Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease.

N-acetylaspartate (NAA) attains one of the highest concentrations of any molecule in the human CNS (1), yet the functions it serves remain controversial. NAA is synthesized from l-aspartate and acetyl CoA in neuronal mitochondria by the enzyme aspartate N-acetyltransferase (Asp-NAT) (EC 2.3.1.17) (2, 3). NAA is found predominantly in neurons, (4) but the catabolic enzyme aspartoacylase (ASPA) is present primarily in oligodendrocytes in the CNS (5). The high concentration of NAA in the CNS and its characteristic peak in water-suppressed proton magnetic resonance spectroscopy (MRS) permits noninvasive determinations of NAA concentrations in the human brain. MRS determinations of NAA levels are commonly used for evaluating the integrity of neurons in a number of neurological disorders, and this method has emerged as a preferred technique for following the clinical course of several CNS pathologies (6–8). MRS studies operate on the assumptions that NAA is synthesized by and accumulated in neurons and that the steady-state NAA levels in the brain can be interpreted as indicating overall neuronal health or integrity (9, 10). Canavan's disease (CD) is a fatal, hereditary leukodystrophy that compromises normal CNS development and is caused by mutations in the gene for the enzyme ASPA (11, 12). ASPA currently is thought to function exclusively to hydrolyze NAA, a neuron-specific amino acid derivative, into l-aspartate and free acetate. However, ASPA is strongly expressed in other tissues, such as kidney, even though the only known substrate, NAA, is present predominantly in the nervous system (13). Despite the established connection between mutations in the gene for ASPA in CD and the lost capacity to deacetylate NAA, the specific connection between ASPA deficiency and the failure of proper CNS development is unclear (14). Further, the precise roles that NAA plays in CNS development and function remain a matter of inquiry. Several hypotheses have been proposed for the roles served by NAA in the nervous system. Because of its high concentration and lack of known actions on neurons or glia, NAA has been proposed to act as an organic osmolyte that removes excess water from neurons by acting as a molecular water pump (15). In this regard, it has been proposed that excess NAA leads to osmotic dysregulation or has other cytotoxic effects that are responsible for the pathology observed in CD patients (16). NAA has also been proposed as a required substrate for the enzymatic synthesis of the neuron-specific dipeptide N-acetylaspartylglutamate (17). There is more compelling evidence that NAA is essential for lipid synthesis and myelination in the CNS, in addition to the well established connection between ASPA mutations and leukodystrophy. In particular, the levels of NAA, ASPA, and Asp-NAT rise with a temporal course similar to those of myelin proteins (18, 19). Further, it has been shown that NAA contributes acetyl groups for the synthesis of lipids, which in turn are incorporated into myelin (20, 21). Finally, radiolabeled NAA is transported down optic nerve axons, and the acetate moiety is incorporated into their ensheathing myelin lipids (22). These and other observations led to our hypothesis that mutations in ASPA result in a deficiency in the supply of NAA-derived acetate, which in turn results in decreased synthesis of myelin-related fatty acids and lipids. Under our hypothesis, it is this lipogenic deficiency that compromises CNS myelination, impairs CNS development, and ultimately results in the white matter degeneration observed in CD (23, 24). In most cell types, such as hepatocytes, the citrate–citrate lyase system provides the acetyl groups for fatty acid synthesis (25). It remains to be demonstrated that the NAA–ASPA system contributes significantly to lipid synthesis in the CNS. We addressed this issue by examining the rate of myelin lipid synthesis during the period of maximal postnatal myelination in ASPA knockout (ASPA–/–) mice, which were developed as a model for CD (26). These mice exhibit pathology similar to that of human CD patients. We also examined acetate levels in the brains of ASPA–/– mice and normal mice, as well as the lipid content in postmortem white matter samples from a 4.75-yearold CD patient compared with the lipid levels in normal human white matter.