Article Figures & Data
Figures
- Figure 1.
Temporal profile of brain inflammation in the 3xTg-AD mice. The time course of intraneuronal and extraneuronal Aβ buildup and its relationship to activated microglia were described in the 3xTg-AD mice. Both intraneuronal and extraneuronal Aβ deposits were determined by 6E10 immunostaining of 3xTg-AD brains at 9 (A), 12 (B), and 24 (C) months of age. Fibrillar amyloid plaques were detected by thioflavin S staining at 9 (D), 12 (E), and 24 (F) months of age. Serial sections were stained with anti-CD45 to detect activated microglia at 9 (H), 12 (I), and 24 (J) months of age. Amyloid burden (G) and CD45-immunopositive-activated microglia (K) were quantitatively analyzed by measuring the percentage of occupied area by thioflavin S (amyloid burden) or CD45-immunopositive staining (microglia burden). Data are presented as mean±SEM (n=5 in each age). Higher-magnification images of CD45-immunopositive staining in the subicular subfield of the hippocampus from a 24-month-old NonTg mouse (L) and 3xTg-AD mouse (M) are shown. GFAP immunostaining revealed no gliosis in layers III–IV of the neocortex of a 24-month-old NonTg mouse (N), whereas there was a marked increase in GFAP-immunoreactive astrocytes detected in 24-month-old 3xTg-AD mice (O).
- Figure 2.
Activated microglia colocalize with plaques in aged 3xTg-AD mice. Activated microglia detected by CD45 immunoreactivity (brown) colocalize with extraneuronal Aβ-immunopositive deposits (blue) in the cortex (A), amygdala (B), and hippocampus (C) in 24-month-old 3xTg-AD mice. Serial brain sections of the hippocampus were stained with thioflavin S (D). Arrows point to activated microglia around thioflavin S-positive plaques. Additional double-staining fluorescent analysis using thioflavin S (green) and Iba1 (red) to detect microglia demonstrates that 3xTg-AD mice (E) show similar microglia and plaque interactions as observed in the hippocampus of the human AD brain (F). Arrows point to microglia associated with the cores of amyloid plaques in the human AD brain. G, Double fluorescent labeling of GFAP-immunopositive astrocytes (green) and amyloid-containing plaques (red) detected by 6E10 in 24-month-old 3xTg-AD mice shows colocalization of astrocytes around plaques.
- Figure 3.
LPS activates microglia in the 3xTg-AD mice. LPS (0.5 mg/kg body weight) or PBS was injected intraperitoneally 4-month-old 3xTg-AD mice twice per week for 6 weeks, and the brains were analyzed by immunohistochemistry. CD45 immunoreactivity was compared in the hippocampus of PBS- and LPS-treated 3xTg-AD mice. The brains from PBS-treated mice show only basal levels of microglial activation (A), whereas LPS-treated mice show significantly increased activated microglia (B). At least five mice per group were analyzed. C, Quantitative RT-PCR of IL-1β and IL-6 was performed in total RNA extracts from PBS- or LPS-treated 3xTg-AD mice brains. In each mouse, the expression levels of mRNA were normalized relative to the levels of GAPDH mRNA (Ct values for the PBS- and LPS-treated brain are 14.54 ± 0.22 and 14.55 ± 0.18, respectively), and fold differences were calculated relative to the PBS-treated group for each IL. **p < 0.01 compared with the PBS-treated mice group (n = 3 in group).
- Figure 4.
Effect of LPS-induced inflammation on APP processing in the 3xTg-AD mice. A, Immunoblotting of APP, C99, and C83 in PBS- or LPS-treated 3xTg-AD mice, withβ-actin used as a protein loading control. B, Densitometric analysis of steady-state levels of full-length APP, C99, or C83 between PBS- and LPS-treated 3xTg-AD mice. Data are presented as mean ± SEM (n = 4 each group). C, Total Aβ40 and Aβ42 from detergent-soluble and -insoluble fractions of PBS- and LPS-treated 3xTg-AD mice were measured by ELISA. □, PBS-treated 3xTg-AD mice; ▪, LPS-treated 3xTg-AD mice. Data are presented as mean ± SEM (n = 8 each group). D, Fluorescent intensity of 6E10-immunopositive neurons in the cortex and hippocampus was quantitatively analyzed using Quantity One software. Data are presented as mean intensity ± SEM (n = 5 each group). E, Representative immunofluorescent staining of APP and Aβ fragments by 6E10 in the neocortex and hippocampus of 3xTg-AD mice treated with PBS or LPS. a.u., Arbitrary units; HC, hippocampus; CX, cortex.
- Figure 5.
Inflammation-induced enhancement of tau phosphorylation. A, Immunoblotting analysis of total tau (HT7) and phosphorylated tau (AT8) in PBS- and LPS-treated 3xTg-AD mice, with β-actin used as a protein loading control. Data represent at least five mice per group. B, Immunohistochemical staining of total human tau and phosphorylated tau in hippocampal neurons of 3xTg-AD mice treated with PBS and LPS. The inset in the bottom right panel is a magnified view of AT8-immunopositive neurons in the hippocampus.
