neurons in addition to that of Tau-positive neurons. Indirect effect of Tau 410 monomer on Tau-negative neurons was suppressed by PQBP1-binding mutation or by tamoxifen-induced Pqbp1-cKO, while the direct effect of Tau 410 polymers on Tau-positive neurons was not affected by PQBP1-binding mutation or by tamoxifen-induced Pqbp1-cKO. TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Values in each group are presented as mean ± SEM. N = 4 fields. #P < 0.05, ##P < 0.01 in Tukey’s HSD test. c Direct effect of monomer and polymerized Tau 410 on primary neurons. Non-polymerized Tau 410 was not toxic to neurons in the absence of microglia. N = 5 visual fields. n.s.P > 0.05 in Tukey’s HSD test. d Direct and indirect pathways from extracellular tau to neuronal damage. Tau polymer directly damages neurons, while tau monomer is incorporated into LPL-negative non-DAM microglia (Supplementary Fig. 8), and indirectly damages neurons via cytokines. Though not included in this study, DAM around Aβ plaques might play a different role to prevent tau seeding. Box plots show the median, quartiles, and whiskers that represent data outside the 25th to 75th percentile range.CC BY · PubMed Central
Fig. 10 PQBP1 depletion in microglia recovers memory functions impaired by Tau. a Memory dysfunction after injection of various species of tau in non-polymerized or polymerized state was evaluated by Y-maze test and Morris water maze test. Protocol from injection to memory function tests in various conditions is shown in Fig. 6a. Y-maze test: N number in each group is indicated in Source data. In Morris water maze test, values in each group are presented as mean ± SEM; N = 6 mice/group. #P < 0.05, ##P < 0.01, ###P < 0.001 in Dunnett’s test. Box plots show the median, quartiles. and whiskers that represent data outside the 25th to 75th percentile range. b PQBP1-depndent and PQBP1-independent pathways mediating microglia activation to neuronal cell death or neuronal cell death to microglia activation. Different types of tau species trigger distinct pathways.
Fig. 10 PQBP1 depletion in microglia recovers memory functions impaired by Tau. a Memory dysfunction after injection of various species of tau in non-polymerized or polymerized state was evaluated by Y-maze test and Morris water maze test. Protocol from injection to memory function tests in various conditions is shown in Fig. 6a. Y-maze test: N number in each group is indicated in Source data. In Morris water maze test, values in each group are presented as mean ± SEM; N = 6 mice/group. #P < 0.05, ##P < 0.01, ###P < 0.001 in Dunnett’s test. Box plots show the median, quartiles. and whiskers that represent data outside the 25th to 75th percentile range. b PQBP1-depndent and PQBP1-independent pathways mediating microglia activation to neuronal cell death or neuronal cell death to microglia activation. Different types of tau species trigger distinct pathways.CC BY · PubMed Central

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Authors
Jin, M., Shiwaku, H., Tanaka, H., Obita, T., Ohuchi, S., Yoshioka, Y., Jin, X., Kondo, K., Fujita, K., Homma, H., Nakajima, K., Mizuguchi, M., & Okazawa, H.
Affiliations
  • Tokyo Medical and Dental University (JP)
  • University of Toyama (JP)
  • Soka University (JP)
Corresponding author
Okazawa, Hitoshi
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-11-15
Volume
12
Issue
1
Pages
6565-6565
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-26851-2
PMC
PMC8592984
OpenAlex
W3214109787
Cited by
223
References
97
Citations by year
2026: 43 · 2025: 49 · 2024: 66 · 2023: 37 · 2022: 28
FWCI
13.21
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • MEXT | Japan Society for the Promotion of Science (16H02655, 19H01042)
  • Ministry of Education, Culture, Sports, Science and Technology (22110001, 22110002)
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
Keywords
tau protein, microglia, PQBP1, cGAS-STING pathway, neuroinflammation, neurodegeneration, NF-κB, innate immune response, HIV, cognitive impairment, intracellular receptors
MeSH terms
STING Protein, Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase, Animals, Brain, DNA-Binding Proteins, Encephalitis, Female, HIV, Humans, Immunity, Innate, Male, Membrane Glycoproteins, Membrane Proteins, Mice, Inbred C57BL, Nucleotidyltransferases, Tamoxifen, NF-kappa B, Microglia, Mice, Knockout, Neurodegenerative Diseases, Mice

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