Tissue-nonspecific alkaline phosphatase promotes neuronal cell proliferation and differentiation: metabolomic reveals glutathione and taurine as molecular correlates

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme whose substrates are various phosphorylated extracellular molecules including pyridoxal phosphate (vitamin B6) and adenine nucleotides. Dysfunctions of TNAP result in hypophosphatasia, a rare disease characterized by defective bone mineralization and impaired brain functions. In the brain, TNAP expression peaks during development and is associated with various steps of neurogenesis. However, the influence of TNAP activity on neurogenesis remains poorly understood in its cellular and molecular aspects. Here we used the SK-N-SH D human neuroblastoma cell line as a cell culture model to further investigate the involvement of TNAP in neuronal precursor proliferation and neuronal differentiation. We also used 1H-NMR-based metabolomics to investigate the molecular correlates of TNAP action on SK-N-SH D cell proliferation and differentiation. We first observed an increase in alkaline phosphatase (AP) activity when the cells were placed in differentiation medium. We next found that inhibiting TNAP with a specific inhibitor (MLS-0038949) impeded neuroblastoma cell proliferation. TNAP inhibition also hindered neuronal differentiation, as evidenced by a decrease in the number of neurite-bearing cells. In contrast, neurite length was not affected by TNAP inhibition, suggesting that TNAP controls neurite sprouting, but not neurite outgrowth per se. The metabolomic results indicate that proliferation and differentiation are associated with a decrease in the amounts of proteinogenic amino acids as well as that of compounds potentially involved in lipid production. This analysis also revealed that proliferation and differentiation are associated with increased glutathione levels and decreased amounts of hypotaurine and taurine, supporting proposals that organosulfur compounds play an important role in these processes. Since pyridoxine was present in the culture media, these results suggest that TNAP is involved in neurogenesis through mechanisms in addition to its role in vitamin B6 metabolism and may instead involve the ectonucleotidase activity (or an unidentified activity) of TNAP.