Bimodal occurrence of aspartoacylase in myelin and cytosol of brain (original) (raw)
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Immunohistochemical localization of aspartoacylase in the rat central nervous system
The Journal of Comparative Neurology, 2004
Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cellspecific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (ϳ37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotinperoxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.
Extensive aspartoacylase expression in the rat central nervous system
Glia, 2011
Aspartoacylase (ASPA) catalyzes deacetylation of N-acetylaspartate (NAA) to generate acetate and aspartate. Mutations in the gene for ASPA lead to reduced acetate availability in the CNS during development resulting in the fatal leukodystrophy Canavan disease. Highly specific polyclonal antibodies to ASPA were used to examine CNS expression in adult rats. In white matter ASPA expression was associated with oligodendrocyte cell bodies, nuclei and some processes, but showed a dissimilar distribution pattern to myelin basic protein and oligodendrocyte specific protein. Microglia expressed ASPA in all CNS regions examined, as did epiplexus cells of the choroid plexus. Pial and ependymal cells and some endothelial cells were ASPA positive, as were unidentified cellular nuclei throughout the CNS. Astrocytes did not express ASPA in their cytoplasm. In some fiber pathways and nerves, particularly in the brainstem and spinal cord, the axoplasm of many neuronal fibers expressed ASPA, as did some neurons. Acetyl coenzyme A synthase immunoreactivity was also observed in the axoplasm of many of the same fiber pathways and nerves. All ASPA-immunoreactive elements were unstained in brain sections from tremor rats, an ASPA-null mutant. The strong expression of ASPA in oligodendrocyte cell bodies is consistent with a lipogenic role in myelination. Strong ASPA expression in cell nuclei is consistent with a role for NAA-derived acetate in nuclear acetylation reactions, including histone acetylation. Expression of ASPA in microglia may indicate a role in lipid synthesis in these cells, whereas expression in axons suggests that some neurons can both synthesize and catabolize NAA.
N-Acetylation of L-Aspartate in the Nervous System: Differential Distribution of a Specific Enzyme
Journal of Neurochemistry, 1985
L-Aspartate N-acetyltransferase. a nervous system enzyme that mediates the synthesis of N-acetyl-L-aspartic acid, has been characterized. In the presence of acetyl-CoA, L-aspartate was acetylated 10-fold more efficiently than L-glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10-fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10-15 pmoVminlmg tissue) and retina the lowest detectable activity ( 1 -1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membraneassociated and was solubilized by treatment with Triton X-100. Key Words: N-Acetyltransferase-N-Acetylaspartic acid-Enzyme distribution-Enzyme specificity-TLC-HPLC.
Proceedings of The National Academy of Sciences, 2005
Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N-acetylaspartate (NAA) into L-aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelinassociated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAAderived acetate, is involved in the pathogenesis of CD.
Identification and distribution of aspartoacylase in the postnatal rat brain
Neuroreport, 2003
Aspartoacylase metabolizes N-acetylaspartic acid to produce L-aspartate and acetate. An aspartoacylase de¢ciency in humans is responsible for Canavan disease, a lethal autosomal recessive leukodystrophy.The role of aspartoacylase in the mammalian brain is unclear. Here we have generated and characterized a highly spe-ci¢c polyclonal antibody against aspartoacylase which recognizes a 37 kDa monomer and a dimer in normal but not in aspartoacylase-de¢cient rat tissue. Aspartoacylase protein expression sharply increases at P14, peaks at P28 and plateaus thereafter. Biochemical analysis reveals immunoreactivity in cytosolic but not in membrane fractions. Histologically, most abundant expression was observed in white matter tracts and thalamus. On the cellular level, aspartoacylase immunoreactivity is restricted to oligodendrocyte somata in both white and gray matter. NeuroReport 14 :1837^1840
Journal of Molecular Neuroscience, 1999
N-acetyl-l-aspartate (NAA) is an important osmolyte in the vertebrate brain and eye, and its cyclical metabolism is accomplished in two separate compartments. In the brain, NAA is synthesized primarily in neurons, and after its regulated release, NAA is hydrolyzed by aspartoacylase, which is present in a glial-associated compartment. However, the precise nature of this hydrolytic compartment has remained obscure. It has been proposed that one role of aspartoacylase in the central nervous system (CNS) is as part of a molecular water pump (MWP) that uses the NAA intercompartmental cycle to remove nerve cell metabolic water against a water gradient and that oligodendrocytes comprise the second compartment in this metabolic sequence. The absence of aspartoacylase activity in Canavan disease (CD), a rare early onset genetic spongiform leukodystrophy, is associated with CNS edema, intramyelinic swelling and a progressive loss of oligdendrocytes. In order to evaluate the MWP hypothesis and its possible relationship to the etiology of CD further, both oligodendrocytes and astrocytes obtained from neonatal rat brain were grown in culture and tested for the presence of aspartoacylase activity. The results of this study show for the first time that aspartoacylase activity is expressed only in oligodendrocytes. The meaning of this observation in understanding the function of the NAA metabolic cycle is discussed.
