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 lM) 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.

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 β-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.

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Astrocytes are the most widely distributed cells in the brain, and astrocyte apoptosis may play an important role in the pathogenesis of neurodegenerative diseases. Folate is required for the normal development of the nervous system, but its effect on astrocyte apoptosis is unclear. In this study, we hypothesized that folic acid (the therapeutic form of folate) decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. Primary cultures of astrocytes were incubated for 12 days with various concentrations of folic acid (0–40 μmol/L), then cell proliferation, apoptosis, intracellular folate concentration, intracellular homocysteine (Hcy) concentration, intracellular reactive oxygen species (ROS) levels, telomeric DNA oxidative damage, and telomere length were determined. The results showed that folic acid deficiency decreased intracellular folate, cell proliferation, and telomere length, whereas it increased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In contrast, folic acid dose-dependently increased intracellular folate, cell proliferation, and telomere length but it decreased Hcy concentration, ROS levels, telomeric DNA oxidative damage, and apoptosis. In conclusion, folic acid inhibited apoptosis in astrocytes. The underlying mechanism for this protective effect may be that folic acid decreased oxidative stress and thereby prevented telomeric DNA oxidative damage and telomere attrition.