Effects of DSP4 on the Noradrenergic Phenotypes and Its Potential Molecular Mechanisms in SH-SY5Y Cells

Abstract

Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4’s precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.

Effects of Antidepressants on DSP4/CPT-Induced DNA Damage Response in Neuroblastoma SH-SY5Y Cells

“…An interesting correlation among these diseases is that the dysfunctional LC-NE system is deeply involved in the pathological development of these diseases (Zubenko & Moossy 1988, German et al 1992, Zarow et al 2003). Consistently, our previous study demonstrated that DSP4 reduced the expression of dopamine β-hydroxylase and norepinephrine transporter, two noradrenergic phenotypes, in SH-SY5Y cells, which are mediated by action in DDR (Wang et al 2014). Further work showed that CPT treatment also resulted in great DDR in SH-SY5Y cells (Wang 2013) and in primary cultures from the rat LC, in which DSP4 exhibited the same effect as in SH-SY5Y cells (unpublished data).…”

“…Our previous study demonstrated that DSP4, used as a neurotoxin, induced DDR in SH-SY5Y cells (Wang et al 2014). To test the effects of antidepressants on DDR induced by DSP4, SH-SY5Y cells were pretreated with different antidepressants for 1 h before 4 h DSP4 (50 μM) treatment.…”

“…Previously, we showed that DSP4 could arrest SH-SY5Y cells predominately in S-phase (Wang et al 2014). To test effects of antidepressants on S-phase arrest caused by DSP4, two parallel experiments were carried out.…”

“…Different antidepressants were used in this study: fluoxetine, reboxetine, desiprimine (DMI), paroxetine, imipramine, amitriptyline, l-deprenyl, and pargyline. The concentration of DSP4 was based on our previous data (Wang et al 2014). The concentration of CPT was based on published paper from this department (Serrano et al 2013).…”

“…While some components of the cell cycle machinery were found to be upregulated after exposure to severe conditions, such as oxidative stress (Kruman et al 2004, Murray 2004, Currais et al 2009), many cytotoxic and genotoxic agents including neurotoxins arrest the cell cycle at different phases (Doi 2011). Previously, we demonstrated that DSP4 induced the DDR in SH-SY5Y cells, resulting in cell cycle arrest predominantly in S-phase (Wang et al 2014). Our previous study demonstrated that CPT also induced the DNA damage response (DDR) in SH-SY5Y cells (Wang 2013) and primary cultured LC neurons (Wang et al 2015).…”