Potentiation of nerve growth factor-induced neurite outgrowth by fluvoxamine: role of sigma-1 receptors, IP3 receptors and cellular signaling pathways

Abstract

Background: Selective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists.

Methods and findings: The effects of three SSRIs (fluvoxamine, sertraline, paroxetine) and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), and dehydroepiandrosterone (DHEA)-sulfate) on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP(3)) receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP(3) receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-gamma), phosphatidylinositol 3-kinase (PI3K), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways blocked the potentiation of NGF-induced neurite outgrowth by SA4503.

Conclusion: These findings suggest that stimulation of sigma-1 receptors and subsequent interaction with IP(3) receptors, PLC-gamma, PI3K, p38MAPK, JNK, and the Ras/Raf/MAPK signaling pathways are involved in the mechanisms of action of sigma-1 receptor agonists such as fluvoxamine and SA4503.

Conflict of interest statement

Competing Interests: Dr. Kenji Hashimoto reports receiving the speaker fees from Solvay, Meiji Seika, Asteras, Eli Lilly, Janssen, Pfizer, and Otsuka. Dr. Masaomi Iyo reports receiving the speaker fees from Janssen, Meiji Seika., Dainippon-Sumitomo, GlaxoSmithKline, Novartis, Eli Lilly, Pfizer, and Otsuka. Ms. Tomoko Nishimura and Ms. Tamaki Ishima report no competing interests.

Figures

Figure 1. Effects of NGF on neurite outgrowth in PC12 cells.

Incubation of NGF (0, 2.5, 5.0, 10.0, 20.0 or 40.0 ng/ml) caused neurite outgrowth in PC12 cells in a concentration dependent manner. The data were shown as percentage of the number of cells with neurite outgrowth in the number of total cells. The data show the mean±S.E.M (n = 8).

Figure 2. Effects of three SSRIs (fluvoxamine, sertraline, paroxetine) on NGF-induced neurite outgrowth in PC12 cells.

Fluvoxamine (The data show the mean±S.E.M (n = 6)), paroxetine (The data show the mean±S.E.M (n = 6)), sertraline (The data show the mean±S.E.M (n = 6)). Fluvoxamine (but not sertraline or paroxetine) significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100 (1.0 µM). Number is the concentration (µM) of drugs. *P+++P<0.001 as compared with fluvoxamine (10.0 µM) plus NE-100 group.

Figure 3. Effects of three sigma-1 receptor agonists (SA4503, PPBP, DHEA-sulfate) on NGF-induced neurite outgrowth in PC12 cells.

SA4503 (The data show the mean±S.E.M (n = 5–15)), PPBP (The data show the mean±S.E.M (n = 8)), DHEA-sulfate (The data show the mean±S.E.M (n = 8)). These drugs (0.1, 1.0 or 10.0 µM) significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by these drugs was significantly blocked by co-administration of the selective sigma-1 receptor antagonist NE-100 (1.0 µM). Number is the concentration (µM) of drugs. *P+++P<0.001 as compared with drugs (10 µM) plus NE-100 group.

Figure 4. Representative photographs of MAP-2 immunocytochemistry in PC12 cells.

(A) Control (NGF (2.5 ng/ml) alone) (B) NGF+SA4503 (1.0 µM), (C) NGF+SA4503 (1.0 µM)/NE-100 (1.0 µM), (D) NGF+Fluvoxamine (10.0 µM), (E) NGF+Fluvoxamine (10.0 µM)/NE-100 (1.0 µM), (F) NE-100 (1.0 µM). Incubation of SA4503 (1.0 µM) or fluvoxamine (10 µM) potentiated NGF-induced neurite outgrowth in PC12 cells. The potentiating effects of SA4503 or fluvoxamine on the NGF-induced neurite outgrowth were antagonized by co-administration of NE-100 (1.0 µM). Furthermore, NE-100 (1.0 µM) alone did not alter NGF-induced neurite outgrowth.

Figure 5. Effects of the IP3 receptor antagonist xestospongin C on potentiation of NGF-induced neurite outgrowth by SA4503.

The potentiating effects of SA4503 (1.0 µM) on the NGF-induced neurite outgrowth were antagonized by co-administration of xestospongin C (1.0 µM). Furthermore, xestospongin C (1.0 µM) alone did not alter NGF-induced neurite outgrowth. The data show the mean±SEM (n = 12). ***p

Figure 6. Effects of the specific inhibitors of PLC-γ, PI3K, p38MAPK, and JNK on potentiation of NGF-induced neurite outgrowth by SA4503.

The potentiating effects of SA4503 (1.0 µM) on the NGF-induced neurite outgrowth were antagonized by co-administration of the PLC-γ inhibitor (U73122; 1.0 µM), the PI3K inhibitor (LY294002; 1.0 µM), the p38MAPK inhibitor (SB203580; 1.0 µM), and the JNK inhibitor (SP600125; 1.0 µM). The data show the mean±SEM (n = 4–12). ***p

Figure 7. Effects of the specific inhibitors of Ras, Raf, MEK1/2, and MAPK on potentiation of NGF-induced neurite outgrowth by SA4503.

The potentiating effects of SA4503 (1.0 µM) on the NGF-induced neurite outgrowth were antagonized by co-administration of the Ras inhibitor (GW5074; 1.0 µM), the Raf inhibitor (lovastatin; 1.0 µM), the MEK1/2 inhibitor (MEK1/2 inhibitor; 1.0 µM), and the MAPK inhibitor (PD98059; 1.0 µM). The data show the mean±SEM (n = 4 or 8). *P

Figure 8. Proposed mechanism for potentiation of NGF-induced neurite outgrowth by sigma-1 receptor agonists.

NGF binding to cell-surface TrkA receptor results in activated receptor complexes, which contain adaptors such as SHC (SH2-containing protein), GRB2 (growth-factor-receptor bound protein 2) and Gab (GRB2-associated binding) proteins. These proteins recruit the protein SOS, increasing Ras-guanosine triphosphate (Ras-GTP) levels by catalyzing nucleotide exchange on Ras. Ras also activates the PI3K-PDK1 (3-phosphoinosisitide-dependent protein kinase 1)- Akt pathway. The activation of TrkA by NGF leads to activation of multiple signaling pathways, including PLC-γ, PI3K, p38MAPK, and JNK signaling pathways and Ras/Raf/MEK/ERK/MAPK pathways. The activation of sigma-1 receptors on the endoplasmic reticulum (ER) by sigma-1 receptor agonists (e.g., SA4503 and fluvoxamine) might interact with IP3 receptors on the ER. Ca2+ released by the interaction between sigma-1 receptors and IP3 receptors may play a role in the potentiation of NGF-induced neurite outgrowth in PC12 cells. Subsequently, several signaling pathways are implicated in the potentiation of NGF-induced neurite outgrowth by sigma-1 receptor agonists.