Cellular and developmental tissue distribution of NAA catabolic enzyme, aspartoacylase. Insights into NAA Function

Introduction NAA (N-acetyl-aspartate) is the second most abundant molecule in the CNS (after glutamate), its MRS signal is commonly used as a putative marker of viable neurones, and yet little is known about its function. Furthermore, recent data from cell culture has raised the possibility of an additional cellular contribution of NAA from nonneuronal origins, principally in isolated mature oligodendrocytes, the myelinating cells of the CNS (1). Proposed roles for NAA include the source of acetyl groups for lipogenesis (in myelination), and involvement in osmotic regulation and neuromodulation (2). In order to gain further insight into the role of NAA in the brain, we have determined the level of aspartoacylase (the first enzyme in the metabolism of NAA) in tissue from developing and mature rat brain, and primary cultures of glial and neuronal cells derived from the CNS. Methods Tissue harvest: Tissues from various regions of the rat brain, at key ages during postnatal development and maturation, were analysed for aspartoacylase activity. The regions examined were: cerebral cortex, corpus callosum, cerebellum, hippocampus, brain stem, olfactory bulb and optic nerve at ages from postnatal day 1 (P1) to adult (i.e. P1, P2, P4, P7, P14, P21 and adult). This work was carried out under UK Home Office procedures. Cells: Several purified cell-types isolated from the CNS were grown in cultures. These included cortical astrocytes, cortical and cerebellar neurones and various cell-types derived from the O-2A progenitor lineage. Aspartoacylase assay: The aspartoacylase assay was based on previous method of Hagenfeldt et al (3), with several modifications in order to assay small quantities of biological material. The specific activity (mU) is defined as nmol of aspartate produced per mg protein per min. Results Tissue activity: Highest activities of aspartoacylase were found in white matter areas of the brain, in which the activity increased markedly after 7 days in the developing rat brain (Figure 1). Gray matter did not show much activity or a strong trend. All brain areas had some activity in adults, the highest being in white matter, although this was lower than the activity at the height of myelination (Table 1). Cellular activity: Type-2 astrocytes from primary O-2A cells have the highest activity, followed by mature oligodendrocytes (myelinating cells; Table 2). Neurons have no activity. Primary type-1 astrocytes also express a moderate activity in culture (Table 3). Discussion Our data demonstrate three main findings: a) no aspartoacylase was detected in cultured neurones, b) white-matter tissue invariably expressed higher levels of aspartoacylase than grey matter, and c) aspartoacylase concentrations increased dramatically following the onset of myelination in the developing brain. Thus the enzyme catalysing NAA metabolism appears to be located in a different cellular compartment from the principle NAA store, namely the neurones. We would hypothese a duality of NAA function. Firstly one could suggest a simple scavenging role for aspartoacylase, preventing potential neurotoxicity such as that seen in Canavan's Disease, where a mutation of aspartoacylase results in accumulation of NAA in the brain leading to spongy degeneration of the white matter tracts. However, a more plausible explanation may be that NAA utilisation is separate from its site of storage. In particular, NAA could be released from the neurones to provide substrate for myelination by the oligodendrocytes during development or in response to white matter damage. Neuronal stores of NAA may be involved in the re-myelination process following injury. This would explain the reversible decrease in NAA sometimes observed in MS lesions. We note however at the tissue level that gray matter contains high levels of NAA, but low levels of aspartoacylase. Therefore, a secondary role for NAA in modulating neuronal homeostasis throughout the CNS would not be inconsistent with our data. References 1. Bhakoo and Pearce, J. Neurochem 74 254-62 (2000). 2. Birken and Oldendorf, Neurosci Biobehav Rev 13 23-31 (1989). 3. Hagenfeldt et al, J. Inherit. Metab. Disease 10 135-41 (1987).