Formyl peptide receptor agonist FPRa14 induced differentiation of Neuro2a mouse neuroblastoma cells in culture

P. J. Cussell, M. S. Howe, M. Gomez Escalada, N. G. Milton, A. W. Paterson. Leeds Beckett University, Leeds, United Kingdom.

Introduction: The N-formyl peptide receptor (FPR) is a G protein-coupled receptor that has been identified within many neuronal tissues [1]. Accumulating evidence suggests a neuronal function for FPR. Ligands for FPRs are structurally diverse and include molecules associated with neurodegeneration [2]. FPR activation has been demonstrated to stimulate the differentiation of neural stem cells [3]. In this study Neuro2a neuroblastoma cells were exposed to the agonist FPRa14 in order to assess the effects on differentiation.

Method: Neuro2a cells were seeded into 24-well microtitre plates and exposed to FPRa14 (1-10μM). Images (x40 magnification) were taken 24h following agonist administration and visually assessed for morphological changes. Further differentiation assays were conducted with cells pre-incubated with the FPR antagonist Boc-MLF (40μM) prior to agonist administration. Data are given as mean ± SEM and analysis was performed via 2-way ANOVA with Dunnett’s test where applicable.

Results: The synthetic FPR agonist FPRa14 induced a significant response in mouse neuroblastoma N2a cells, with % cell differentiation increasing in a dose-dependent manner. Three distinct differentiated phenotypes were observed, the two non-archetypal forms of which were only observed with higher FPRa14 concentrations (≥6μM). At these higher concentrations of FPRa14 toxic responses were also observed via MTT assay (26.0 - 56.1% cell inhibition). Pre-incubating N2a cultures with the antagonist Boc-MLF (40μM) prior to agonist administration significantly reduced differentiation to near untreated control levels.

Fig.1 (A): The effect of FPRa14 (2-10μM and 10μM following pre-incubation with Boc-MLF (40μM)) on the % of differentiated N2a cells. * = P<0.05 relative to appropriate incubation control, # = P<0.05 relative to the appropriate FPRa14 concentration (n=171-278). (B): Key highlighting examples of the four classes of cell morphology observed and recorded during FPRa14 neurite outgrowth assays

Conclusions: These results suggest that the N2a differentiation response observed has an FPR-dependent component. The demonstration of neuronal differentiation mediated via an FPR agonist in this study represents a significant finding and suggests a role for FPR in the CNS. This finding could potentially lead to novel therapies for a range of neurological conditions including neuroblastoma.

References:

[1] Migeotte et al. (2006). Cytokine Growth Factor Rev, 17, 501-19.

[2] Li, Y. & Ye, D. (2013). J Mol Med, 91, 781-9.

[3] Wang et al. (2016). Sci Rep, 6, 25946