Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells

N-acetylaspartate (NAA) is synthesized predominantly in neurons, and brain homogenate subfractionation studies suggest that biosynthesis occurs at both microsomal (cytoplasmic) and mitochondrial sites. We investigated NAA synthesis in SH-SY5Y human neuroblastoma cells using distinct metabolic precursors that are preferentially metabolized in mitochondria and cytoplasm. Incorporation of ¹⁴C-aspartate and ¹⁴C-malate into NAA was examined in the presence and absence of an inhibitor (aminooxyacetic acid, AOAA) of aspartate aminotransferase (AAT), a central enzyme involved in the biosynthesis of aspartate in mitochondria, and degradation of aspartate in the cytoplasm. AOAA increased the incorporation of ¹⁴C-aspartate into NAA, reflecting direct aspartate → NAA synthesis in an extramitochondrial location. As expected, AOAA decreased incorporation of ¹⁴C-malate into NAA, reflecting NAA synthesis in mitochondria via the malate → oxaloacetate → aspartate → NAA pathway. When ¹⁴C-malate was used as substrate, the ¹⁴C-NAA/¹⁴C-aspartate ratio was over 20-fold higher than the ratio obtained with ¹⁴C-aspartate. Because NAA can only be synthesized from aspartate, the higher ¹⁴C-NAA/¹⁴C-aspartate (product/substrate) ratio obtained with ¹⁴C-malate suggests greater NAA biosynthetic activity. We conclude that NAA biosynthesis occurs in both the cytoplasm and mitochondria of SH-SY5Y cells, and that the contribution from the mitochondrial compartment is quantitatively larger than that in the extramitochondrial compartment.