Despite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19-7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation-associated apoptosis (+16%). Decreased production (-58%) of S-adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (-57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long-lasting consequences.

Vitamin B12 (cobalamin, Cbl) is indispensable for proper brain development and functioning, suggesting that it has neurotrophic effects beside its well-known importance in metabolism. The molecular basis of these effects remains hypothetical, one of the reasons being that no efficient cell model has been made available for investigating the consequences of B12 cellular deficiency in neuronal cells. Here, we designed an approach by stable transfection of NIE115 neuroblastoma cells to impose the anchorage of a chimeric B12-binding protein, transcobalamin-oleosin (TO) to the intracellular membrane. This model produced an intracellular sequestration of B12 evidenced by decreased methyl-Cbl and Sadenosylmethionine and increased homocysteine and methylmalonic acid concentrations. B12 deficiency affected the proliferation of NIE115 cells through an overall increase in catalytic protein phosphatase 2A (PP2A), despite its demethylation. It promoted cellular differentiation by improving initial outgrowth of neurites and, at the molecular level, by augmenting the levels of proNGF and p75 NTR . The up-regulation of PP2A and pro-nerve growth factor (NGF) triggered changes in ERK1/2 and Akt, two signaling pathways that influence the balance between proliferation and neurite outgrowth. Compared with control cells, a 2-fold increase of p75 NTR -regulated intramembraneous proteolysis (RIP) was observed in proliferating TO cells (P < 0.0001) that was associated with an increased expression of two tumor necrosis factor (TNF)-␣ converting enzyme (TACE) secretase enzymes, Adam 10 and Adam 17. In conclusion, our data show that B12 cellular deficiency produces a slower proliferation and a speedier differentiation of neuroblastoma cells through interacting signaling pathways that are related with increased expression of PP2A, proNGF, and TACE.homocysteine ͉ neurotrophin V itamin B12 (B12, also named cobalamin, Cbl) deficiency has long been associated with pernicious anaemia (1) and neurological disorders that range from minor behavior changes to severe neurodegenerative disorders (2). Molecular mechanisms are still lacking to explain how the deficiency can bring about all of the symptoms observed. Indeed, only two enzymes are B12-dependent in mammalian cells: the mitochondrial enzyme L-methylmalonyl-CoA mutase (EC 5.4.99.2) and the cytoplasmic homocysteine methyltransferase, also referred as methionine synthase (EC 2.1.1.13). Inferences are thus based on the two direct consequences of B12 deficiency: the accumulation of methylmalonic acid (MMA) and homocysteine (Hcy). Until now, the consequences of B12 deficiency in the brain have been difficult to evidence because of the experimental limitations of the classical cell models. Indeed, the minute amount of vitamin B12 needed by cells can be provided in vitro by B12 from the FCS, which can bind the ''autocrine'' B12 carrier protein, transcobalamin (TC) (3, 4).To delineate the role of B12 in neurological disorders, we designed a neuronal cell model made deficient in B12 through the stable ...

The methyl donors folate (vitamin B9) and vitamin B12 are centrepieces of the one-carbon metabolism that has a key role in transmethylation reactions, and thus in epigenetic and epigenomic regulations. Low dietary intakes of folate and vitamin B12 are frequent, especially in pregnant women and in the elderly, and deficiency constitutes a risk factor for various diseases, including neurological and developmental disorders. In this respect, both vitamins are essential for normal brain development, and have a role in neuroplasticity and in the maintenance of neuronal integrity. The consequences of a methyl donor deficiency (MDD) were studied both in vivo in rats exposed in utero, and in vitro in hippocampal progenitors (H19-7 cell line). Deficiency was associated with growth retardation at embryonic day 20 (E20) and postnatally with long-term brain defects in selective areas. mRNA and protein levels of the transcription factor Stat3 were found to be decreased in the brains of deprived fetuses and in differentiating progenitors (62 and 48% for total Stat3 protein, respectively), along with a strong reduction in its phosphorylation at both Tyr705 and Ser727 residues. Vitamin shortage also affected upstream kinases of Stat3 signaling pathway (phospho-Erk1/2, phospho-Src, phospho-JNK, and phospho-p38) as well as downstream target gene products (Bcl-2 and Bcl-xL), thus promoting apoptosis. Conversely, the expression of the Stat3 regulator miR-124 was upregulated in deficiency conditions (≥65%), and its silencing by using siRNA partly restored Stat3 signaling in hippocampal neurons by increasing specifically the phosphorylation of Erk1/2 and Src kinases. Furthermore, miR-124 siRNA improved the phenotype of deprived cells, with enhanced neurite outgrowth. Taken together, our data suggest that downregulation of Stat3 signaling by miR-124 would be a key factor in the deleterious effects of MDD on brain development.