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. 2003 Aug 13;23(19):7385-94.
doi: 10.1523/JNEUROSCI.23-19-07385.2003.

p75 Neurotrophin Receptor Protects Primary Cultures of Human Neurons Against Extracellular Amyloid Beta Peptide Cytotoxicity

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Free PMC article

p75 Neurotrophin Receptor Protects Primary Cultures of Human Neurons Against Extracellular Amyloid Beta Peptide Cytotoxicity

Yan Zhang et al. J Neurosci. .
Free PMC article

Abstract

The cytotoxicity of extracellular amyloid beta peptide (Abeta) has been clearly demonstrated in many cell types. In contrast, primary human neurons in culture are resistant to extracellular Abeta-mediated toxicity. Here, we investigate the involvement of p75 neurotrophin receptor (p75NTR) in Abeta-treated human neurons. We find that Abeta1-40 and Abeta1-42, but not the reverse control peptide, Abeta40-1, rapidly increase the levels of p75NTR in a specific and dose-dependent manner. In contrast to observations in cell lines, enhanced expression of p75NTR in human neurons via a herpes simplex virus amplicon vector does not increase the susceptibility of neurons to Abeta. Unexpectedly, inhibition of p75NTR expression with an antisense expression construct or incubation of the cells with an antibody to the extracellular domain of p75NTR sensitizes human neurons to extracellular nonfibrillar or fibrillar Abeta1-42 cytotoxicity. Unlike intracellular Abeta, extracellular Abeta toxicity is independent of p53 and Bax activity. However, Abeta toxicity is inhibited by caspase inhibitors and the glycogen synthase kinase 3beta inhibitor lithium. Neuroprotection against Abeta is phosphatidylinositide 3-kinase dependent but Akt independent. These results are consistent with a neuroprotective role for p75NTR against extracellular Abeta toxicity in human neurons.

