Negative regulation of microRNA-132 in expression of synaptic proteins in neuronal differentiation of embryonic neural stem cells

https://doi.org/10.1016/j.neuint.2016.04.013Get rights and content

Highlights

  • miR-132 expression was regulated via MAPK/ERK signaling pathway during differentiation of eNSCs.

  • Blockade of ERK activation increased synaptic proteins in the eNSCs differentiation.

  • Inhibition of miR-132 function led to increase postsynaptic proteins during the eNSCs differentiation.

Abstract

MicroRNAs (miRs) play important roles in neuronal differentiation, maturation, and synaptic function in the central nervous system. They have also been suggested to be implicated in the pathogenesis of neurodegenerative and psychiatric diseases. Although miR-132 is one of the well-studied brain-specific miRs, which regulates synaptic structure and function in the postnatal brain, its function in the prenatal brain is still unclear. Here, we investigated miR-132 function during differentiation of rat embryonic neural stem cells (eNSCs). We found that miR-132 expression significantly increased during the fetal rat brain development and neural differentiation of eNSCs in vitro. Furthermore, miR-132 expression was increased during differentiation through MAPK/ERK1/2 pathway. Inhibition of ERK1/2 activation resulted in increased levels of synaptic proteins including PSD-95, GluR1 and synapsin I. Silencing of miR-132 also increased PSD-95 and GluR1. Considering that miR-132 increases synaptic proteins in differentiated cortical neurons, our result shows a novel function of miR-132 as a negative regulator for synaptic maturation in the neuronal differentiation of eNSCs.

Introduction

Neural stem cells (NSCs) have the ability of self-renewing and differentiating into neurons and glial cells (astrocytes and oligodendrocytes) in the developing and adult brain. MicroRNAs (miRs) are small (∼22 nt) non-coding RNAs that act as post-translational regulators through target mRNA degradation and/or translational repression by binding to the 3’ untranslated region of mRNAs (Bartel, 2004). miRs are essential for developmental and physiological processes including cell apoptosis, differentiation and metabolism in animals and plants (Carrington and Ambros, 2003). The miRs studies in the developing brain and differentiating cultured embryonic NSCs (eNSCs) have demonstrated that brain-enriched miRs are important regulators for neuronal differentiation (Krichevsky et al., 2003, Sempere et al., 2004, Miska et al., 2004, Smith et al., 2010, Kawahara et al., 2012, Meza-Sosa et al., 2014). Moreover miRs offer novel insights into brain-related disease research. Recently, the crucial roles of miRs are increasingly reported in the pathogenesis of neurodegenerative and psychiatric diseases (Im and Kenny, 2012, Rao et al., 2013).

As a single microRNA can repress several targeting mRNAs, multiple functions of a miR has been shown, depending not only on cell types but also on spatial and temporal factors. For example, our group recently reported that glial miR-134 enhanced by bFGF positively regulated astroglial cell maturation (Numakawa et al., 2015). On the other hand, it was demonstrated that miR-134 targets Chordin-like 1 (Chrdl-1) and Doublecortin (Dcx) in the developing brain, and it has important roles in the stage-specific modulation of eNSC proliferation, neuron migration and neuronal differentiation via interacting with Chrdl-1 and Dcx (Gaughwin et al., 2011).

In this study, to investigate further functions of miRs specifically expressed during fetal neurodevelopment, we investigated miRs expression during differentiation of rat eNSCs in vitro. Among examined miRs, miR-132 showed the most dramatic increase during the differentiation into neuron from eNSCs. miR-132 is a well-studied miR in the central nervous system (CNS). It functions in neurotrophin signaling pathway including brain-derived neurotrophic factor (BDNF) and enhances neurite outgrowth (Wanet et al., 2012). Interestingly, miR-132 has been implicated in depression disorder (Li et al., 2013) in addition to neurodegenerative disorders such as Alzheimer's disease (AD) (Wong et al., 2013) and Parkinson's disease (PD) (Lungu et al., 2013). Methyl-CpG-binding protein 2 (MeCP2), a transcriptional repressor, was reported as a target of miR-132 (Klein et al., 2007). Mutation in MeCP2 causes progressive neurological disorder Rett syndrome (RTT) in female as a result of deficits in synaptic maturation. Whereas functions of miR-132 associated with postnatal CNS have been well studied, its role in the prenatal brain still uncertain. Here we examined the function and the induction mechanism of miR-132 during neuronal differentiation of eNSCs in vitro. Our results suggested that miR-132 negatively regulated the expression of synaptic proteins in the neuronal maturation of eNSCs.

Section snippets

Animals

All animals used in this study were Wistar rats, and were treated according to the guidelines of the Animal Ethics Committee for the care and use of animals in the National Institute of Neuroscience, National Center of Neurology and Psychiatry, Japan. Brain samples for the quantitative PCR were harvested from embryonic stage day 14.5 and 19.5 (E14.5 and E19.5) rats (CLEA JAPAN, Tokyo, Japan). The tissues were frozen in liquid nitrogen quickly and stored at −125 °C until RNA extraction.

Neural stem cell culture, differentiation, and transfection

eNSCs

The expression of miRs in the developing fetal brain

To investigate changes in miRs expression levels, we analyzed five miRs (miR-9, -124a, -132, -134 and -206) in the developing fetal brains by real-time qPCR. These miRs are suggested to be involved in psychiatric and/or neurological disorders (Mouillet-Richard et al., 2012, Lee et al., 2012, Rao et al., 2013). Total RNAs were isolated from the rat telencephalon (TE) at E14.5, cerebral cortex (CTX) and hippocampus (HIP) at E19.5. BDNF mRNA expression levels was increased, especially in HIP, with

Discussion

Previous studies have reported positive effects of miR-132 on neuronal functions including synaptic plasticity, dendrite outgrowth, and cell survival (Edbauer et al., 2010, Magill et al., 2010, Luikart et al., 2011, Wanet et al., 2012, Remenyi et al., 2013, Hwang et al., 2014). Down-regulation of miR-132 has also been implicated in several neurological disorders such as AD (Wong et al., 2013), PD (Lungu et al., 2013), schizophrenia (Miller et al., 2012) and depression (Li et al., 2013). Most of

Acknowledgements

This work was supported by grants from Takeda Science Foundation (T.N.), from the Grant-in-Aid for Challenging Exploratory Research (Grant number: 25640019) (T.N.), the Grant-in-Aid for Scientific Research (B) (24300139) (T.N.) and the Grant-in-Aid for Young Scientists (B) (26830061) (A.Y.) in the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Health and Labor Sciences Research Grants (H.K.), Intramural Research Grant for Neurological and Psychiatric

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