Cholinesterase inhibitor rivastigmine enhances nerve growth factor-induced neurite outgrowth in PC12 cells via sigma-1 and sigma-2 receptors

Abstract

Rivastigmine (Riv) is a potent and selective cholinesterase (acetylcholinesterase, AChE and butyrylcholinesterase, BuChE) inhibitor developed for the treatment of Alzheimer's disease (AD). To elucidate whether Riv causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. At concentrations of 0-100 μM, Riv was non-toxic in PC12 cells. Riv caused dose-dependent (10-100 μM) enhancement of NGF-induced neurite outgrowth, which was completely inhibited by the TrkA antagonist GW-441756. By contrast, Riv-mediated enhancement of neurite outgrowth was not blocked by the acetylcholine receptor antagonists, scopolamine and hexamethonium. However, the sigma-1 receptor (Sig-1R) antagonist NE-100 and sigma-2 receptor (Sig-2R) antagonist SM-21 each blocked about half of the Riv-mediated enhancement of NGF-induced neurite outgrowth. Interestingly, the simultaneous application of NE-100 and SM-21 completely blocked the enhancement of NGF-induced neurite outgrowth by Riv. These findings suggest that both Sig-1R and Sig-2R play important roles in NGF-induced neurite outgrowth through TrkA and that Riv may contribute to neuronal repair via Sig-1R and Sig-2R in AD therapy.

Fig 1. Influence of Riv on PC12 cell viability.

PC12 cells were treated with Riv for 24 h. Cell viability was determined using a WST-1 assay, and the results are expressed as a percentage of the control value (0 μM). Experiments were repeated at least three times, and the values represent the mean of three experiments ± SD. *p < 0.05, **p < 0.01 versus control.

Fig 2. Riv enhances NGF-induced neurite outgrowth in PC12 cells.

PC12 cells were treated for 24 h with NGF with or without Riv. (A) Culture images at a magnification of 400×. Scale bar: 50 μm. (B) Neurite length was determined as indicated in the Materials and Methods section. Values represent the mean ± SD for all PC12 cells contained within five randomly chosen fields for each condition. All experiments were repeated at least three times **p < 0.01 versus NGF. (C) Neurite lengths in the presence of various concentrations of NGF (0–250 ng/mL).

Fig 3. Influence of Riv on NGF-induced phosphorylation of Akt and ERK1/2 in PC12 cells.

PC12 cells underwent various durations of treatment (0, 10, 30, or 60 min) with NGF in the presence or absence of Riv (100 μM) (A, C) Total protein lysates were collected and subjected to western blotting for detection of phosphorylation status of Akt and ERK1/2. (B, D) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) (B), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) (D). Results are presented as the mean ± SD of three independent experiments.

Fig 4. Effect of TrkA receptor antagonist on Riv-mediated enhancement of NGF-induced neurite outgrowth in PC12 cells.

PC12 cells were pre-incubated in the presence or absence of GW-441756 (1 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth assays. For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. **p < 0.01 compared with NGF, ^††p < 0.01 compared with NGF+Riv).

Fig 5. Effect of AChR antagonists on Riv-mediated enhancement of NGF-induced neurite outgrowth in PC12 cells.

(A) PC12 cells were pre-incubated in the presence or absence of Scop (100 μM) for 4 h. (B) PC12 cells were pre-incubated in the presence or absence of HEX (100 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth assays. For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. **p < 0.01 compared with NGF.

Fig 6. Effect of Sig-1R antagonist on Riv-mediated enhancement of NGF-induced neurite outgrowth in PC12 cells.

PC12 cells were pre-incubated in the presence or absence of NE-100 (10 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth assays. For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. **p < 0.01 compared with NGF, ^††p < 0.01 compared with NGF+Riv.

Fig 7. Effect of Sig-2R antagonist and additional Sig-1R antagonist on Riv-mediated enhancement of NGF-induced neurite outgrowth and phosphorylation of Akt and ERK1/2 in PC12 cells.

(A) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) for 4 h. (B, C) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) and NE-100 (10 μM) for 4 h. NGF and Riv were added, and cells were incubated for an additional 24 h before neurite outgrowth and western blotting assays. (C) Total protein lysates were collected and subjected to western blotting for the assessment of the phosphorylation status of Akt and ERK1/2. (D, E) The intensity of the p-Akt polypeptide band was normalized to that of the total Akt band (p-Akt/Akt) (D), while the intensity of the p-ERK1/2 polypeptide band was normalized to that of the total ERK1/2 band (p-ERK1/2/ERK1/2) (E). For each condition, values are reported as the mean ± SD for all PC12 cells included within five randomly chosen fields. **p < 0.01 compared with NGF, ^††p < 0.01 compared with NGF+Riv.

Fig 8. Effect of Sig-1R and Sig-2R agonist/antagonist on NGF-induced neurite outgrowth in PC12 cells.

(A) PC12 cells were pre-incubated in the presence or absence of NE-100 (10 μM) for 4 h. NGF and PRE-084 (10 μM) were added, and cells were incubated for an additional 24 h before neurite outgrowth assays. (B) PC12 cells were pre-incubated in the presence or absence of SM-21 (10 μM) for 4 h. NGF and PB28 (10 μM) were added, and cells were incubated for an additional 24 h before neurite outgrowth assays. For each condition, values are reported as the mean ± SD for all PC12 cells within five randomly chosen fields **p < 0.01 compared with NGF, ^††p < 0.01 compared with NGF+PRE-084 or NGF+PB28.

Fig 9. Influence of knockdown of Sig-1R and Sig-2R on Riv-mediated enhancement of neurite outgrowth in PC12 cells.

PC12 cells were transfected with 10 nM siRNA (Sig-1R and Sig-2R siRNA). After 2 days of transfection, NGF and Riv were added, and cells were incubated for 24 h. For each condition, values are the mean ± SD for all PC12 cells within five randomly chosen fields. *p < 0.05; **p < 0.01 compared with control siRNA NGF. N.S., not significant.