Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor

Abstract

Alzheimer's disease is characterized by the accumulation of neurotoxic amyloidogenic peptide Abeta, degeneration of the cholinergic innervation to the hippocampus (the septohippocampal pathway), and progressive impairment of cognitive function, particularly memory. Abeta is a ligand for the p75 neurotrophin receptor (p75(NTR)), which is best known for mediating neuronal death and has been consistently linked to the pathology of Alzheimer's disease. Here we examined whether p75(NTR) is required for Abeta-mediated effects. Treatment of wild-type but not p75(NTR)-deficient embryonic mouse hippocampal neurons with human Abeta(1-42) peptide induced significant cell death. Furthermore, injection of Abeta(1-42) into the hippocampus of adult mice resulted in significant degeneration of wild-type but not p75(NTR)-deficient cholinergic basal forebrain neurons, indicating that the latter are resistant to Abeta-induced toxicity. We also found that neuronal death correlated with Abeta(1-42) peptide-stimulated accumulation of the death-inducing p75(NTR) C-terminal fragment generated by extracellular metalloprotease cleavage of full-length p75(NTR). Although neuronal death was prevented in the presence of the metalloprotease inhibitor TAPI-2 (tumor necrosis factor-alpha protease inhibitor-2), Abeta(1-42)-induced accumulation of the C-terminal fragment resulted from inhibition of gamma-secretase activity. These results provide a novel mechanism to explain the early and characteristic loss of cholinergic neurons in the septohippocampal pathway that occurs in Alzheimer's disease.

Figure 1.

Aβ_1–42-induced toxicity of hippocampal neurons requires p75^NTR. A, The percentage survival of cells after treatment with Aβ_1–42 or control peptide Aβ_1–16. Wild-type (WT) but not p75^NTR-deficient (KO) neurons were sensitive to Aβ_1–42-induced toxicity. Aβ_1–16 had no effect on neuronal survival. B, Treatment of wild-type hippocampal neuronal cultures with a p75^NTR-blocking antibody (Ab1554) significantly inhibited Aβ_1–42 toxicity. n = 4 experiments. *p < 0.05; ***p < 0.001.

Figure 2.

Aβ_1–42 toxicity regulates and requires extracellular cleavage of p75^NTR. A, Anti-p75^NTR Western blots of hippocampal neuronal cultures treated for 3 h with Aβ_1–42 and the γ-secretase inhibitor compound E (CpdE). All conditions contain β-clasto-lactacystin. B, These treatments result in a significant increase in the amount of C-terminal fragment (CTF) as a ratio of full-length (FL) p75^NTR as quantified by densitometry. C, D, Aβ_1–42 toxicity (C) but not staurosporine (stauro) toxicity (D) is significantly inhibited after treatment with the metalloprotease inhibitor TAPI-2. x2, Double TAPI-2 treatment. n = 3 experiments. E, Aβ_1–42 toxicity is promoted in the presence of CpdE. n = 2 experiments. *p < 0.05; ***p < 0.001.

Figure 3.

Aβ_1–42 inhibits γ-secretase cleavage of p75^NTR. A–C, Western blot (A) and quantification of the amount of the C-terminal fragment (CTF; B) and intracellular domain fragments (ICD; C) of p75^NTR in lysates of HEK293 cells transfected with full-length (FL) p75^NTR before overnight treatment with Aβ_1–42 and/or 3 h treatment with PMA and/or compound E (CpdE). All cultures were treated with β-clasto-lactacystin. D, Western blot of p75^NTR C-terminal fragment mimic ΔE14 in lysates of transfected HEK293 cells after overnight treatment with Aβ_1–42 or 3 h treatment with CpdE or PMA. E, F, Luciferase activity in lysates of HEK293 cells coexpressing ΔE14-Gal4 (E) or C99-Gal4 (F) together with pUAS (black bars) after treatment with Aβ_1–16, Aβ_1–42, or CpdE. Gray bars indicate positive-control (pCMV-Gal- and pUAS-transfected cells) and negative-control (cells transfected with pUAS alone) conditions. The total amount of total protein in lysates (white bars; in micrograms per microliter) is also shown in E (n = 6 samples from 2 experiments). *p < 0.05; **p < 0.01; ***p < 0.001. n = 4 experiments for all other conditions.

Figure 4.

Aβ_1–42 toxicity of basal forebrain cholinergic neurons in vivo is prevented by p75^NTR deficiency. The hippocampi of wild-type (WT) and p75^NTR knock-out (KO) mice were injected with Aβ, and after 14 d the number of ChAT-positive cells in each hemisphere was counted. A, The number of wild-type ChAT-positive neurons in the hemisphere ipsilateral to the Aβ_1–42 injection site was significantly reduced compared with that in uninjected or Aβ_1–16-injected animals. The numbers of ChAT-positive neurons in p75^NTR-deficient mice after injection with Aβ_1–16 or Aβ_1–42 were not significantly different, and were similar also to that found in uninjected wild-type mice. n ≥ 4 per condition; ***p < 0.001. B, C, Photomicrographs of ChAT-positive cells in the basal forebrain of wild-type (B) and p75^NTR-deficient (C) mice 14 d after injection with Aβ.