Chikkathur N. Madhavarao

Immunohistochemical Localization of Aspartoacylase in the Rat Central Nervous System

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 cell‐specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (∼37 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/biotin‐peroxidase 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. J. Comp. Neurol. 472:318–329, 2004. Published 2004 Wiley‐Liss, Inc.

Characterization of the N‐acetylaspartate biosynthetic enzyme from rat brain

Aspartate N‐acetyltransferase (Asp‐NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp‐NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp‐NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size‐exclusion high performance liquid chromatography. Asp‐NAT activity and the optical density pattern of the eluted protein from size‐exclusion column indicated a single large protein (∼670 kDa), which on sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi‐subunit complex Asp‐NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3‐[(3‐cholamidopropyl)dimethylammonia]‐1‐propanesulfonate (CHAPS) treatment followed by size‐exclusion chromatography. Asp‐NAT showed an order of magnitude difference in Km between l‐aspartate (l‐Asp, ∼0.5 mm) and acetyl CoA (∼0.05 mm). Asp‐NAT showed high specificity towards l‐Asp with 3% or less activity towards l‐Glu, l‐Asn, l‐Gln and Asp‐Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.

Methamphetamine-induced neuronal protein NAT8L is the NAA biosynthetic enzyme: implications for specialized acetyl coenzyme A metabolism in the CNS.

N-acetylaspartate (NAA) is a concentrated, neuron-specific brain metabolite routinely used as a magnetic resonance spectroscopy marker for brain injury and disease. Despite decades of research, the functional roles of NAA remain unclear. Biochemical investigations over several decades have associated NAA with myelin lipid synthesis and energy metabolism. However, studies have been hampered by an inability to identify the gene for the NAA biosynthetic enzyme aspartate N-acetyltransferase (Asp-NAT). A very recent report has identified Nat8l as the gene encoding Asp-NAT and confirmed that the only child diagnosed with a lack of NAA on brain magnetic resonance spectrograms has a 19-bp deletion in this gene. Based on in vitro Nat8l expression studies the researchers concluded that many previous biochemical investigations have been technically flawed and that NAA may not be associated with brain energy or lipid metabolism. In studies done concurrently in our laboratory we have demonstrated via cloning, expression, specificity for acetylation of aspartate, responsiveness to methamphetamine treatment, molecular modeling and comparative immunolocalization that NAT8L is the NAA biosynthetic enzyme Asp-NAT. We conclude that NAA is a major storage and transport form of acetyl coenzyme A specific to the nervous system, thus linking it to both lipid synthesis and energy metabolism.

Canavan disease and the role of N-acetylaspartate in myelin synthesis

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.

Nuclear-cytoplasmic localization of acetyl coenzyme A synthetase-1 in the rat brain

Acetyl coenzyme A synthetase‐1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. By using an affinity‐purified antibody generated against an 18‐mer peptide sequence of AceCS1 and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons, and oligodendrocytes. Some nonneuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18‐day‐old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially up‐regulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons. J. Comp. Neurol. 518:2952–2977, 2010.

Aspartoacylase is a regulated nuclear-cytoplasmic enzyme

Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N‐acetyl‐L‐aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes. Immunohistochemistry showed cytoplasmic and nuclear ASPA staining, the specificity of which was demonstrated by its absence from tissues of the Tremor rat, an ASPA‐null mutant. Subcellular fractionation analysis revealed low enzyme activity against NAA in nuclear fractions from normal rats. Whereas two recent reports have indicated that ASPA exists as a dimer, size‐exclusion chromatography of subcellular fractions showed ASPA is an active monomer in both subcellular fractions. Western blotting detected ASPA as a single 38 kD band. Because ASPA is small enough to passively diffuse into the nucleus, we constructed, expressed, and detected in COS‐7 cells a green fluorescent protein‐human ASPA (GFP‐hASPA) fusion protein larger than the permissible size for the nuclear pore complex. GFP‐hASPA was enzymatically active and showed mixed nuclear‐cytoplasmic distribution. We conclude that ASPA is a regulated nuclear‐cytoplasmic protein that may have distinct functional roles in the two cellular compartments.—Hershfield, J. R., Madhavarao, C. N., Moffett, J. R., Benjamins, J. A., Garbern, J. Y., Namboodiri, A. Aspartoacylase is a regulated nuclear‐cytoplasmic enzyme. FASEB J. 20, E1482–E1494 (2006)

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N‐acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N‐acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH‐SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2′‐O‐dibutyryl cAMP) and protein kinase C (PMA; phorbol‐12‐myristate‐13‐acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l‐[3H]glutamine, whereas PMA‐induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l‐[3H]glutamine, suggesting precursor–product and mass–action relationships between NAA and NAAG. Incorporation of l‐[3H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l‐[3H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l‐[3H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l‐[3H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.

N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes

Several reports during the last three decades have indicated that biosynthesis of N-acetylaspartate (NAA) occurs primarily in the mitochondria. But a recent report by Lu et al. in this journal [2004; 122: 71-78] and subsequent two reports that cited those data suggested a predominant microsomal localization of the NAA biosynthetic enzyme, which is surprising in view of what is known about the biological functions of NAA. Therefore we reinvestigated this issue in rat brain homogenates using a similar fractionation procedure used by Lu et al. but without the loss of enzyme activity that they have encountered. We found that about 70% of the total Asp-NAT activity in the crude supernatant was present in the mitochondrial fraction which is about 5 times more than that in the microsomes. We found similar results in the case of the enzyme from bovine brain. In subsequent studies, we also have found that Asp-NAT activity in the bovine brain is very similar to that in the rat brain in substrate specificity and chromatographic characteristics including the high molecular weight pattern (approx. 670 kD) on size-exclusion HPLC.

Mutational analysis of aspartoacylase : Implications for Canavan Disease

Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells.

N‐Acetylaspartate (NAA) and N‐acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D2 receptors. NAAG is reported to be an agonist of the metabotropic glutamate 2/3 receptor, which is linked to neurotransmitter release modulation, including glutamate release. Alterations in NAAG metabolism have been implicated in the development of schizophrenia possibly via dysregulation of glutamate neurotransmission. In the present study we have used high performance liquid chromatography to determine the effects of the antipsychotic drugs haloperidol and clozapine on NAA and NAAG levels in SH‐SY5Y human neuroblastoma cells, a model system used to test the responses of dopaminergic neurons in vitro. The results indicate that the antipsychotic drugs haloperidol and clozapine increase both NAA and NAAG levels in SH‐SY5Y cells in a dose and time dependant manner, providing evidence that NAA and NAAG metabolism in neurons is responsive to antipsychotic drug treatment.