Aspartoacylase Deficiency in the White Matter of Human Immunodeficiency Virus Encephalitis: Novel Mechanism in Axonal Damage (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.
Bimodal occurrence of aspartoacylase in myelin and cytosol of brain
Journal of Neurochemistry, 2007
The growing use of N-acetylaspartate as an indicator of neuronal viability has fostered interest in the biological function(s) of this unusual amino acid derivative. In considering the various physiological roles that have been proposed for this relatively abundant molecule one is obliged to take into account its unusual metabolic compartmentalization, according to which synthesis and storage occur in the neuron and hydrolytic cleavage in the oligodendrocyte. The latter reaction, catalyzed by aspartoacylase (ASPA), produces acetyl groups plus aspartate and has been proposed to occur in both soluble and membranous subfractions of white matter. Our study supports such bimodal occurrence and we now present immunoblot, proteomic, and biochemical evidence that the membrane-bound form of ASPA is intrinsic to purified myelin membranes. This was supported by a novel TLC-based method for the assay of ASPA. That observation, together with previous demonstrations of numerous lipid-synthesizing enzymes in myelin, suggests utilization of acetyl groups liberated by myelin-localized ASPA for lipid synthesis within the myelin sheath. Such synthesis might be selective and could explain the deficit of myelin lipids in animals lacking ASPA.
Neurobiology of Disease, 2005
Canavan disease (CD) is a neurodegenerative disorder characterized by the spongy degeneration of the white matter of the brain. Aspartoacylase (ASPA) gene mutation resulting enzyme deficiency is the basic cause of CD. Whether the ASPA defect in CD affects the spinal cord has been investigated using the ASPA gene knockout mouse. Luxol fast blue-hematoxylin and eosin staining in the spinal cord of the knockout mouse showed vacuolation in both white matter and gray matter areas of cervical, thoracic, lumbar, and sacral segments of the spinal cord. However, more vacuoles were seen in the gray matter than the white matter of the spinal cord. ASPA activity in the cervical, thoracic, lumbar, and sacrococcygeal regions of the spinal cord was significantly lower in the knockout mouse compared to the wild type. The enzyme defect in the knockout mouse was also confirmed using the Western blot method. These observations suggest that the ASPA gene defect in the mouse leads to spinal cord pathology, and that these changes may be partly involved in the cause of the physiological/behavioral abnormalities seen in the knockout mouse, if documented also in patients with CD.
Acta Neuropathologica, 1990
The encephalopathy resulting from direct infection of the brain by human immunodeficiency virus (HIV), which correlates clinically with the AIDS dementia complex, has been reported as being localized to the white matter where it induces myelin loss, gliosis and perivascular infiltration by mononuclear macrophages and multinucleated giant cells. Damage to the cortical grey matter in HIV encephalopathy was investigated in nine randomly selected HIV-positive cases with or without clinical or morphological evidence of encephalopathy and in five age-matched controls, using routine histology and immunohistochemical methods [glial fibrillary acidic protein (GFAP), microglia and HIV antibodies]. Increased numbers of GFAP-expressing astrocytes and Ricinus communis agglutinin 1-120-expressing microglial cells were found in all the HIV-positive cases (including asymptomatic) and their severity could be correlated with the severity of the encephalopathy in the white matter; the increase in number of cells expressing GFAP was diffuse and the intensity of the staining higher than that of microglial cells. The subpial region was the most severely involved. It is suggested that involvement of the cortical grey matter is more common in HIV infection than previously suspected and that clinical evidence of a dementing process in AIDS is not necessarily due only to white matter lesions.
Characterization of Human Aspartoacylase: The Brain Enzyme Responsible for Canavan Disease †
Biochemistry, 2006
Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coliexpressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.
Molecular Therapy, 2005
Canavan disease is an early onset leukodystrophy associated with psychomotor retardation, seizures, and premature death. This disorder is caused by mutations in the gene encoding the enzyme aspartoacylase (ASPA). Normally, ASPA is enriched in oligodendrocytes and ASPA deficiency results in elevated levels of its substrate molecule, N-acetylaspartate (NAA), brain edema, and dysmyelination. Using adeno-associated virus, we permanently expressed ASPA in CNS neurons of the tremor rat, a genetic model of Canavan disease, and examined the efficacy of the treatment by monitoring NAA metabolism, myelination, motor behavior, and seizures. Assessment of ASPA protein and enzyme activity in whole brain hemispheres showed restoration to normal levels as long as 6 months after treatment. This finding correlated with a reduction of NAA levels, along with a rescue of the seizure phenotype. However, gross brain pathology, such as dilated ventricles and spongiform vacuolization, was unchanged. Moreover, hypomyelination and motor deficits were not resolved by ASPA gene transfer. Our data suggest that NAA-mediated neuronal hyperexcitation but not oligodendrocyte dysfunction can be compensated for by neuronal ASPA expression.
