Elsevier

Brain Research

Volume 1101, Issue 1, 26 July 2006, Pages 36-42
Brain Research

Research Report
Nicotine regulates SH-SY5Y neuroblastoma cell proliferation through the release of brain-derived neurotrophic factor

https://doi.org/10.1016/j.brainres.2006.05.023Get rights and content

Abstract

Nicotine has been shown to produce some beneficial effects in neurodegenerative disorders, and several studies have suggested that these effects may be mediated in part through the action of the neurotrophic factor BDNF. To further elucidate the interaction between nicotine and BDNF, we examined the effect of nicotine on the proliferation of the neuroblastoma cell line SH-SY5Y, which, following differentiation with retinoic acid, expresses both nicotinic receptors and the receptor for BDNF, TrkB. Both nicotine and the nicotinic alpha-7 selective agonist AR-17779 significantly increased cell proliferation albeit with bell-shaped dose–response kinetics. The blockade of this effect with either the alpha-7 nicotinic antagonist methyllycaconitine or the non-selective nicotinic antagonist mecamylamine indicated that the effect was mediated by nicotinic receptors. Prior addition of neutralising BDNF antibodies or of the tyrosine kinase inhibitor K252A (200 nM) completely blocked nicotine-induced proliferation, suggesting the involvement of TrkB signalling in the mediation of the effect. Nicotine also enhanced both the secretion of BDNF from the SH-SY5Y and cell surface density of TrkB receptors. These effects were abolished by pretreatment with MLA. These data indicate that activation of nicotinic receptors has effects upon the BDNF–TrkB pathway, inducing cell proliferation by promoting the release of BDNF, which in turn activates TrkB receptors.

Introduction

There is growing evidence suggesting a role for nicotine in neuroprotection (Singh et al., 2004). While post-mortem studies have shown substantial reductions in the density of central nicotinic receptors in patients with Parkinson's disease (PD) (Perry et al., 2000, Quik and Kulak et al., 2002) and Alzheimer's disease (AD) (Perry et al., 1995), epidemiologic studies have shown that cigarette smoking and nicotine injection can have beneficial effects on cognition in PD and AD (Singh et al., 2004). Such effects, however, cannot always be replicated (Alves et al., 2004, Lemay et al., 2003, Lemay et al., 2004). Similarly, PET studies performed on AD patients at early stages of the disease have demonstrated a significant reduction in [11C]-nicotine binding in frontal cortex and hippocampus (Nordberg et al., 1995).

In animals, a large number of studies have demonstrated that nicotine increases the expression of nicotinic receptors as well as improving cognitive function in animal models of neurodegenerative disease (Ryan et al., 2001).

However, the mechanisms underlying neuroprotection by nicotine and nicotinic agonists (Singh et al., 2004) remain poorly understood. Recently, a growing body of evidence has implicated neurotrophic factors in the mediation of the long-term effects of nicotine. In particular, Maggio et al. (Maggio et al., 1998, Maggio et al., 1997) have reported that the neuroprotective effect of nicotine in 2 animal models of PD is accompanied by a corresponding increase in BDNF levels in the striatum. Similarly, intrahippocampal administration of nicotine transiently increased nerve growth factor (NGF) expression and TrkB message levels in the CA1 and in dentate gyrus (French et al., 1999). Exposure of PC12 cells to nicotine also resulted in an increase in NGF receptor expression (Terry and Clarke, 1994). On the other hand, decreased expression of BDNF and of its full-length receptor, TrkB, was observed in the frontal cortex and hippocampus of patients with AD (Ferrer et al., 1999).

Hence, these data support a role for the BDNF signalling pathway in the mediation of the neuroprotective effects of nicotine. However, the cellular mechanisms for protection triggered by nicotine and its agonists remain unexplored and their elucidation may provide insights into the development of more effective neuroprotective agents.

The neuroblastoma cell line SH-SY5Y natively expresses nicotinic receptor alpha-3, alpha-5, alpha-7, beta-2 and beta-4 subunits (Gould et al., 1992, Wang et al., 1996). Furthermore, when treated with retinoic acid, they differentiate into neuronal cells and express functional TrkB receptors (Kaplan et al., 1993, Lucarelli et al., 1994, Ruiz-Leon and Pascual et al., 2003). It has been shown that, at that stage, proliferation of the differentiated cells can be driven through the BDNF–TrkB pathway (Ruiz-Leon and Pascual et al., 2003). In the present study, the proliferation of the differentiated neuroblastoma was used as a quantifiable index of the functionality of the BDNF–TrkB pathway to investigate the effects of nicotinic agents on BDNF signalling.

Section snippets

Effect of nicotine on [14C]-thymidine incorporation in SH-SY5Y cells

Nicotine produced a time-dependent increase in [14C]-thymidine incorporation into the SH-SY5Y cells compared with controls. The effect of nicotine on cell growth was significant after an exposure of 42 h then plateaued after 48 h of exposure. For all subsequent experiments, [14C]-thymidine incorporation was estimated after an exposure to nicotine of 48 h (Fig. 1) when cells in both control and treated conditions were still growing.

Nicotine produced a bell-shaped dose–response curve on cell

Discussion

The goal of the present study was to define to what extent nicotine interacts with the BDNF–TrkB pathway. The neuroblastoma cell line SH-SY5Y endogenously expresses native nicotinic subunits such as alpha-3, alpha-5, alpha-7, beta-2 and beta-4 subunits (Gould et al., 1992), which form a number of different receptors subtypes and therefore provide a suitable model for studying neuronal mechanisms and identifying the receptor subtype involved in nicotine effects. Moreover, upon differentiation

Conclusion

In conclusion, the present data showed that nicotine interacts with the neurotrophin pathway by increasing BDNF secretion and by recruiting TrkB receptors onto the cell surface. Given the difficulty in producing agonists capable of crossing the blood–brain barrier and activating TrkB receptors, drugs such as nicotinic agonists capable of enhancing the release and signalling of neurotrophins may represent an alternative strategy for modulation of the BDNF pathway.

Cell culture

SH-SY5Y neuroblastoma cells were grown in a 1:1 mixture of Eagle's minimal essential medium–F12 medium, containing foetal calf serum (10%), glutamine (2 mM), penicillin (50 U/ml) and streptomycin (50 μg/ml) in a humidified incubator (95% air/5% CO2) at 37 °C. Cells were subcultured at a 1:10 ratio, left to attach for 2 days and treated with retinoic acid (10 μM) for an additional 5 days. Only cells between passages P6 to P20 were used in the studies.

Drugs

Drugs used were: nicotine, mecamylamine,

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