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Year : 2016  |  Volume : 11  |  Issue : 10  |  Page : 1564-1565

Valproic acid as a microRNA modulator to promote neurite outgrowth

1 Neuroepigenetics Laboratory, Singapore Institute for Clinical Sciences, Agency for Science and Technology (A*STAR), Singapore, Singapore
2 Neuroepigenetics Laboratory, Singapore Institute for Clinical Sciences, Agency for Science and Technology (A*STAR), Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

Date of Acceptance09-Sep-2016
Date of Web Publication4-Nov-2016

Correspondence Address:
Judy C. G. Sng
Neuroepigenetics Laboratory, Singapore Institute for Clinical Sciences, Agency for Science and Technology (A*STAR), Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1673-5374.193227

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How to cite this article:
Oikawa H, Sng JC. Valproic acid as a microRNA modulator to promote neurite outgrowth. Neural Regen Res 2016;11:1564-5

How to cite this URL:
Oikawa H, Sng JC. Valproic acid as a microRNA modulator to promote neurite outgrowth. Neural Regen Res [serial online] 2016 [cited 2022 Jan 24];11:1564-5. Available from: http://www.nrronline.org/text.asp?2016/11/10/1564/193227

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).

In our recent study, we propose to add a new function of VPA to modulate miRNAs to affect neurite outgrowth (Oikawa et al., 2015). We demonstrated VPA induced miRNA-124 that down-regulated a novel protein target, guanine nucleotide binding protein (Gi protein), alpha inhibiting 1, or sometimes termed as Gia1 or GNAI1. GNAI1 is primarily expressed in the brain and functions to inhibit adenylate cyclase activity and cAMP dependent pathways. By using a global miRNA microarray analysis, we observed 188 miRNAs differentially changed after 200 mg/kg VPA intraperitoneal infusion over a period of two days. A previous report also supported our findings that VPA induced miRNAs (Hunsberger et al., 2012). Most of the miRNAs were up-regulated (136 out of 188 miRNAs) while 52 miRNAs were down-regulated. We first validated our microarray data with RT-qPCR method in both mouse cortical tissues and mouse primary neuronal culture. We then narrowed it down on four up-regulated miRNAs, miRNA-22, 33a, 124 and 132 and by far, these four produced the most robust results.

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 a molecular mechanism through VPA that induces BDNF under miRNA-124 control and its target protein, GNAI1.

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.
Figure 1 Cross-talk of epigenetic modulation by valproic acid in neurons.
We propose that new function of valproic acid (VPA) as a microRNA modulator and epigenetic modulation of VPA in brain. From this miRNA-124 cascade, brain-derived neurotrophic factor (BDNF) transcription is doubly supported by acetylated histone H3 (AcH3) increment of protein kinase A (PKA) and cAMP response element binding (CREB) phosphorylation. By combining with histone deacetylase 1 (HDAC1) inhibition cascade, secretion of BDNF by VPA is supported. Consequently, it is promoted that neurite outgrowth, neuroprotection, neurogenesis, neuronal differentiation, and neuroregeneration.

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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.[12]

  References Top

Ballas N, Grunseich C, Lu DD, Speh JC, Mandel G (2005) REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. Cell 121:645-657.  Back to cited text no. 1
Chwang WB, Arthur JS, Schumacher A, Sweatt JD (2007) The nuclear kinase mitogen- and stress-activated protein kinase 1 regulates hippocampal chromatin remodeling in memory formation. J Neurosci 27:12732-12742.  Back to cited text no. 2
Fiore R, Khudayberdiey S, Saba R, Schrtt G (2011) MicroRNA function in the nervous system. Prog Mol Biol Transl Sci 102:47-100.  Back to cited text no. 3
Foti SB, Chou A, Moll AD, Roskams AJ (2013) HDAC inhibitors dysregulate neural stem cell activity in the postnatal mouse brain. Int J Dev Neurosci 31:434-447.  Back to cited text no. 4
Goh WW, Oikawa H, Sng JC, Sergot M, Wong L (2012) The role of miRNAs in complex formation and control. Bioinformatics 28:453-456.  Back to cited text no. 5
Hunsberger JG, Fessler EB, Wang Z, Elkahloun AG, Chuang DM (2012) Postinsultvalproic acid-regulated microRNAs potential targets for cerebral ischemia. Am J Transl Res 4:316-332.  Back to cited text no. 6
Jessberger S, Nakashima K, Clemenson GD Jr, Mejia E, Mathews E, Ure K, Ogawa S, Sinton CM, Gage FH, Hsieh J (2007) Epigenetic modulation of seizure-induced neurogenesis and cognitive decline. J Neurosci 27:5967-5975.  Back to cited text no. 7
Lubin FD, Sweatt JD (2007) The IkappaB kinase regulates chromatin structure during reconsolidation of conditioned fear memories. Neuron 55:942-952.  Back to cited text no. 8
Nishino S, Ohtomo K, Numata Y, Sato T, Nakahata N, Kurita M (2012) Divergent effects of lithium and sodium valproate on brain-derived neurotrophic factor (BDNF) production in human astrocytoma cells at therapeutic concentrations. Prog Neuropsychopharmacol Biol Psychiatry 39:17-22.  Back to cited text no. 9
Oikawa H, Goh WW, Lim VK, Wong L, Sng JC (2015) Valproic acid mediates miR-124 to down-regulate a novel protein target, GNAI1. Neurochem Int 91: 62-71.  Back to cited text no. 10
Wayman GA, Davare M, Ando H, Fortin D, Varlamova O, Cheng HY, Marks D, Obrietan K, Soderling TR, Goodman RH, Impey S (2008) An activity-regulated microRNA controls dendritic plasticity by downregulating p250GAP. Proc Natl Acad Sci U S A 105:9093-9098.  Back to cited text no. 11
Yu JY, Chung KH, Deo M, Thompson RC, Turner DL (2008) MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation. Exp Cell Res 314:2618-2633.  Back to cited text no. 12


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