Myelin Lipid Abnormalities in the Aspartoacylase-Deficient Tremor Rat
Neurochemical Research, 2009
The high concentration of N-acetylaspartate (NAA) in neurons of the central nervous system and its growing clinical use as an indicator of neuronal viability has intensified interest in the biological function of this amino acid derivative. The biomedical relevance of such inquiries is highlighted by the myelin-associated pathology of Canavan disease, an inherited childhood disorder resulting from mutation of aspartoacylase (ASPA), the NAA-hydrolyzing enzyme. This enzyme is known to be localized in oligodendrocytes with bimodal distribution in cytosol and the myelin sheath, and to produce acetyl groups utilized in myelin lipid synthesis. Loss of this acetyl source in Canavan disease and rodent models such as the tremor rat are thought to account for the observed myelin deficit. This study was undertaken to further define and quantify the specific lipid abnormalities that occur as a result of ASPA deficit in the tremor rat. Employing mass spectrometry together with high performance thin-layer chromatography, we found that myelin from 28-day-old animals showed major reduction in cerebrosides (CB) and sulfatides (Sulf) with unsubstituted fatty acids, and equal if not greater changes in myelin from 7-month-old tremors. Cerebrosides with 2-hydroxyfatty acids showed little if any change at either age; Sulf with 2-hydroxyfatty acids showed no significant change at 28 days, but surprisingly a major increase at 7 months. Two species of phosphatidylcholine, 32:0 and 34:1, also showed significant increase, but only at 28 days. One form of phosphatidylethanolamine, PE36:1, was reduced a modest amount at both ages, whereas the plasmalogen form did not change. The dysmyelination that results from inactivation of ASPA is thus characterized by selective decreases as well as some increases in specific lipids. Keywords Tremor rat Á Myelin lipids Á N-acetylaspartate Á Axon-myelin interaction Á Myelin-localized enzymes Á Aspartoacylase Abbreviations ASPA Aspartoacylase BHT 2,6-di-tert-butyl-4-methylphenol C Chloroform CB Cerebrosides CNS Central nervous system EPG Ethanolamine phosphoglyceride HT Heterozygote Special issue article in honor of Dr. George DeVries. Fatty acid designations (e.g. 18:1) indicate carbon number and number of double bonds.
Canavan disease and the role of N-acetylaspartate in myelin synthesis
Molecular and Cellular Endocrinology, 2006
Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.15) due to mutations. ASPA releases acetate by deacetylation of N-acetylaspartate (NAA), a highly abundant amino acid derivative in the central nervous system. CD results in spongiform degeneration of the brain and severe psychomotor retardation, and the affected children usually die by the age of 10. The pathogenesis of CD remains a matter of inquiry. Our hypothesis is that ASPA actively participates in myelin synthesis by providing NAA-derived acetate for acetyl CoA synthesis, which in turn is used for synthesis of the lipid portion of myelin. Consequently, CD results from defective myelin synthesis due to a deficiency in the supply of the NAA-derived acetate. The demonstration of the selective localization of ASPA in oligodendrocytes in the central nervous system (CNS) is consistent with the acetate deficiency hypothesis of CD. We have tested this hypothesis by determining acetate levels and studying myelin lipid synthesis in the ASPA gene knockout model of CD, and the results provided the first direct evidence in support of this hypothesis. Acetate supplementation therapy is proposed as a simple and inexpensive therapeutic approach to this fatal disease, and progress in our preclinical efforts toward this goal is presented.
Fatty acid synthesizing enzymes intrinsic to myelin
Molecular Brain Research, 2003
A recent study showing incorporation of acetyl groups from neuronal N-acetylaspartate into myelin lipids suggested the presence of fatty acid synthesizing enzymes in myelin that utilize the acetyl groups liberated by myelin-associated aspartoacylase [J. Neurochem. 78 (2001) 736]. We report here detection of the fatty acid synthase (FAS) complex and acetyl-CoA carboxylase (ACC) in purified myelin. The activity of myelin FAS was approximately half that of cytosolic FAS and, unlike the latter, required detergent for activation. Intrinsic association of FAS with myelin was indicated by failure to remove the activity with NaCl or Na-taurocholate. Myelin-associated ACC was approximately 10% of cytosolic ACC in myelin isolated by gradient centrifugation, and this was reduced by half following osmotic shock; this suggested bimodal distribution of myelin ACC, some being loosely associated within inter-lamellar cytoplasmic spaces and the remainder more firmly associated in a manner that resists NaCl / Na-taurocholate treatments. These results, in combination with earlier findings, provide a possible mechanism for the observed incorporation of neuronal NAA acetyl groups into myelin lipids.