Journal: The Journal of Neuroscience

Article Title: p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

doi: 10.1523/JNEUROSCI.2733-10.2011

Figure Lengend Snippet: Expression of p75NTR in the brain. p75NTR expression in the brain was measured by Western blot and immunohistochemistry (n = 10 in each group). A, Brain homogenates of APPSwe/PS1dE9 mice and their wild-type littermates at 3, 6, and 9 months of age were subjected to Western blot analysis probed with rabbit anti-p75NTR polyclonal antibody (G3231) and monoclonal antibody to β-actin. B, Sections of basal forebrain, frontal lobe, and hippocampus from 9-month-old APPSwe/PS1dE9 mice and their wild-type littermates were stained using free-floating immunohistochemistry for p75NTR with rabbit anti-p75NTR polyclonal antibody (Ab9650). C, Representative confocal images for colocalization of p75NTR-positive fibers and fibrillar plaques in brain of 9-month-old APPSwe/PS1dE9 mice, with Ab 9650 for p75NTR (arrowheads), N52 for neurofilament 200 (NF200, arrows), and thioflavine S for fibrillar plaque.

Article Snippet: The blots were probed with the following antibodies: rabbit anti-p75NTR polyclonal antibody (G3231 from Promega for full-length p75NTR and ANT-007 from Alomone for shed extracellular domain), anti-BACE1 monoclonal antibody (MAB931, R&D Systems), anti-insulin-degrading enzyme (IDE) polyclonal antibody (25970, Abcam), anti-neprilysin (NEP) polyclonal antibody (NEP11-A, Alpha Diagnostic), anti-LRP monoclonal antibody (5A6, Abcam), and anti-β actin monoclonal antibody (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: The Journal of Neuroscience

Article Title: p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

doi: 10.1523/JNEUROSCI.2733-10.2011

Figure Lengend Snippet: Extracellular domain of p75NTR attenuates Aβ aggregation. A, Dose-dependent inhibition of Aβ oligomerization by p75/Fc in vitro. Aβ42 (final concentration 20 μm) was incubated with p75/Fc at various molar ratios (1:0.01, 1:0.1, and 1:0.5) or HuIgG (molar ratio, 1:0.5) at 4°C for 24 h. “Aβ non-incubated” is the control Aβ42 peptide without incubation at 4°C for 24 h. “Aβ incubated” is the control Aβ42 peptide (final concentration 20 μm) that was incubated alone at 4°C for 24 h. Bands were visualized by Western blot analysis probed with biotin-conjugated 6E10 antibody. B, Inhibition of Aβ fibrillation by p75/Fc. Twenty-five micromolar Aβ42 peptide (30 μg) was incubated with 12.5 μm p75/Fc or HuIgG in DMEM containing 10 mm HCl at 37°C for 24 h. The same amount of Aβ42 was incubated alone under the same conditions as control. The Aβ fibrils were measured by ThT assay. C, Disaggregation of preformed Aβ fibrils by p75/Fc. Twenty-five micromolar Aβ42 (30 μg) was incubated at a concentration of at 37°C for 1 d to generate fibrils. Preformed fibrils were then incubated with the 12.5 μm p75/Fc or HuIgG for an additional 3 d at 37°C. Aβ42 was incubated alone under the same conditions, along with the experiment as control. The Aβ fibrils were measured by ThT assay. D–L, Electron micrographs showing morphology of Aβ assembly in the presence or absence of p75/Fc. D–F, Aβ oligomerization. Aβ42 was incubated alone (D) or with p75/Fc (E) or HuIgG (F) at 4°C for 24 h. G–I, Aβ fibrillation. Aβ42 was incubated alone (G) or with p75/Fc (H) or HuIgG (I) at 37°C for 24 h. J–L, Disaggregation of Aβ fibrils. Preformed Aβ was incubated alone (J) or with p75/Fc (K) or HuIgG (L) at 37°C for an additional 3 d. Scale bar, 500 nm.

Article Snippet: The blots were probed with the following antibodies: rabbit anti-p75NTR polyclonal antibody (G3231 from Promega for full-length p75NTR and ANT-007 from Alomone for shed extracellular domain), anti-BACE1 monoclonal antibody (MAB931, R&D Systems), anti-insulin-degrading enzyme (IDE) polyclonal antibody (25970, Abcam), anti-neprilysin (NEP) polyclonal antibody (NEP11-A, Alpha Diagnostic), anti-LRP monoclonal antibody (5A6, Abcam), and anti-β actin monoclonal antibody (Sigma-Aldrich).