Figures

Figure 1.
Figure 1.
Aβ upregulates p75NTR in primary cultures of human neurons. A, Western blot analysis of p75NTR or β-actin in proteins from untreated neurons (Control) or neurons treated with 100 nm1-40, Aβ1-42, or reverse peptide control Aβ40-1 for 48 hr and in fetal brain or K562 protein extracts (10 μg/lane). B, Western blot analysis of p75NTR and TNF-R1 with 0.1, 1.0, or 10 μM1-42 treatments of 48 hr. The point zero represents untreated neurons cultured in serum before the addition of peptide. C, Quantitative analysis of p75NTR levels after 48 hr of treatment measured by densitometric analysis of ECL-Western blots. Data represent the means and SEMs of experiments in three independent neuron cultures. D, Quantitative analysis of p75NTR levels with time of 100 nm Aβ treatments in four independent experiments. For Aβ1-40 orAβ1-42 treatments of ≥3 hr, p < 0.01 compared with untreated control or Aβ40-1. E, Western blot analysis of p75NTR in neurons treated with 0.1 or 10 μm staurosporine in serum-containing conditions.
Figure 2.
Figure 2.
p75NTR protects human neurons against extracellular Aβ1-42 toxicity. Western blot analysis of p75NTR in two independent experiments of pHSVPrPUCp75 sense (p75S)-infected primary neurons (A) or pHSVPrPUCp75 antisense (p75AS)-infected primary neurons in the presence and absence of 100 nm1-42 (B). BE, Proteins from a brain extract. Ctl, Control. C, Interference of p75S-mediated expression of p75NTR by cotransduction of neuron cultures with p75AS shown by Western blotting of the transduced cultures with anti-p75NTR and β-actin. D, TUNEL-positive cell death in neurons microinjected with DTR, pHSVPrPUC empty construct (vector), p75S, p75AS, or PrPAS, and either untreated (Ctl) or treated with 100 nm1-42 or Aβ42-1 in the presence of serum. Data represent the mean and SEM of three independent experiments. The difference between microinjection of p75S, p75AS, and PrPAS versus vector microinjections was assessed by one-way ANOVA followed by Scheffé's test; *p < 0.0001. There was no other statistically significant difference between the different groups.
Figure 3.
Figure 3.
Impact of Aβ concentration, fibrillar state, and pro-NGF on neuronal cell death. A, TUNEL-positive cell death of 100 nm versus 10 μm1-42 in vector-, p75S-, or p75AS-microinjected neurons. B, Extracellular toxicity of fibrillized and nonfibrillized Aβ1-42 and Aβ42-1 on p75S- or p75AS-microinjected neurons. One-way ANOVA followed by Scheffé's test compares p75S and p75AS with control (Ctl); *p < 0.0001. C, Western blot analysis of NGF with anti-NGF H20 in protein extracts from 24 hr serum-deprived (-S) or serum-treated (+S) neurons (50 μg/lane), adult or fetal brain (100 μg/lane), and conditioned media (CM) from serum-deprived neurons and serum. Mature recombinant NGF (R-NGF) was added as a control (100 ng/lane). Data in A and B represent the mean and SEM of three independent experiments.
Figure 4.
Figure 4.
Anti-extracellular p75 antibody specifically induces neuronal cell death in the presence of Aβ. A, TUNEL-positive cell death in vector-, p75S-, or p75AS-microinjected neurons treated for 24 hr with anti-p75ec. B, TUNEL-positive cell death in serum-deprived neurons treated with anti-p75ec or with anti-p75ic in the absence [control (Ctl)] or presence of 100 nm extracellular Aβ1-42. The difference between Aβ1-42 alone versus p75 antibodies plus Aβ1-42 was assessed by one-way ANOVA followed by Scheffé's test; *p < 0.003. The increase in cell death with anti-p75ic is not significantly different from control. C, Competition of anti-p75ec-induced Aβ toxicity by R-p75NTR; *p < 0.02 compared with Ctl (one-way ANOVA). D, TUNEL-positive cell death in neurons treated for 24 hr with 100 nm1-42, 0.1 μm H2O2, or 20 μm etoposide in the absence or presence of 1 μg/ml anti-p75ec. Data represent mean and SD.
Figure 5.
Figure 5.
Wortmannin sensitizes neurons to Aβ1-42 toxicity. A, TUNEL-positive neuronal cell death in neurons pretreated 1 hr with 200 nm or 10 μm wortmannin and exposed to 100 nm extracellular Aβ1-42 in the continued presence of wortmannin for 24 hr. Data represent the mean and SEM of three independent experiments. One-way ANOVA followed by Scheffé's compared wortmannin-treated cells with untreated cells; *p < 0.001. B, Western blot analysis of total PI3K in neurons treated with Aβ1-42 and wortmannin. C, Autoradiogram of phosphorylated phosphatidylinositol (PIP) from immunoprecipitated PI3K. The concentration of wortmannin was 200 nm. Wtm, Wortmannin; Ctl, control; ORI, origin.
Figure 6.
Figure 6.
p75NTR does not protect human neurons through Akt activation. A, Western blot analyses with phospho-Akt (pAkt) 473, total Akt, or β-actin in neurons treated with 10 μm wortmannin for 6 hr. B, TUNEL-positive cell death in neurons pretreated 1 hr with 10 μm wortmannin and microinjected with wild-type Akt (Akt WT), constitutively active Akt (Akt active), or dominant-negative Akt (Akt DN) before a 24 hr treatment with extracellular Aβ1-42 and wortmannin. Controls are serum deprived [-serum (-S)] for 96 hr after microinjection with the Akt constructs. Data represent the mean and SEM of three independent experiments. C, Western blot analyses of phospho-Akt (P-Akt) 473 or 308, total Akt, and β-actin in proteins from neurons treated with p75ic or p75ec antibodies in the absence or presence of 100 nm1-42. D, Western blot of phospho-Akt (P-Akt) 308 in proteins from neurons treated with anti-p75ec in the presence of 200 nm or 10 μm wortmannin. Wtm, Wortmannin; Ctl, control.
Figure 7.
Figure 7.
Pathways of extracellular Aβ1-42-induced toxicity. A, Cell death in neurons microinjected with p53WT or p53DN comicroinjected with p75AS or treated with wortmannin in addition to a 100 nm extracellular Aβ1-42 treatment. There was no significant difference with p53 microinjections compared with the absence of p53. B, Cell death in cells microinjected with Bax-neutralizing antibodies N20, 2D2, or 6A7 comicroinjected with Bax cDNA or treated with 100 nm extracellular Aβ1-42 and 200 nm wortmannin. The APP or N20 treatments were compared with the rabbit sera (Rb-sera), and 2D2 and 6A7 were compared with mouse IgG (Mo-IgG) by one-way ANOVA followed by Scheffé's test; *p < 0.05; **p < 0.005. C, Neuronal cell death in neurons microinjected with p75AS and treated with 100 nm1-42 or treated with wortmannin and Aβ1-42 in the absence or presence of 5 μm Boc-D-fmk caspase inhibitor. Recombinant active caspase-6 (R-Csp-6) was microinjected as a positive control for the Boc-D activity. The difference between the absence or presence of Boc-D-fmk was assessed by one-way ANOVA followed by Scheffé's test; *p < 0.0001. Data represent the means and SEMs of three independent experiments. D, Cell death from 100 nm extracellular Aβ1-42 with either p75AS or wortmannin in the absence or presence of 20 mm LiCl2. Data represent the mean and SEM of three independent experiments. The difference between the absence or presence of LiCl2 was assessed by one-way ANOVA and Scheffé's test; *p < 0.0001. Ctl, Control; Wtm, wortmannin.

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