Elsevier

Neuroscience

Volume 181, 5 May 2011, Pages 196-205
Neuroscience

Neurodegeneration, Neuroprotection, and Disease-Oriented Neuroscience
Research Paper
Ethanol-induced neurodegeneration in NRSF/REST neuronal conditional knockout mice

https://doi.org/10.1016/j.neuroscience.2011.02.059Get rights and content

Abstract

The transcription regulator, neuron-restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor (REST), plays an important role in neurogenesis and various neuronal diseases such as ischaemia, epilepsy, and Huntington's disease. In these disease processes, neuronal loss is associated with abnormal expression and/or localization of NRSF. Previous studies have demonstrated that NRSF regulates the effect of ethanol on neuronal cells in vitro, however, the role of NRSF in ethanol-induced neuronal cell death remains unclear. We generated nrsf conditional knockout mice using the Cre-loxP system to disrupt neuronal expression of nrsf and its truncated forms. At postnatal day 6, ethanol significantly increased the expression of REST4, a neuron-specific truncated form of NRSF, in the brains of wild type mice, and this effect was diminished in nrsf conditional knockout mice. The apoptotic effect of ethanol was pronounced in multiple brain regions of nrsf conditional mutant mice. These results indicate that NRSF, specifically REST4, may protect the developing brain from ethanol, and provide new evidence that NRSF can be a therapeutic target in foetal alcohol syndrome (FAS).

Highlights

▶We generated a kind of nrsf conditional knockout mice model in neuron. ▶We studied the nrsf gene function using the conditional knockout mouse model. ▶The relationship between nrsf and the ethanol induced infant mice neuronal apoptosis. ▶Lack of nrsf increases ethanol-induced neurodegeneration in new born mice.

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Section snippets

Generation and characterization of NSE-Cre transgenic mice

The pNSE-Cre vector was constructed (Fig. 1A), in which the promoter for transgene expression is the 1794 bp Ecl136II–HindIII fragment from the rat neuron-specific enolase (NSE) gene (Forss-Petter et al., 1990). Purified and linearized DNA was microinjected into the pronuclei of fertilized mouse eggs isolated from superovulated (C57BL/6 cross with DBA) F1 hybrid mice. Microinjected eggs were implanted into pseudopregnant females to produce transgenic mice. Transgenic founders were identified by

Generation of NSE-Cre transgenic mice and selective deletion of nrsf in the neuron of cKO mice

The Cre-loxP strategy was adapted to knockout nrsf in neuronal cells. First, NSE-Cre transgenic mice were created, in which the rat NSE promoter drove Cre recombinase expression (Fig. 1A). The transcriptional expression of Cre recombinase was limited to the brain (Fig. 1B).

To determine the activity and specificity of the NSE promoter in mouse brain, NSE-Cre transgenic mice were crossed with ROSA26-lacZ reporter mice. Cre recombinase activity was determined in offspring containing both NSE-Cre

Discussion

To elucidate the function of NRSF in the process of EtOH-induced neuronal death, both NSE-Cre transgenic mice and nrsfflox/+ mice were created. Cre recombinase activity was determined in the brain of offspring containing NSE-Cre and ROSA26-lacZ transgenes, and most neurons in the cortex and hippocampus expressed LacZ (Fig. 1C). In our conditional nrsf knockout mouse model, exon 2 of nrsf is deleted specifically by neuronal expressed Cre recombinase. Since exon 2 is the common region in all

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30670438 and 30770659, 90919004), National 863 project (2008AA02Z126), National Key Project (2010CB945501), the Science and Technology Commission of Shanghai Municipality (06DZ19004), E-Institutes of Shanghai Municipal Education Commission (E03003), and the Program for NCET to F.H. We thank Dr. Kehong Zhang for kindly reviewing our work and Cactus Communications Pvt. Ltd (www.editage.com) for providing

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