Article Figures & Data
Figures
- Fig. 1.
β-Amyloid fibrils stimulate activation of NFκB and multiple MAP kinase pathways in THP-1 cells. THP-1 cells were stimulated in serum-free RPMI with fibrillar Aβ25–35 (60 μm) for increasing times (0–60 min). Cell lysates were resolved by 7.5% SDS-PAGE and Western-blotted with selected antibodies. The p65 (RelA) subunit of NFκB was immunoprecipitated from the stimulated cell lysates and resolved by 7.5% SDS-PAGE and Western-blotted. ERK2 levels were evaluated in parallel as a protein loading control. The antibodies used were 4G10 (anti-phosphotyrosine), anti-phospho-ERK, anti-phospho-p38, anti-phospho-IκBα, anti-IκBα, anti-p65NLS, anti-c-fos, anti-phospho-CREB, and anti-ERK2.
- Fig. 2.
Regulation of transcription factor activation through Lyn- and Syk-linked signaling pathways. THP-1 cells were treated in serum-free RPMI with vehicle (DMSO) or 5 μmPP1 (Src inhibitor) or 10 μm piceatannol (Syk inhibitor) for 30 min before stimulation with fibrillar Aβ1–40 (60 μm) for 60 min. Cell lysates were resolved by 7.5% SDS-PAGE and Western-blotted with selected antibodies. The antibodies used were 4G10 (anti-phosphotyrosine), anti-phospho-ERK, anti-phospho-p38, anti-IκBα, anti-c-fos, anti-phospho-CREB, and anti-ERK2.
- Fig. 3.
β-Amyloid-induced TNFα and IL1-β gene expression require Syk activity. THP-1 cells were transiently transfected with a TNFα reporter construct and assayed for promoter activity 48 hr later. The cells were cotransfected with a β-galactosidase reporter construct to control for transfection efficiency. A, During the last 8 hr, cells were incubated in serum-free RPMI alone (black bars) or stimulated for increasing times (0–8 hr) with fibrillar Aβ25–35 (60 μm) (gray bars). B, To determine whether proximal tyrosine kinase and NFκB activities were required for TNFα promoter activity, the cells were incubated with drug/vehicle only (black bars) or with fibrillar Aβ25–35 (60 μm) (light gray bars) or Aβ1–40 (60 μm) (dark gray bars) for the last 5 hr in the presence or absence of PP1 (5 μm), piceatannol (10 μm), or 100 μg/ml SN-50 peptide. The data shown represent the average (±SEM) of three independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (**p < 0.001). C, To determine whether proximal tyrosine activities were required for β-amyloid-dependent IL1-β expression, the cells were incubated with drug/vehicle only or fibrillar Aβ25–35 (60 μm) for 5 hr in the presence or absence of PP1 (5 μm) or piceatannol (10 μm). Cell lyates were resolved by 9% SDS-PAGE and Western-blotted using anti-IL-1β antibody.
- Fig. 4.
Proinflammatory products in conditioned media from Aβ-stimulated THP-1 cells induce neuronal apoptosis. Conditioned Neurobasal media from THP-1 monocytes stimulated for 48 hr without (A, control) or with (B) immobilized Aβ1–40 was applied to mouse cortical neuron cultures (E16, 5 d in vitro) for 48 hr. Neurons were fixed in 4% paraformaldehyde, and endonuclease-cleaved DNA was visualized by TUNEL staining. C, Nuclear extracts were collected from the same conditions, control (C) and conditioned media (CM)-treated neurons after 48 hr treatment, and fragmented DNA was visualized after agarose electrophoresis.
- Fig. 5.
Conditioned media from Aβ-stimulated monocytes produces TNFα-dependent-neuronal apoptosis. Purified cultures of mouse cortical neurons (E16, 5 d in vitro) were incubated for 72 hr in Neurobasal media from unstimulated THP-1 cells or conditioned Neurobasal media (CM) obtained from THP-1 cells stimulated (48 hr) via surface-immobilized Aβ25–35 or Aβ1–40 fibrils (48 pmol/mm2). The incubations also included, as indicated, anti-human TNFα antibody (5 μg/ml), mouse TNFα (100 ng/ml), and anti-human IL-1β antibody (5 μg/ml). The neurons were fixed and stained for neuron-specific MAP2 protein, and surviving neurons were counted. Neurons from four fields/condition were counted in duplicate wells and averaged ± SEM. The mean values shown (±SEM) are representative of four independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (*p < 0.001).