Techniques: Inhibition, In Vitro, Concentration Assay, Incubation, Control, Western Blot, ThT Assay

Journal: The Journal of Neuroscience

Article Title: p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

doi: 10.1523/JNEUROSCI.2733-10.2011

Figure Lengend Snippet: Hippocampus injection of p75/Fc reduces local Aβ plaques. p75/Fc (3 μg in 3 μl) or HuIgG (6 μg in 3 μl, the equivalent molar to p75/Fc) were injected into the left hippocampus of 9-month-old APPSwe/PS1dE9 mice (n = 4 in each group). One week after injection, Aβ plaques in hippocampus were stained using biotin-conjugated 6E10 antibody and quantified. The area fraction of Aβ plaque in hippocampus of the injection side was normalized with the control side. A, Distribution and diffusion of p75/Fc 24 h after injection in the left hippocampus. Sections were stained with antibody to Fc of human IgG. B, C, Representative images of hippocampus Aβ plaque staining 7 d after injection of p75/Fc (B) or HuIgG (C) into the hippocampus of 9-month-old APPSwe/PS1 mice. D, Comparison of Aβ plaque burden in hippocampus between p75/Fc and HuIgG injection groups. E, Expression of p75NTR extracellular domain (ECD) in the brain of wild-type and APPSwe/PS1dE9 mice at age of 9 months. To see the diffusion of injected p75/Fc in the hippocampus, three mice were killed 24 h after injection and brain sections were stained against Fc fragment of human IgG. Extensive diffusion of the protein was observed in the injected hippocampus, whereas little protein diffused into the contralateral hippocampus (arrow). * denotes p < 0.05.

Article Snippet: The blots were probed with the following antibodies: rabbit anti-p75NTR polyclonal antibody (G3231 from Promega for full-length p75NTR and ANT-007 from Alomone for shed extracellular domain), anti-BACE1 monoclonal antibody (MAB931, R&D Systems), anti-insulin-degrading enzyme (IDE) polyclonal antibody (25970, Abcam), anti-neprilysin (NEP) polyclonal antibody (NEP11-A, Alpha Diagnostic), anti-LRP monoclonal antibody (5A6, Abcam), and anti-β actin monoclonal antibody (Sigma-Aldrich).

Techniques: Injection, Staining, Control, Diffusion-based Assay, Comparison, Expressing

Journal: The Journal of Neuroscience

Article Title: p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

doi: 10.1523/JNEUROSCI.2733-10.2011

Figure Lengend Snippet: Deletion of p75NTR does not lessen memory deficits until 9 months of age. Nine-month-old animals were subjected to Morris water maze test for a consecutive 5 d (n = 10 in each group). A, Latency taken to escape from the water in the platform trials. B, Distance taken to escape from the water in the platform trials. C, Swimming speed during the consecutive days of training. D, The number of crosses over the exact location of the hidden platform in the probe trial. E, Percentage of time spent in the quadrant area relative to the total time spent in the pool in the probe trial. The results are means ± SEM.

Article Snippet: The blots were probed with the following antibodies: rabbit anti-p75NTR polyclonal antibody (G3231 from Promega for full-length p75NTR and ANT-007 from Alomone for shed extracellular domain), anti-BACE1 monoclonal antibody (MAB931, R&D Systems), anti-insulin-degrading enzyme (IDE) polyclonal antibody (25970, Abcam), anti-neprilysin (NEP) polyclonal antibody (NEP11-A, Alpha Diagnostic), anti-LRP monoclonal antibody (5A6, Abcam), and anti-β actin monoclonal antibody (Sigma-Aldrich).

Techniques:

Journal: The Journal of Neuroscience

Article Title: p75NTR Regulates Aβ Deposition by Increasing Aβ Production But Inhibiting Aβ Aggregation with Its Extracellular Domain

doi: 10.1523/JNEUROSCI.2733-10.2011

Figure Lengend Snippet: Schematic diagram depicting functions of p75NTR in Aβ metabolism. p75NTR may have two-sided effects on Aβ metabolism. On one hand, p75NTR signaling may increase Aβ production and enhance steady-state levels of Aβ, which may increase AD pathology. In addition to Aβ production, p75NTR also mediates Aβ and proNGF-mediated neurotoxicity such as neuron death and neurite degeneration. On the other hand, the extracellular domain of p75NTR after shedding from the membrane may bind and sequester Aβ, and thus suppress Aβ aggregation and reduce Aβ deposition in the brain. Meanwhile, the extracellular domain of p75NTR may also block the interaction of p75NTR and its ligands (e.g., Aβ or proNGF) by competitive binding, and thus attenuate the p75NTR signaling that leads to the neurotoxicity. ECD denotes extracellular domain of p75NTR.

Article Snippet: The blots were probed with the following antibodies: rabbit anti-p75NTR polyclonal antibody (G3231 from Promega for full-length p75NTR and ANT-007 from Alomone for shed extracellular domain), anti-BACE1 monoclonal antibody (MAB931, R&D Systems), anti-insulin-degrading enzyme (IDE) polyclonal antibody (25970, Abcam), anti-neprilysin (NEP) polyclonal antibody (NEP11-A, Alpha Diagnostic), anti-LRP monoclonal antibody (5A6, Abcam), and anti-β actin monoclonal antibody (Sigma-Aldrich).

Techniques: Membrane, Blocking Assay, Binding Assay