Amyloid beta peptide of Alzheimer's disease downregulates Bcl-2 and upregulates bax expression in human neurons

Abstract

Neuronal apoptosis is a suspected cause of neurodegeneration in Alzheimer's disease (AD). Increased levels of amyloid beta peptide (Abeta) induce neuronal apoptosis in vitro and in vivo. The underlying molecular mechanism of Abeta neurotoxicity is not clear. The normal concentration of Abeta in cerebrospinal fluid is 4 nM. We treated human neuron primary cultures with 100 nM amyloid beta peptides Abeta(1-40) and Abeta(1-42) and the control reverse peptide Abeta(40-1). We find that although little neuronal apoptosis is induced by either peptide after 3 d of treatment, Abeta(1-42) provokes a rapid and sustained downregulation of a key anti-apoptotic protein, bcl-2, whereas it increases levels of bax, a protein known to promote cell death. In contrast, the Abeta(1-40) downregulation of bcl-2 is gradual, although the levels are equivalent to those of Abeta(1-42)-treated neurons by 72 hr of treatment. Abeta(1-40) does not upregulate bax levels. The control, reverse peptide Abeta(40-1), does not affect either bcl-2 or bax protein levels. In addition, we found that the Abeta(1-40)- and Abeta(1-42)- but not Abeta(40-1)-treated neurons had increased vulnerability to low levels of oxidative stress. Therefore, we propose that although high physiological amounts of Abeta are not sufficient to induce apoptosis, Abeta depletes the neurons of one of its anti-apoptotic mechanisms. We hypothesize that increased Abeta in individuals renders the neurons vulnerable to age-dependent stress and neurodegeneration.

Fig. 1.

Neurite degeneration is observed after 72 hr of treatment with Aβ peptides. Phase-contrast micrograph of primary neuron cultures treated with 100 nm Aβ_1–40or Aβ_1–42 and control reverse peptide Aβ_40–1 for 6, 12, and 72 hr. Arrows show neuritic beading, and arrowheads show neurite thickening.

Fig. 2.

Aβ peptides do not cause high levels of cell death in neuron primary cultures. Determination of cell death in Aβ_1–40-, Aβ_1–42-, and Aβ_40–1-treated human primary neurons for 12, 24, 48, and 72 hr by MTT reduction assay expressed as a percentage of time 0 (n = 3 ± SEM) (A), LDH release assay (n = 3 ± SEM) (B), and TUNEL-positive cells in a representative experiment (C).

Fig. 3.

Aβ peptides downregulate bcl-2 protein and upregulate bax protein levels in primary neuron cultures.A, Western blot analysis of one representative experiment of bcl-2, bax, and actin protein levels in neurons treated with Aβ_1–40, Aβ_1–42, and Aβ_40–1 for 0, 6, 12, 24, 48, and 72 hr.B, Densitometric quantitation by phosphorimager of bcl-2 protein levels in neuron cultures normalized to levels in 0 hr culture (mean of three independent experiments, ± SEM) shows rapidly decreasing bcl-2 protein levels in Aβ_1–42(p < 0.01, from 6–72 hr) and a gradual decrease in Aβ_1–40-treated neurons (p < 0.01, at 72 hr only) compared with Aβ_40–1-treated neurons. C, Densitometric quantitation by phosphorimager of bax protein levels in neuron cultures normalized to levels in untreated (0 hr) sister culture (mean of three independent experiments, ± SEM) shows rapidly increasing bax protein levels in Aβ_1–42 (p < 0.05, between 24–72 hr) but no difference in Aβ_1–40- compared with Aβ_40–1-treated neurons.

Fig. 4.

Susceptibility to H₂O₂ in neurons pretreated with Aβ_1–40 and Aβ_1–42but not Aβ_40–1. TUNEL labeling detects increased apoptosis in neurons pretreated for 48 hr with Aβ_1–40and Aβ_1–42 and then submitted to 0.1 μmH₂O₂ and 1.0 μmH₂O₂ for 48 hr. In contrast, sister cultures treated with the control peptide Aβ_40–1 do not show increased sensitivity to low levels of oxidative stress. Data represent an average of four experiments.