- Figure 6.
Site-specific phosphorylation of tau after LPS exposure. A, Immunoblotting of AT180- and PHF1-positive phospho-tau in LPS- or PBS-treated 3xTg-AD mice brain, with β-actin used as a protein loading control. B, Densitometric analysis of steady-state levels of PHF1 or AT180 between PBS- and LPS-treated 3xTg-AD mice. Data are presented as mean ± SEM (n = 5). **p < 0.01. C, Representative immunohistochemical staining of AT180-positive tau in hippocampal neurons of 3xTg-AD mice treated with PBS and LPS. D, Six-month-old NonTg mice were treated with 0.5 mg/kg LPS or PBS intraperitoneally twice per week for 6 weeks. After the treatment period, the hippocampus and cortex were isolated, and proteins were analyzed by immunoblotting for AT8-positive phosphorylated tau. a.u., Arbitrary units.
- Figure 7.
LPS-induced inflammation causes increased p25 formation. A, Immunoblotting for p35/p25, cdk5, phosphorylated/total GSK-3β, phosphorylated/total p38–MAPK, and phosphorylated/total JNK of PBS- and LPS-treated 3xTg-AD mice, withβ-actin used as a protein loading control. Densitometric analysis revealed a significant (p < 0.05) increase in p25 levels in LPS-treated 3xTg-AD mice (n = 5; data not shown). B, Immunoblotting for p35/p25 and cdk5 of PBS- and LPS-treated NonTg mice, with β-actin used as a protein loading control. Densitometric analysis revealed a significant (p < 0.05) increase in p25 levels in LPS-treated NonTg mice (n = 4; data not shown).
- Figure 8.
Roscovitine inhibits the cdk5-dependent phosphorylation of tau in LPS-treated 3xTg-AD mice. 3xTg-AD mice were treated with roscovitine (300 nmol per day) or vehicle (50% DMSO) through an intraventricular infusion during the last 2 weeks of the 6 week LPS treatment. The hippocampus was isolated and tested for phosphorylation status of tau by immunoblotting. The steady-state levels of human APP and tau were not altered by roscovitine treatment. Densitometric analysis revealed the steady-state levels of AT8- and AT180-positive tau resulted in nearly a twofold and threefold reduction in roscovitine treatment, respectively, whereas PHF1-positive tau levels were not affected by LPS (data not shown). Membranes were reprobed with β-actin to control the equal protein loading (n = 3 each group).
Tables
Antibody Immunogen Host Specificity Application Source 6E10 aa 1-17 of Aβ Mouse H, M IHC, IF, WB Signet (Dedham, MA) CT20 aa 751-770 of human APP Rabbit H, M WB Calbiochem GFAP Bovine GFAP Rabbit H, M, R IHC, IF DAKO (Carpinteria, CA) CD11b Mouse B-cells Rat M IHC Serotec (Raleigh, NC) CD45 Mouse B-cells Rat M IHC Serotec Iba1 C terminus of Iba1 Rabbit H, M, R IF WAKO (Richmond, VA) HT7 aa 159-163 of tau Mouse H IHC, WB Innogenetics (Alpharetta, GA) AT8 Peptides containing phospho-S202/T205 Mouse H, M, R IHC, IF, WB Innogenetics AT180 Peptides containing phospho-T231/S235 Mouse H, M, R IHC, WB Innogenetics PHF1 Peptides containing phospho-S396/S404 Mouse H, M, R IHC, WB a β-Actin C terminus of actin Rabbit Wide range WB Sigma Aβ40 aa 35-40 of Aβ Mouse H, M, R, D ELISA Mayo Clinic (Jacksonville, FL) Aβ42 aa 35-42 of Aβ Mouse H, M, R, D WB Mayo Clinic p35/p25 C terminus of human p35 Rabbit H, M, R WB Santa Cruz Biotechnology (Santa Cruz, CA) cdk5 aa 16 of human cdk5 Rabbit H, M, R WB Calbiochem JNK Human JNK2 Rabbit H, M, R WB Cell Signaling (Beverly, MA) Phospho-JNK Phosphopeptides around The183/Tyr185 Mouse H, M, R WB Cell Signaling GSK-3β N terminus of rat GSK-3β Mouse H, M, R, D, C WB BD Transduction (San Diego, CA) Phospho-GSK-3β Phosphopeptides around Tyr216 Mouse H, M, R, D WB BD Transduction Phospho-GSK-3β Phosphopeptides around Ser9 Rabbit H, M, R WB Cell Signaling p38 Human p38 Rabbit H, M, R WB Cell Signaling Phospho-p38 Phosphopeptides around Thr180/Tyr182 Rabbit H, M, R WB Cell Signaling -
H, Human; M, mouse; R, rat; D, dog; C, chicken; IHC, immunohistochemistry; IF, immunofluorescent staining; WB, Western blotting.
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↵ a Generous gift from P. Davis (Albert Einstein College of Medicine, Bronx, NY).
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