Presentation Title

Regulation of Cellular Cobalamin Acquisition and Processing by Nrf2 and mTORC1 Downstream of Neurotrophic Factors

Speaker Credentials

Ph.D. student

Speaker Credentials

BS

College

College of Pharmacy

Location

Nova Southeastern University, Davie, Florida, USA

Format

Poster

Start Date

16-2-2018 12:15 PM

End Date

16-2-2018 1:15 PM

Abstract

Objective. Define the roles of Nrf2 and mTORC1 in regulating cobalamin content downstream of neurotrophic factors in SH-SY5Y neuroblastoma cells. Background. Vitamin B12 (cobalamin) serves as a cofactor for methionine synthase, which catalyzes the regeneration of methionine from homocysteine. Cellular cobalamin processing requires proper lysosomal acidification and the availability of cytoplasmic glutathione. Mechanistic target of rapamycin complex 1 (mTORC1) abrogates lysosomal acidification and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) promotes glutathione production. Both mTORC1 and Nrf2 are activated downstream of neurotrophic factor-induced receptor tyrosine kinase signaling. We seek to understand the individual roles of Nrf2 and mTORC1 in regulating cobalamin content in this setting. Methods. SH-SY5Y cells were pre-treated with temsirolimus (TEMS) or treated with either neuregulin-1 (NRG-1) or brain-derived neurotrophic factor (BDNF). RT-qPCR was performed to measure gene expression. Cobalamin analysis was done via HPLC. Results. Both 1 nM and 100 nM NRG-1 decreases LMBRD1 (probable lysosomal cobalamin transporter) expression, but only 1 nM NRG-1 decreased ATP6V1H (V-type proton ATPase subunit H) mRNA. 10 nM BDNF decreased both LMBRD1 and ATP6V1H mRNA levels. 10 nM BDNF may selectively increase adenosylcobalamin. Furthermore, pre-treatment of cells with 100 nM TEMS and treatment with 100 nM NRG-1 increased cobalamin content over either agent alone. Conclusion. Our research suggests that different neurotrophic factors play unique roles in regulating cobalamin processing, NRG-1 may have opposing effects at various concentrations, and that mTORC1 activation can limit cobalamin content in SH-SY5Y cells. Grants. This research was partially funded by the A2 Milk Company.

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Feb 16th, 12:15 PM Feb 16th, 1:15 PM

Regulation of Cellular Cobalamin Acquisition and Processing by Nrf2 and mTORC1 Downstream of Neurotrophic Factors

Nova Southeastern University, Davie, Florida, USA

Objective. Define the roles of Nrf2 and mTORC1 in regulating cobalamin content downstream of neurotrophic factors in SH-SY5Y neuroblastoma cells. Background. Vitamin B12 (cobalamin) serves as a cofactor for methionine synthase, which catalyzes the regeneration of methionine from homocysteine. Cellular cobalamin processing requires proper lysosomal acidification and the availability of cytoplasmic glutathione. Mechanistic target of rapamycin complex 1 (mTORC1) abrogates lysosomal acidification and Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) promotes glutathione production. Both mTORC1 and Nrf2 are activated downstream of neurotrophic factor-induced receptor tyrosine kinase signaling. We seek to understand the individual roles of Nrf2 and mTORC1 in regulating cobalamin content in this setting. Methods. SH-SY5Y cells were pre-treated with temsirolimus (TEMS) or treated with either neuregulin-1 (NRG-1) or brain-derived neurotrophic factor (BDNF). RT-qPCR was performed to measure gene expression. Cobalamin analysis was done via HPLC. Results. Both 1 nM and 100 nM NRG-1 decreases LMBRD1 (probable lysosomal cobalamin transporter) expression, but only 1 nM NRG-1 decreased ATP6V1H (V-type proton ATPase subunit H) mRNA. 10 nM BDNF decreased both LMBRD1 and ATP6V1H mRNA levels. 10 nM BDNF may selectively increase adenosylcobalamin. Furthermore, pre-treatment of cells with 100 nM TEMS and treatment with 100 nM NRG-1 increased cobalamin content over either agent alone. Conclusion. Our research suggests that different neurotrophic factors play unique roles in regulating cobalamin processing, NRG-1 may have opposing effects at various concentrations, and that mTORC1 activation can limit cobalamin content in SH-SY5Y cells. Grants. This research was partially funded by the A2 Milk Company.