DNA methylation analysis about differentiation genes in cerebral infarcted rat

Abstract

Transient ischemic attack has long been identified as a risk factor causing massive damage such as tissue loss and neuronal cell death in the brain. Emerging therapeutic strategies have, therefore, focused on the development of neuroprotective and neural regenerative approaches to modifying the central nervous system and restoring neurological function through neuroprotectors treatment, transgene insertion or cell therapy, etc. Epigenetic regulation can lead to biological effects and maintain of functional neurogenesis in the mammalian brain. The level of DNA methylation, a key mechanism of epigenetic changes is developed due to transfer of methyl groups to various genomic regions during embryogenesis of mammals. For early postnatal development of the brain, DNA methylation plays a key role in regulating the differentiation of NSCs into neuronal cells.Agmatine, as a promising neuromodulator formed by decarboxylation of arginine decarboxylase (ADC), was contributed to tissue regeneration in ischemic injured models according to many previous studies. Recently, it has been reported that agmatine treatment enhanced neurogenesis of subventricular zone neural stem cells by increasing the expression of extracellular-signal-regulated kinases (ERK) 1/2 protein, and also neuronal differentiation of cortical NSCs by increasing the methylation of bone morphogenetic protein (BMP) 2 and decreasing the methylation of Neurog2. Already it was demonstrated that retrovirus delivered human ADC genes in NSCs (ADC-NSCs), endogenously synthesized agmatine, reduced cell apoptosis against oxidative stress in vitro but the characterization and cell fate of ADC-NSCs is not yet known. The present study is to characterize and explore the factors for the neuronal differentiation of ADC-NSCs in vitro. And this study investigated that the ADC-NSCs transplanted middle cerebral artery occlusion (MCAO) injured animals would investigate regulating the DNA methylation of genes associated with neurogenesis.NSCs were infected with empty retrovirus vector (LXSN) or retrovirus vector containing hADC gene 7 days in vitro. The expression of stemness and neuronal cell lineage was determined in control NSCs, NSCs infected with LXSN (LXSN-NSCs) and ADC-NSCs. It was increased the cellular proliferation and the expression of stemness in ADC-NSCs compared with control NSCs and LXSN-NSCs. Also, ADC-NSCs induced the increase of microtubule-associated protein (MAP) 2 and Olig2 expressions by determining immunocytochemistry and Western blot.For in vivo study, the rats were transplanted with NSCs, LXSN-NSCs or ADC-NSCs at 1 week after MCAO for 1 hour, and performed behavioral tests to analyze functional recovery. Immunohistochemistry and immunoblotting were performed to determine the expressions of neuronal cell lineage and DNA methylation. It was confirmed by performing polymerase chain reaction for determining the relationship between DNA methylation and differentiation related genes. The functional recovery was increased in ADC-NSCs transplanted animals after MCAO injury. Besides, the expressions of MAP2, a neural marker, and Olig2, an oligodendrocyte marker, and DNA methyltransferases were significantly increased in ADC-NSCs transplanted rats. However, methylated SRY (sex determining region Y)-box (SOX) 2, Nestin and platelet-derived growth factor α receptor (PDGFRα) genes directly involving in neuronal differentiation were less expressed in ADC-NSCs group compared with the other groups.This investigation was demonstrated that the levels of DNA methylation were altered in ADC-NSCs after transient focal cerebral ischemia relating neuronal differentiation. ADC-NSCs transplantation into ischemic damaged niche may contribute to tissue regeneration relating with the regulation of DNA methylation in transient focal cerebral ischemia.