PERSPECTIVE

Valproic acid as a microRNA modulator to promote neurite outgrowth

Valproic acid (VPA) has been a first-choice drug for clinical treatment of epilepsy and manic disorder. For decades, its pharmacological action was believed to act on inhibition of gamma-aminobutyric acid (GABA) transaminase, in turn, increasing GABA in inhibitory synapses. However, in recent years, VPA has been investigated on other therapeutic actions. Those investigations demonstrate that VPA shows neuroprotective effects by promoting neurogenesis, neuronal differentiation, and neuroregeneration (Foti et al., 2013). Those VPA efficacies had been reported to induce epigenetic changes such as inhibiting histone deacetylases (Tremolizzo et al., 2005; Jessberger et al., 2007) that modulate gene expression changes transcriptionally. To unravel other epigenetic modulators, our research group, together with others, have recently reported that VPA treatment induces microRNAs (miRNAs) expression in the brain (Goh et al., 2011; Hunsberger et al., 2012; Oikawa et al., 2015).

miRNAs are functional nucleic acid molecule about 22 nucleotides in length and that is encoded in the genome. These small chain lengths RNAs are classified into non-cording RNA and silence RNA and subsequent post-transcriptional regulation of gene expression, without changes in DNA sequence. The humane genome has encoded over 1,000 miRNAs, and many miRNAs are expressed in the central nervous system (CNS). miRNAs in the brain have been reported to regulate neuronal development, differentiation, synaptogenesis, and plasticity (Fiore et al., 2011). miRNA-132 has been reported to regulate the cAMP response element binding (CREB) protein pathway that repressed translation of the Rho family GTPase-activating protein, p250GAP and neuronal morphogenesis in developing neurons (Wayman et al., 2008). miRNA-124 contributes to the control of neurite outgrowth during neuronal differentiation through regulation of cytoskeleton to also partially effect via Rho GTPase family pathway (Yu et al., 2008). In fact, VPA up-regulated a network of inter-related miRNAs that are intimately linked with neural network development. This fact has more strongly supported the potential of critical period reactivation. Our previous study also shows that miRNAs interact with components of the protein-protein interaction networks that affect dendritic growth and synaptic plasticity in VPA treated mouse brain (Goh et al., 2011).

We next detected differential protein changes by using iTRAQ method. Approximately more than half of the proteins were up-regulated (83 out of 147 proteins) while 64 proteins were down-regulated. Based on our hypothesis that miRNA functions in RNA silencing and post-transcriptional regulation of gene expression, we focused on down-regulated proteins by VPA. A combination of three different in silico algorithm analyses: Target Scan, PicTar, and DIANA, predicted the likely candidate miRNAs that target to down-regulate proteins from our iTRAQ result and miRNA-124 was found to have the highest probability to target at GNAI1 protein. miRNA-124 is one of the richest miRNA in the brain and it is known to cause neuronal differentiation, maturation, and neurogenesis in normal brain growth.

Next, we investigated if GNAI1 changes were indeed caused by VPA treatment. By treating mouse cortical tissue and primary neuronal culture with VPA treatment, GNAI1 protein was significantly decreased. However, no change in Gnai1 mRNA was observed. VPA down-regulated the expression level of miRNA-124 target protein GNAI1 without Gnai1 mRNA reduction and this suggests that GNAI1 protein was silenced post-transcriptionally by VPA induced miRNA-124. To strengthen our in silico prediction, we then investigated GNAI1 protein’s regulation by using selective miRNA-124 inhibitor. We performed both experiments in primary neuronal culture and found that miRNA-124 inhibitor markedly increased the GNAI1 protein expression whereas the miRNA-124 mimic significantly decreased the GNAI1 protein expression.

As the function of GNAI1 is to inhibit adenylate cyclase activity, we tested our hypothesis by checking the amount of cAMP concentration in primary neuron culture with the following parameters: with or without VPA, with or without miRNA-124 inhibitor, and with or without Gnai1 siRNA. Our results showed that cAMP levels were markedly increased by VPA stimulation, markedly decreased by using selective miRNA-124 inhibitor, and significantly increased in Gnai1 siRNA. We also looked at other downstream effectors and investigated brain-derived neurotrophic factor (BDNF) expression level as our proteomics data suggested VPA aids in neutrophic growth. Bdnf mRNA was concomitantly expressed with cAMP activity in each treatment but the up-regulation of GNAI1 and Bdnf were abolished with the application of selective miRNA-124 inhibitor and even with addition of VPA. Conversely, under the application of selective miRNA-124 mimic, GNAI1 was significantly decreased and BDNF was significantly increased by VPA treatment for primary neuron culture. These results suggest that VPA treatment enhanced miRNA-124-GNAI1-BDNF pathway.