- Fig. 6.
Conditioned media from Aβ-stimulated monocytes and microglia produces TNFα/iNOS-dependent-neuronal apoptosis. Purified cultures of mouse cortical neurons (E16, 5 din vitro) were treated for 72 hr in Neurobasal media from unstimulated microglia (B) or THP-1 cells (A) or conditioned Neurobasal media (CM) obtained from microglia (B) or THP-1 cells (A) stimulated (48 hr) via surface-immobilized Aβ25–35 or Aβ1–40 fibrils. The incubations also included, as indicated, 10 μm AMT.HCl, 5 μm 1400W.2HCl, 20 μm Vinyl-l-NIO, and anti-mouse TNFα antibody (5 μg/ml). To terminate experiments, the neurons were fixed and stained for neuron- specific MAP2 protein, and surviving neurons were counted. Neurons from four fields/condition were counted in duplicate wells and averaged ± SEM. The mean values shown (±SEM) are representative of four independent experiments. Unpaired ANOVA was performed with Tukey-Kramer post-comparison to evaluate statistical significance (*p < 0.001).
- Fig. 7.
Aβ-stimulated conditioned media-dependent neuronal apoptosis involves increased iNOS expression. Mouse cortical neuron cultures (E16, 5 d in vitro) were treated for 72 hr in the absence (control) or presence of conditioned Neurobasal media (CM) obtained from THP-1 cells stimulated 48 hr with surface-immobilized Aβ1–40 fibrils with and without 5 μg/ml anti-human TNFα antibody or 100 ng/ml mouse TNFα. A–D, After treatment, neuronal cultures were fixed in 4% paraformaldehyde and stained using an anti-iNOS antibody. Antibody binding was visualized with goat anti-rabbit FITC-conjugated secondary antibody. E, Neuronal cultures were also collected after stimulation, and lysates were resolved by 7.5% SDS-PAGE and Western-blotted using the anti-iNOS antibody. Blots were stripped and reprobed using anti-ERK2 antibody to confirm equal protein loading.
- Fig. 8.
Aβ-stimulated conditioned media-dependent neuronal apoptosis is characterized by increased nitrotyrosine expression. Mouse cortical neuron cultures (E16, 5 d in vitro) were treated for 72 hr in the absence (c,control) or presence of conditioned Neurobasal media (CM) obtained from THP-1 cells or microglia-stimulated (48 hr) with surface-immobilized Aβ1–40 fibrils. Neuronal cultures were incubated in Neurobasal media from unstimulated THP-1 cells (A) or microglia (B), conditioned Neurobasal media from Aβ-stimulated THP-1 cells (C) or microglia (D), 10 μm 1400W.2HCl only (F), and Aβ-stimulated THP-1 cell (E) conditioned Neurobasal media + 10 μm 1400W.2HCl. After treatment, neuronal cultures were fixed in 4% paraformaldehyde and stained using an anti-nitrotyrosine antibody. Antibody binding was visualized with goat anti-rabbit FITC-conjugated secondary antibody. G, Neuronal cultures were also lysed after stimulation, and nitrotyrosine-containing proteins were immunoprecipitated and resolved by 7.5% SDS-PAGE and Western-blotted using the anti-nitrotyrosine antibody.
- Fig. 9.
Aβ-stimulated microglial and monocytic proinflammatory products cause TNFα/iNOS-dependent neuronal apoptosis. Binding of Aβ fibrils to microglia initiates a tyrosine kinase-dependent signaling response involving Src family members and Syk as membrane proximal signaling elements. Src kinases, such as Lyn, mediate the activation of several tyrosine kinase activities associated with adhesion and phagocytosis of Aβ fibrils. In parallel, Syk kinase activity specifically regulates increased cytokine production in response to Aβ stimulation. A separate cytokine regulatory pathway using the proinflammatory transcription factor NFκB is also activated on Aβ stimulation to operate independent of Src and Syk activation. Aβ-stimulated microglia secrete TNFα plus an additional secretory factor(s) to produce neuronal apoptosis. The apoptosis requires iNOS activity and correlates with increased expression of iNOS and peroxynitrite.
