NEUREGULIN-1 PROMOTES REDOX-DEPENDENT NEURONAL COBALAMIN METABOLISM BYSTIMULATING CYSTEINE-DEPENDENT GLUTATHIONE SYNTHESIS

POSTER PRESENTATIONS

Objective. To investigate the ability of the neurotrophic factor neuregulin-1 (NRG-1) to influence the status of vitamin B12 (cobalamin; Cbl) in cultured human neuroblastoma cells. Background. Neuregulin-1 (NRG-1) is an epidermal growth factor (EGF)-like growth factor that plays critical roles in development of the central nervous system by influencing neuronal differentiation, regulation of neurotransmitter receptor expression and oligodendrocyte development. Methionine synthase (MS) catalyzes the conversion of homocysteine (HCY) to the essential amino acid methionine, utilizing a methyl group derived from 5-methyltetrahydrofolate. Methionine can further receive an adenosyl moiety from ATP to form S-adenosylmethionine (SAM), which is the universal methyl donor providing methyl groups to more than 250 different methylation reactions including DNA and histone methylation. MS activity depends upon its cofactor cobalamin (Cbl), also known as vitamin B12. In a recent study we observed more than five-fold lower Cbl levels in postmortem human frontal cortex of schizophrenia subjects when compared to samples from age-matched controls. While the factors leading to this abnormally low Cbl status remain unknown, in vivo studies of EGF-knockout neurodegenerative mouse models revealed crosstalk between Cbl metabolism and the EGF system, and EGF can regulate the neurotrophic effects of Cbl in brain, suggesting the possibility that NRG-1 may play a role in regulating neuronal Cbl metabolism. We previously showed that growth factor stimulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway promotes EAAT3-mediated cysteine uptake and subsequently increases GSH levels in neurons. Since NRG-1 is capable of initiating the PI3K/Akt signaling cascade, we hypothesized that NRG-1 may promote neuronal Cbl metabolism by stimulating EAAT3-mediated cysteine uptake and increasing GSH synthesis, making more GSCbl available for active Cbl species formation. Using the SH-SY5Y human neuroblastoma cell line, we examined the influence of NRG-1 on levels of six individual Cbl species. Methods. Cbl extraction and measurement were performed under dim-red light. After pretreatment culture media was removed and SH-SY5Y cells were washed 3X with 4 mL of Dulbecco’s phosphate buffered saline (DPBS). Cells were then lysed, scraped and collected in a 1.5 mL microcentrifuge tube. 100 microliters of cell lysate was aliquoted for protein quantification by Lowry protein assay and 900 microliters of the remaining cell lysate was mixed and incubated with 1.35 mL ice-cold absolute ethanol for 10 min. Protein precipitates were removed by centrifugation at 10,600 RPM for 3 min. The supernatant was dried in a speedvac, resuspended in 300 microliters DPBS and filtered through 0.22 micron syringe-driven filter unit. 200 microliters of sample was added to a conical micro autosampler vial, blown with nitrogen, capped and kept at 4 degrees C in the autosampler (ESA model 542). 30 microliters of Cbl sample was injected into an ESA CoulArray HPLC system equipped with an Agilent Eclipse XD8-C8 (3×150 mm; 3.5 microns) reverse- phase C8 column and an Agilent Eclipse XDB-C8 (4.6×12.55 mm; 5 microns) guard column. A dual mobile phase gradient elution was used: mobile phase A contains 0.1% acetic acid in water, adjusted to pH 3.5 with 6.0 N ammonium hydroxide and mobile phase B contains 0.1% acetic acid in acetonitrile. The system was run at a flow rate of 0.6 mL/min at ambient temperature with the following gradients: 0-2 min 0% B, 2-12 min 10% B, 12-15 min 15% B and 15-35 min 20% B. Cbls were measured by electrochemical detection at an operating potential of 1,000 mV. Peak area analysis was performed by CoulArray software and standard curves were generated for each Cbl species. Cbl levels were normalized against protein content. Results. We found that NRG-1 stimulates neuronal synthesis of bioactive Cbl species adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) in SH-SY5Y neuronal cells by both promoting conversion of inactive to active Cbl species and increasing neuronal Cbl uptake. Formation of active Cbls is glutathione (GSH)-dependent and NRG-1-initiated increase is dependent upon its stimulation of cysteine uptake by excitatory amino acid transporter-3 (EAAT3), leading to increased GSH. The stimulatory effect of NRG-1 on cellular Cbl uptake is associated with increased expression of megalin, which facilitates Cbl transport in ileum and kidney. MeCbl is a required cofactor for methionine synthase (MS) and we demonstrate the ability of NRG-1 to increase MS activity, as well as affecting levels of methionine methylation cycle metabolites. The ability of lithium to promote megalin-related transport activity has been previously described. Lithium treatment (10 mM) for 1 or 4 hrs caused a significant increase in cysteine and GSH levels along with an increase in methylation capacity and a decrease in Cbl content. Conclusion. Our results identify novel neuroprotective roles for NRG-1 in terms of stimulating antioxidant production and synthesis of active Cbl species, as well as novel actions of lithium on redox and methylation status. These findings provide a potential mechanistic link between impaired NRG-1 signaling and neurological disorders such as schizophrenia. Grants. This work was partially supported by a grant from the Autism Research Institute