Folic acid deficiency exacerbates the inflammatory response of astrocytes after ischemia-reperfusion by enhancing the interaction between IL-6 and JAK-1/pSTAT3.

AIM: To demonstrate the role of IL-6 and pSTAT3 in the inflammatory response to cerebral ischemia/reperfusion following folic acid deficiency (FD). METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in adult male Sprague-Dawley rats in vivo, and cultured primary astrocytes were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to emulate ischemia/reperfusion injury in vitro. RESULTS: Glial fibrillary acidic protein (GFAP) expression significantly increased in astrocytes of the brain cortex in the MCAO group compared to the SHAM group. Nevertheless, FD did not further promote GFAP expression in astrocytes of rat brain tissue after MCAO. This result was further confirmed in the OGD/R cellular model. In addition, FD did not promote the expressions of TNF-α and IL-1ß but raised IL-6 (Peak at 12 h after MCAO) and pSTAT3 (Peak at 24 h after MCAO) levels in the affected cortices of MCAO rats. In the in vitro model, the levels of IL-6 and pSTAT3 in astrocytes were significantly reduced by treatment with Filgotinib (JAK-1 inhibitor) but not AG490 (JAK-2 inhibitor). Moreover, the suppression of IL-6 expression reduced FD-induced increases in pSTAT3 and pJAK-1. In turn, inhibited pSTAT3 expression also depressed the FD-mediated increase in IL-6 expression. CONCLUSIONS: FD led to the overproduction of IL-6 and subsequently increased pSTAT3 levels via JAK-1 but not JAK-2, which further promoted increased IL-6 expression, thereby exacerbating the inflammatory response of primary astrocytes.

Homocysteine can aggravate depressive like behaviors in a middle cerebral artery occlusion/reperfusion rat model: a possible role for NMDARs-mediated synaptic alterations.

BACKGROUND: Post-stroke depression (PSD), the most frequent psychiatric complication following stroke, could have a negative impact on the recuperation of stroke patients. Hyperhomocysteinemia (HHCY) has been reported to be a modifiable risk factor of stroke. OBJECTIVE: The study tries to explore the effect of HHCY on PSD and the role of N-methyl-d-aspartate receptors (NMDARs)-mediated synaptic alterations. METHODS: Forty-five adult male Sprague-Dawley rats were randomly allocated into five groups: sham operation group, middle cerebral artery occlusion group (MCAO), HCY-treated MCAO group HCY and MK-801 co-treated MCAO group and MK-801-treated MCAO group. 1.6 mg/kg/d D, L-HCY was administered by tail vein injection for 28 d prior to SHAM or MCAO operationand up to 14 d after surgery. The MK-801 (3 mg/kg) was administered by intraperitoneal injection 15 min prior to MCAO operation. RESULTS: HCY treatment aggravated depressive-like disorders of post-stroke rats by the open field test and sucrose preference test. Further, HCY significantly decreased central monoamines levels in the MCAO rats by HPLC. The transmission electron microscopy results showed that the number of synapses and the area of postsynaptic density decreased in the hippocampus of the HCY-treated MCAO rats. Additionally, HCY augmented ischemia-induced up-regulation of NMDARs, decreased the levels of synaptic structure-related marker PSD-95and the synaptic transmission-associated synaptic proteins (VGLUT1, SNAP-25 and Complexin Ι/ΙΙ). These effects of HCY were partly reversed by the NMDA antagonist MK-801. CONCLUSIONS: The current study suggested that NMDARs-mediated synaptic plasticity may be involved in the adverse effect of HCY on PSD.

Homocysteine restrains hippocampal neurogenesis in focal ischemic rat brain by inhibiting DNA methylation.

Ischemic stroke represents a major cause of mortality worldwide. An elevated level of homocysteine (Hcy) is recognized as a powerful risk factor of ischemic stroke. We previously reported that Hcy induces cytotoxicity and proliferation inhibition in neural stem cells (NSCs) derived from the neonatal rat hippocampus in vitro. However, the toxic potential of Hcy on NSCs and its underlying mechanisms are not entirely clear in ischemic brain. Since DNA methylation is critical for establishing the diverse cell fates in the central nervous system, we hypothesized that negative effect of Hcy (an intermediate in the one-carbon metabolism) on neurogenesis might be link to DNA methylation in ischemic stroke. In our study, the rats in Hcy intervention group were intraperitoneally injected with 2% Hcy solution (5 mL/kg/d) for 7 consecutive days before MCAO surgery until they were sacrificed. Our study indicated that Hcy inhibited NSCs self-renewal capacity, which was exhibited by lowering the number of DCX+/BrdU+ and NeuN+/BrdU+ in ischemic brain hippocampus. A reduction in the activity of the DNA methyltransferases (DNMTs), total methylation level and the number of 5mC+/NeuN+ and DCX+/5mC+ cells was observed in Hcy-treated ischemic brains. Additionally, Hcy also induced an increase in S-adenosylhomocysteine (SAH), and a decrease in the ratio of S-adenosylmethionine (SAM) to SAH. These results suggest that the alterations in DNA methylation may be an important mechanism by which Hcy inhibits neurogenesis after stroke. Hcy-induced DNA hypomethylation may be mainly caused by a reduction in the DNMT activity which is regulated by the concentrations of SAM and SAH. Maintaining normal DNA methylation by lowering Hcy level may possess therapeutic potential for promoting neurological recovery and reconstruction after stroke.

