Elsevier

Neuroscience Research

Volume 65, Issue 3, November 2009, Pages 236-244
Neuroscience Research

Alteration of NRSF expression exacerbating 1-methyl-4-phenyl-pyridinium ion-induced cell death of SH-SY5Y cells

https://doi.org/10.1016/j.neures.2009.07.006Get rights and content

Abstract

Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST) and its neuron-specific truncated form REST4 participates in the pathological processes of nervous system diseases, such as global ischemia, epilepsy, Huntington disease and so on. In this paper, we investigated the changes of NRSF and REST4 in a cellular model of Parkinson's disease (PD). Our results showed that neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) treatment triggered the mRNA and protein expression of NRSF and REST4, and caused both NRSF and REST4 proteins relocalized between the nucleus and cytoplasm in human dopaminergic SH-SY5Y cells. Redistribution of NRSF and REST4 derepressed the expression of target genes at late time points. Furthermore, alteration of NRSF and REST4 expression by overexpression or RNAi techniques elicited deleterious effects on cell viability of SH-SY5Y treated with toxic MPP+.

Introduction

Transcriptional abnormality of neuronal genes plays an important role in the process of nervous system diseases. NRSF/REST (neuron-restrictive silencer factor or repressor element-1 silencing transcription factor), an essential zinc-finger transcription factor, restricts the expression of a group of neuron-specific genes in non-neuronal cells through histone deacetylation, chromatin remodeling, methylation and other mechanisms (Ballas and Mandel, 2005, Lunyak et al., 2002, Palm et al., 1998). Examples of such neuron-specific genes are mu-opioid receptor (Andria and Simon, 2001), AMPA receptor (AMPAR) subunit GluR2 (Calderone et al., 2003), synapsin I (Nishimura et al., 1996, Schoenherr and Anderson, 1995) and BDNF (Zuccato et al., 2007), Superior Cervical Ganglia 10 (SCG10) (Eggen and Mandel, 1997), neuron-glia cell adhesion molecule (Ng-CAM) (Eggen and Mandel, 1997) and neuronal β−III tubulin. Recently, lines of researches indicated that NRSF participated in the pathogenesis of nervous system diseases, such as global ischemia, epilepsy and degenerative Huntington disease (HD). That changes in the expression or localization of NRSF consequently affect the expression of downstream genes may be an important mechanism of neuronal death (Formisano et al., 2007, Spencer et al., 2006, Zuccato et al., 2003). Parkinson's disease (PD) is a common neurodegenerative disease in the central nervous system, and featured by the progressive loss of dopaminergic neurons in the substantia nigra (SN). Tyrosine hydroxylase (TH) is the rate-limiting enzyme of dopamine synthesis. In the promoter region of human TH gene, there exists a neuron-restrictive silencer element (NRSE), which could bind NRSF. The outcome of such a combination is silencing TH expression (Kim et al., 2008). Moreover, PD-related gene ubiquitin carboxyl-terminal hydroxylase L1 (UCH-L1) has been confirmed to be one of NRSF target genes (Barrachina et al., 2007). Therefore, we speculated NRSF might be relevant to PD. REST4, a neuron-specific truncated form of NRSF, could resist the silencing function of NRSF (Shimojo et al., 1999); however, the function of REST4 is controversial (Magin et al., 2002).

1-methyl-4-phenyl-pyridinium ion (MPP+) is a conventional neurotoxin inducing dopaminergic cell death. By treating SH-SY5Y cells with MPP+, we found both the expression and cellular localization of NRSF and REST4 were changed, which in turn caused expression changes of downstream genes. Alteration of NRSF and REST4 expression by overexpression or RNAi techniques decreased viability of SH-SY5Y cells under the treatment of MPP+.

Section snippets

Materials

1-methyl-4-phenyl-pyridinium ion (MPP+) was a product of Sigma (D048, St. Louis, USA.).

Real-time polymerase chain reaction (PCR)

Total RNA was extracted from SH-SY5Y cells using Trizol reagent (Invitrogen, Carlsbad, USA). Reverse transcription was carried out using random primers and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA). Real-time PCR was performed for quantification of NRSF, REST4, brain derived neurotrophic factor (BDNF), TH, synapsin I, UCH-L1, Atp1b3 and β-actin mRNA on Rotor-Gene 3000

MPP+ treatment increased the expression of NRSF and REST4 in SH-SY5Y cells

To investigate the effect of MPP+ on the expression of NRSF and REST4 in human dopaminergic SH-SY5Y cells, real-time PCR was performed to detected the mRNA expression levels after treatment with 1 mM MPP+ for 3, 8, 20 or 48 h. At early time points, 3 and 8 h after exposure to MPP+, the transcriptional expression of NRSF and REST4 decreased dramatically compared to PBS controls (P < 0.05). However, their mRNA expression recovered or exceeded the basal levels at 20 and 48 h after the MPP+

Discussion

The expression and localization of NRSF and REST4 are well-regulated in cells (Calderone et al., 2003, Formisano et al., 2007, Spencer et al., 2006, Zuccato et al., 2003). Changes of NRSF or REST4 play important roles in the pathological process of nervous system diseases. For examples, global ischemia triggers NRSF expression, which suppresses GluR2 promoter activity and gene expression in neurons destined to die (Calderone et al., 2003). In the epilepsy model, the interplay between NRSF and

Acknowledgements

This work was supported by National Natural Science Foundation of China (30770659, 90919004); Shanghai Metropolitan Fund for Research and Development (06DZ19004 and 07DJ14005); National “863” Project (2008AA02Z126); Program of IOBS and Program for NCET to F.H.

References (22)

  • M. Bian et al.

    Expression of Cbl-interacting protein of 85 kDa in MPTP mouse model of Parkinson's disease and 1-methyl-4-phenyl-pyridinium ion-treated dopaminergic SH-SY5Y cells

    Acta Biochim. Biophys. Sin (Shanghai)

    (2008)
  • Cited by (0)

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