We postulated that the cAMP level increased by the inhibition of GNAI1 at downstream cascade. Indeed, VPA treatment also increased PKA expression level and CREB phosphorylation in primary neuronal culture. Similarly, Gnai1 siRNA treatment increased PKA expression level and CREB phosphorylation. However, with the selective miRNA-124 inhibitor, PKA and CREB phosphorylation expression levels were not affected. These results suggest that the inhibition of GNAI1 at the downstream of miRNA-124-GNAI1 pathway enhanced PKA-pCREB-BDNF cascade. In summary, we have identified amolecular mechanism through VPA that induces BDNF under miRNA-124 control and its target protein, GNAI1.

Figure 1 Cross-talk of epigenetic modulation by valproic acid in neurons.

As a follow-up to our study, we used VPA in adult mice and looked at the dendritic arborization in primary visual cortex after VPA treatment. Surprisingly, VPA induced dendritic branching and cortical rewiring in a circuitry, theoretically, should not have such dynamic rewiring at adulthood. We also show that it has potential in reactivating critical period plasticity (Lim et al., in preparation). One of the epigenetic mechanistic actions of VPA is also acting as a HDAC inhibitor. Neurodevelopment, synaptic plasticity, and memory formation are dynamically regulated by histone acetylation and histone deacetylation (Ballas et al, 2005; Jessberger et al, 2007; Lubin and Sweatt, 2007). Additionally, it has been shown that PKA-induced histone H3 acetylation could play a role in synaptic plasticity (Chwang et al., 2007). To modulate histone acetylation within mammalian brain, we and other groups proposed treating the adult brain with VPA, though its exact mechanism is still relatively unknown. It could be transcription of the BDNF protein sustained by the persistence of acetylation of histone H3 or by the inhibition of histone deacetylases. There could be crosstalk between miRNA and HDACs to consequently control transcription of the BDNF protein via the pCREB and PKA acting through histone H3 acetylation (Figure 1). BDNF is well established to direct growth and differentiation in the developing nervous system, and promote neurogenesis, dendritic spine reorganization, and activity-dependent plasticity in adult brain (Nishino et al., 2012). It is speculated that the transcribed BDNF enhances the neurite extension and neurite outgrowth as a functional neurotrophic factor (Figure 1). How they specifically target at each other or crosstalk is of interest in the neurochemistry field. We would like to propose the role and the function of VPA as a miRNA inducer, in addition to being an HDAC inhibitor, and that it may become the prospective innovative therapeutic drug of various refractory CNS diseases.

This work was supported by the Agency for Science and Technology (A★STAR) intramural funding for the Integrative Neuroscience Programme, Singapore Institute for Clinical Sciences.

Hirotaka Oikawa, Judy C. G. Sng*

縮小網(wǎng)店服務(wù)范圍的面積，增加一定網(wǎng)店的數(shù)量，來進行網(wǎng)點的擴張。通過網(wǎng)點拆分方法，原來一個網(wǎng)點所負責(zé)的服務(wù)范圍現(xiàn)在由多個新網(wǎng)點來服務(wù)，原來的網(wǎng)點小了，但是服務(wù)范圍擴張了，配送的效率也提升了，可以有效解決網(wǎng)點覆蓋區(qū)域不夠廣泛的問題，減少了人員和資源的浪費，節(jié)約了資本，提高了服務(wù)水平。并撤銷布局失誤和市場資源匱乏以及在市場競爭中被淘汰的網(wǎng)點，合并那些布局不合理，但還擁有一部分市場資源的網(wǎng)點，優(yōu)化企業(yè)管理，提高企業(yè)運行效率和企業(yè)競爭力。

Neuroepigenetics Laboratory, Singapore Institute for Clinical Sciences, Agency for Science and Technology (A*STAR), Singapore, Singapore (Oikawa H, Sng JC)

Department of Pharmacology, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore, Singapore (Sng JC)

*Correspondence to: Judy C. G. Sng, Ph.D., phcsngj@nus.edu.sg.

Accepted: 2016-09-09

orcid: 0000-0002-6740-6940 (Judy C. G. Sng)

How to cite this article: Oikawa H, Sng JC (2016) Valproic acid as a microRNA modulator to promote neurite outgrowth. Neural Regen Res 11(10)∶1564-1565.

Open access statement: This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

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