Abstract
Canavan disease is caused by mutations in the gene encoding aspartoacylase (ASPA), a deacetylase that catabolizes N-acetylaspartate (NAA). The precise involvement of elevated NAA in the pathogenesis of Canavan disease is an ongoing debate. In the present study, we tested the effects of elevated NAA in the brain during postnatal development. Mice were administered high doses of the hydrophobic methyl ester of NAA (M-NAA) twice daily starting on day 7 after birth. This treatment increased NAA levels in the brain to those observed in the brains of Nur7 mice, an established model of Canavan disease. We evaluated various serological parameters, oxidative stress, inflammatory and neurodegeneration markers and the results showed that there were no pathological alterations in any measure with increased brain NAA levels. We examined oxidative stress markers, malondialdehyde content (indicator of lipid peroxidation), expression of NADPH oxidase and nuclear translocation of the stress-responsive transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF-2) in brain. We also examined additional pathological markers by immunohistochemistry and the expression of activated caspase-3 and interleukin-6 by Western blot. None of the markers were increased in the brains of M-NAA treated mice, and no vacuoles were observed in any brain region. These results show that ASPA expression prevents the pathologies associated with excessive NAA concentrations in the brain during postnatal myelination. We hypothesize that the pathogenesis of Canavan disease involves not only disrupted NAA metabolism, but also excessive NAA related signaling processes in oligodendrocytes that have not been fully determined and we discuss some of the potential mechanisms.
Highlights
Canavan disease is a progressive autosomal genetic leukodystrophy caused by mutations in the gene that codes for the deacetylase enzyme aspartoacylase (ASPA; Matalon et al, 1988)
methyl ester of NAA (M-NAA) was obtained from Bachem, Switzerland, methanol was from Merck, AG50W X8 resin was from BioRad and paraformaldehyde, gavage needles, MDA assay kits and Tween 20 were from Sigma Aldrich
Following 30 days of M-NAA treatment of the young, wild type mice, we examined a number of potential etiological factors in the vacuolization characteristic of Canavan disease ranging from reactive oxygen species (ROS) production to immune activation, as well as various pathological consequences including vacuole formation, glial fibrillary acidic protein (GFAP) immunoreactivity and lipid peroxidation
Summary
Canavan disease is a progressive autosomal genetic leukodystrophy caused by mutations in the gene that codes for the deacetylase enzyme aspartoacylase (ASPA; Matalon et al, 1988). The gene mutations cause loss of ASPA enzyme function which results in an inability to catabolize N-acetylaspartate (NAA), one of the most concentrated metabolites in the human brain. The primary documented metabolic effects of mutations in the gene for ASPA in Canavan disease patients are a lack of NAA deacetylation leading to buildup of NAA in the brain (Kvittingen et al, 1986) and greatly increased excretion of NAA in urine (Hagenfeldt et al, 1987). A primary question that remains unresolved is whether the etiology involves toxic buildup of NAA or impaired brain metabolism associated with the inability to catabolize NAA, or both. The precise involvement of NAA in the pathogenesis of Canavan disease remains controversial primarily because of the uncertainties surrounding the physiological roles of NAA in brain development and function
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