Folic acid stimulation of neural stem cell proliferation is associated with altered methylation profile of PI3K/Akt/CREB.

Proliferation of neural stem cells (NSCs) is required for development and repair in the nervous system. NSC amplification in vitro is a necessary step towards using NSC transplantation therapy to treat neurodegenerative diseases. Folic acid (FA) has been shown to act through DNA methyltransferase to stimulate NSC proliferation. To elucidate the underlying mechanism, the effect of FA on the methylation profiles in neonatal rat NSCs was assessed by methylated DNA immunoprecipitation (MeDIP) and methylated DNA immunoprecipitation-DNA microarray (MeDIP-Chip). Differentially methylated regions (DMRs) were determined by quantitative differentially methylated regions analysis, and genes carrying at least three DMRs were selected for pathway analysis. Gene network analysis revealed links with steroid biosynthesis, fatty acid elongation and the PI3K/Akt/CREB, neuroactive ligand-receptor interaction, Jak-STAT and MAPK signaling pathways. Moreover, Akt3 acted as a hub in the network, in which 14 differentially methylated genes converged to the PI3K/Akt/CREB signaling pathway. These findings indicate that FA stimulates NSC proliferation by modifying DNA methylation levels in the PI3K/Akt/CREB pathway.

Homocysteine induces cytotoxicity and proliferation inhibition in neural stem cells via DNA methylation in vitro.

Mild to moderate hyperhomocysteinemia has been implicated in neurodevelopmental disorders and neurodegenerative diseases in human studies. Although the molecular mechanisms underlying the effects of homocysteine (Hcy) neurotoxicity on the nervous system are not yet fully understood, inhibition of neural stem cell (NSC) proliferation and alterations in DNA methylation may be involved. The aim of the present study was to characterize the effects of Hcy on DNA methylation in NSCs, and to explore how Hcy-induced changes in DNA methylation patterns affect NSC proliferation. We found that D,L-Hcy (30-1000 µm) but not L-cysteine inhibited cell proliferation and reduced levels of global DNA methylation in NSCs from neonatal rat hippocampus and increased cell injury. High levels of Hcy also induced an increase in S-adenosylhomocysteine (SAH), a decrease in the ratio of S-adenosylmethionine (SAM) to SAH, and a reduction in protein expression of the DNA methyltransferases DNMT1, DNMT3a and DNMT3b and their enzymatic activity. Moreover, the DNMT inhibitor zebularine reduced the global DNA methylation level and inhibited NSC proliferation. Our results suggest that alterations in DNA methylation may be an important mechanism by which high levels of Hcy inhibit NSC viability in vitro. Hcy-induced DNA hypomethylation may be caused by a reduction in the DNMT activity which is regulated by the cellular concentrations of SAM and SAH, or their protein expression levels. Our results also suggest that Hcy may play a role in the pathogenesis of certain nervous system diseases via a molecular mechanism that involves negative regulation of NSC proliferation and alterations in DNA methylation.

Folic acid acts through DNA methyltransferases to induce the differentiation of neural stem cells into neurons.

The present study investigated the roles of folic acid and DNA methyltransferases (DNMTs) in the differentiation of neural stem cells (NSCs). Neonatal rat NSCs were grown in suspended neurosphere cultures and identified by their expression of SOX2 protein and capacity for self-renewal. Then NSCs were assigned to five treatment groups for cell differentiation: control (folic acid-free differentiation medium), low folic acid (8 µg/mL), high folic acid (32 µg/mL), low folic acid and DNMT inhibitor zebularine (8 µg/mL folic acid and 150 nmol/mL zebularine), and high folic acid and zebularine (32 µg/mL folic acid and 150 nmol/mL zebularine). After 6 days of cell differentiation, immunocytochemistry and western blot analyses were performed to identify neurons by ß-tubulin III protein expression and astrocytes by GFAP expression. We observed that folic acid increased DNMT activity which may be regulated by the cellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and the abundance of neurons but decreased the number of astrocytes. Zebularine blocked these effects of folic acid. In conclusion, folic acid acts through elevation of DNMT activity to increase neuronal differentiation and decrease astrocytic differentiation in NSCs.