Neuropathogenesis of human immunodeficiency virus infection
Seminars in Neuroscience, 1991
In general, the acquired immunodeficiency syndrome (AIDS) is considered to be a disease of the immune system ; however, other organ systems, including the nervous system, are targets • the human immunodeficiency virus (HIV). It is now well established that infection with HIV frequently results in a wide range of degenerative neurological abnormalities affecting the nervous system in many AIDS patients. A wide body • experimental evidence indicates that HIV is present in the nervous system of AIDS patients with neurological complications, thus implicating HIV in the etiology of HIVassociated neurological dysfunction. This may occur either as a result of direct HIV infection of glia and possibly neurons or because of indirect mechanisms mediated by the toxic action • viral or cellular products in the nervous system; however, the exact pathogenic mechanisms leading to HIV-associated neurological dysfunction remain obscure. Key words : human immunodeficiency virus /virus latency / developing human nervous system / neurotoxicity / cytokines / autoimmunity Clinical ramifications of HIV neuropathogenesis AIDS dementia complex is the most frequent and important cause of neurological morbidity in patients with AIDS and it is estimated that 30-40% of adults at the time of diagnosis of AIDS exhibit clinical features of AIDS dementia complex. 1,2 In general, patients with AIDS dementia complex present with an array of abnormalities in cognitive, motor and behavioral functions. As the disease progresses, a dementia accompanied by subacute encephalitis, vacuolar spinal cord degeneration and meningitis are often observed (Figure 1) and, ultimately, nearly all aspects of cognition, motor function and behavior are affected. 2 The course of dementia is variable
Pediatric Research, 1993
In this study, we tested the hypothesis that human immunodeficiency virus (H1V)-1-derived peptides augment the neurotoxicity of excitatory amino acid agonists in vivo in postnatal day (PND) 7 rats. Stereotaxic intracerebral injections of the excitatory amino acid agonist N-methyl-maspartate (NMDA), alone or coinjected with an HIV-derived recombinant fusion peptide envelope gag (env-gag) were performed in PND-7 rats [group I: 5 nmol NMDA, n = 20; group 11: 5 nmol NMDA + low-dose (1 or 50 ng) env-gag, n = 27; group 111: 5 nmol NMDA + high-dose (100 ng) env-gag, n = 201, and brain injury was evaluated on PND 12. Based on histopathology scoring and measurements of hippocampal cross-sectional areas in the injected and contralateral hemispheres, coinjection of 100 ng of env-gag with 5 nmol of NMDA markedly increased the severity of resulting injury (p < 0.002, comparing histopathology scores; p c 0.003, comparing interhemispheric differences in hippocampal areas). These data suggest that in the developing nervous system HIV neurotoxicity may result, at least in part, from overactivation of excitatory amino acid receptors, and that perinatal rodent models may provide clinically relevant insights about the pathophysiology of HIV-mediated brain injury. (Pediatr Res 34: 192-198, 1993) Abbreviations EAA, excitatory amino acid HIV, human immunodeficiency virus NMDA, N-methyl-maspartate env-gag, envelope-gag PND, postnatal day Current estimates suggest that 25 to 40% of infants born to HIV-infected women develop clinical and/or serologic evidence of HIV infection in the first year of life (l), and progressive encephalopathy is a prominent and devastating complication of congenital HIV infection (2). Relatively little is known about the cellular and molecular mechanisms that cause neuronal injury
Biochemistry, 2008
Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-L-aspartate. Canavan disease (CD) 1 is a fatal autosomal recessive disorder that affects the central nervous system (1) and for which there is currently no effective treatment. The symptoms for CD can be noticed as early as 3-6 months of age and include rapid increase of the head circumference (megalocephaly), lack of head control, reduced visual responsiveness, abnormal muscle tone (i.e., floppiness or stiffness), and mental retardation (2). CD patients usually do not live past the first decade of their lives. Canavan disease is caused by a defect in the capability of the brain to metabolize N-acetyl-L-aspartate (NAA) (3), one of the most abundant amino acids in the brain (4). Aspartoacylase, whose function is to catalyze the deacetylation of NAA (Figure 1), is located primarily in the white matter of the brain, more specifically in the cytosol of the oligodendrocytes (5). Analysis of the acy2 gene that encodes aspartoacylase taken from DNA isolated from CD patients has revealed more than 50 different mutations, including numerous deletions, missense mutations, and premature terminations (6). In most cases, the missense mutations result in nonconservative amino acid substitutions, leading to an altered enzyme that either is not expressed at all or is expressed but has little or no catalytic activity (6). Aspartoacylase was originally proposed to be member of the esterase family since it was shown to be inactivated by diisopropylfluorophosphate, a classic serine protease inactivator (7). On † This work was supported by a grant from the National Institutes of Health (NS45664), and C.X. was supported by Grant GM71790. ‡ The atomic coordinates for apo-aspartoacylase (2O53) and the tetrahedral intermediate analogue complex of human aspartoacylase (2O4H) have been deposited at the RCSB Protein Data Bank.