N-acetylcysteine prevents 4-hydroxynonenal- and amyloid-beta-induced modification and inactivation of neprilysin in SH-SY5Y cells

Abstract

As one of the dominant amyloid-beta peptide (Abeta) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Abeta production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Abeta, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Abeta treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 microM alleviated HNE adduction after HNE or Abeta treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimer's disease.

Figure 1

NAC prevents HNE-adduction of NEP. After incubation with vehicle, HNE (20 μM) with/without NAC (10, 100 μM) for 24 hours, SH-SY5Y cells were harvested and equal amounts of protein were analyzed by western blot to determine NEP protein levels. HNE-NEP adducts were detected by anti-NEP IP followed by western blot. (A) Representative NEP western blot with quantification. (B) Representative anti-NEP immunoprecipitation followed by HNE and NEP western blotting. Data was quantified and expressed as HNE/NEP ratio. The data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared to vehicle-treated control. # p < 0.05, ## p < 0.01 compared to HNE treated group.

Figure 2

NAC partially blocks NEP and HNE-NEP adducts after Aβ_1–42 peptide treatment. After incubation with vehicle, Aβ_1–42 (1 μM) with/without NAC (10, 100 μM) for 24 hours, SH-SY5Y cells were harvested and equal amounts of protein were analyzed by western blot to determine NEP protein levels. HNE-NEP adducts were detected by anti-NEP IP followed by western blot as shown. (A) Representative NEP western blot and quantification. (B) Representative blot of NEP immunoprecipitation followed by HNE and NEP western blotting. Quantified data was presented as HNE/NEP ratio. The data are expressed as mean ± SEM from 3 independent experiments. **p < 0.01 compared to vehicle-treated control. # p < 0.05 compared to Aβ_1–42 treated group.

Figure 3

NEP and HNE colocalize in the cytoplasm of HNE-treated SH-SY5Y cells. Cells were untreated (a–d), treated with 10 μM HNE (e–h) or 100 μM NAC (i–l) for 24 hours prior to fixation and staining with anti-NEP (red) (a, e, i) or anti-HNE (green) (b, f, j). The nuclei were counterstained with DAPI (c, g, k). Colocalization was determined by confocal imaging (d, h, l) and is represented by yellow.

Figure 4

NEP and HNE colocalize in the cytoplasm of Aβ-treated SH-SY5Y cells. Cells were untreated (a–c), treated with 10 μM HNE (d–f) or 1 μM Aβ_1–42 (g–i) for 24 hours prior to fixation and staining with anti-NEP (green) (a, d, g) or anti-HNE (red) (b, e, h). Colocalization was determined by confocal imaging (c, f, i) and is represented by yellow.

Figure 5

NAC prevents HNE-induced reduction of NEP activity in SH-SY5Y cells. After 24-hour incubation with vehicle, HNE (10 μM), or together with NAC (10 and 100 μM), cells were harvested. NEP activity in the cell lysates (A) or whole cells (B) was analyzed by fluorescence resonance energy transfer (FRET). Average NEP activity represented by fluorescence intensity was measured from 0 to 60 minutes with a reading interval of 5 minutes. Specific NEP activity was calculated by subtracting residual fluorescent intensity after incubation with the NEP inhibitor thiorphan. (A) Specific NEP activity of cell lysates after HNE with/without NAC treatments. (B) Whole cell specific NEP activity after HNE with/without NAC treatments. (C) Dependence of the mean NEP activity on increasing substrate concentration in SH-SY5Y lysates. Values are the mean ± SEM expressed as the percentage of control value (A, B). Three independent experiments were performed in triplicate. ** p < 0.01, vs control. # p < 0.05, ## p < 0.01, vs HNE treatment.

Figure 6

NAC alleviates Aβ_1–42-induced reductions in NEP specific activity in SH-SY5Y cells. After 24-hour incubation with vehicle, Aβ_1–42 (1 μM) or together with NAC (10 and 100 μM), cells were harvested, NEP activity in the cell lysates (A) or whole cells (B) was analyzed by fluorescence resonance energy transfer (FRET). Average NEP activity represented by fluorescence intensity was measured from 0 to 60 minutes with a reading interval of 5 minutes. Specific NEP activity was calculated by subtracting residual fluorescent intensity after incubation with the NEP inhibitor thiorphan. (A) Specific NEP activity of cell lysates after Aβ with/without NAC treatments. (B) Whole cell specific NEP activity after Aβ with/without NAC treatments. (C) Dependence of the mean NEP activity on increasing substrate concentration in SH-SY5Y lysates. Values are the mean ± SEM expressed as the percentage of control value (A, B). Three independent experiments were performed in triplicate. ** p < 0.01, vs control. # p < 0.05, ## p < 0.01, vs Aβ treatment.