A radiometric assay for aspartoacylase activity in cultured oligodendrocytes

doi:10.1016/S0003-2697(02)00225-7

Analytical Biochemistry

Volume 308, Issue 2, 15 September 2002, Pages 314-319

https://doi.org/10.1016/S0003-2697(02)00225-7 Get rights and content

Abstract

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [¹⁴C]N-acetylaspartate (NAA) from l-[¹⁴C]Asp; (b) use of [¹⁴C]NAA as substrate in the assay; and (c) separation and quantitation of the product l-[¹⁴C]Asp using a TLC system. This method can detect as low as 10 pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.

Section snippets

Materials

Solvents and unlabeled l-Asp were bought from Sigma (St.Louis, MO). Tris-HCl buffer, pH 8.0, from Digene (Beltsville, MD) and CaCl₂ from Quality Biological (Gaithersberg, MD). l-[¹⁴C]Asp and l-[¹⁴C]Glu (specific activity of 207 mCi/mmol) were purchased from Amersham Pharmacia Biotech. Aluminum-supported flexible thin-layer chromatography (TLC) plates were purchased from Whatman Ltd, (Maidstone, Kent, England). Phosphor image exposure cassettes were bought from Molecular Dynamics. All other

Results and discussion

Fig. 1 shows the phosphor image of the TLC separation of the substrates [¹⁴C]NAA and [¹⁴C]NAG (Rf, 0.8) and their corresponding products l-[¹⁴C]Asp (Rf, 0.3) and l-[¹⁴C]Glu (Rf, 0.5). We have found that increasing the percentage of methanol in the solvent up to 30% increased the Rf in all cases, thus clearly separating both products from origin and also increasing the absolute separation between l-Asp and l-Glu from NAA and NAG, respectively. Comparison of the enzyme activity using the

Acknowledgements

This work was supported by a RO1 grant from NIH (NS39387) to M.A.A.N.

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N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange column to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using l-aspartate oxidase. Generated H₂O₂ is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 μM (10 pmol/well) and 3.3 μM (33 pmol/well), respectively, with a linear range to 100 μM. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (−10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.
Therapeutic development for Canavan disease using patient iPSCs introduced with the wild-type ASPA gene
2022, iScience
Canavan disease (CD) is a devastating neurological disease that lacks effective therapy. Because CD is caused by mutations of the aspartoacylase (ASPA) gene, we introduced the wild-type (WT) ASPA gene into patient iPSCs through lentiviral transduction or CRISPR/Cas9-mediated gene editing. We then differentiated the WT ASPA-expressing patient iPSCs (ASPA-CD iPSCs) into NPCs and showed that the resultant ASPA-CD NPCs exhibited potent ASPA enzymatic activity. The ASPA-CD NPCs were able to survive in brains of transplanted CD mice. The engrafted ASPA-CD NPCs reconstituted ASPA activity in CD mouse brains, reduced the abnormally elevated level of NAA in both brain tissues and cerebrospinal fluid (CSF), and rescued hallmark pathological phenotypes of the disease, including spongy degeneration, myelination defects, and motor function impairment in transplanted CD mice. These genetically modified patient iPSC-derived NPCs represent a promising cell therapy candidate for CD, a disease that has neither a cure nor a standard treatment.
Non-genetic therapeutic approaches to Canavan disease
2016, Journal of the Neurological Sciences
Citation Excerpt :
However, it is not yet known how triheptanoin supplies substrates to oligodendrocytes, but triheptanoin was chosen based on its ability to produce TCA cycle intermediates while also providing substrates for lipogenesis. Since oligodendrocytes are an important source of CNS ASPA [71], if not the most important [34–36,72–74], repopulation of the brain of a CD patient with functional oligodendrocytes could potentially rectify much of the disease phenotype through the restoration of normal NAA metabolism. Neural stem cells have been successfully used to generate oligodendrocytes in the CD mouse model [75] and these oligodendrocytes expressed a myelin-specific enzyme indicative of myelin-producing ability, but clinical outcomes of such a treatment have yet to be investigated.
Canavan disease (CD) is a rare leukodystrophy characterized by diffuse spongiform white matter degeneration, dysmyelination and intramyelinic oedema with consequent impairment of psychomotor development and early death. The molecular cause of CD has been identified as being mutations of the gene encoding the enzyme aspartoacylase (ASPA) leading to its functional deficiency. The physiological role of ASPA is to hydrolyse N-acetyl-l-aspartic acid (NAA), producing l-aspartic acid and acetate; as a result, its deficiency leads to abnormally high central nervous system NAA levels. The aim of this article is to review what is currently known regarding the aetiopathogenesis and treatment of CD, with emphasis on the non-genetic therapeutic strategies, both at an experimental and a clinical level, by highlighting: (a) major related hypotheses, (b) the results of the available experimental simulatory approaches, as well as (c) the relevance of the so far examined markers of CD neuropathology. The potential and the limitations of the current non-genetic neuroprotective approaches to the treatment of CD are particularly discussed in the current article, in a context that could be used to direct future experimental and (eventually) clinical work in the field.
Evidence for mitochondrial and cytoplasmic N-acetylaspartate synthesis in SH-SY5Y neuroblastoma cells
2009, Neurochemistry International
N-acetylaspartate (NAA) is synthesized predominantly in neurons, and brain homogenate subfractionation studies suggest that biosynthesis occurs at both microsomal (cytoplasmic) and mitochondrial sites. We investigated NAA synthesis in SH-SY5Y human neuroblastoma cells using distinct metabolic precursors that are preferentially metabolized in mitochondria and cytoplasm. Incorporation of ¹⁴C-aspartate and ¹⁴C-malate into NAA was examined in the presence and absence of an inhibitor (aminooxyacetic acid, AOAA) of aspartate aminotransferase (AAT), a central enzyme involved in the biosynthesis of aspartate in mitochondria, and degradation of aspartate in the cytoplasm. AOAA increased the incorporation of ¹⁴C-aspartate into NAA, reflecting direct aspartate → NAA synthesis in an extramitochondrial location. As expected, AOAA decreased incorporation of ¹⁴C-malate into NAA, reflecting NAA synthesis in mitochondria via the malate → oxaloacetate → aspartate → NAA pathway. When ¹⁴C-malate was used as substrate, the ¹⁴C-NAA/¹⁴C-aspartate ratio was over 20-fold higher than the ratio obtained with ¹⁴C-aspartate. Because NAA can only be synthesized from aspartate, the higher ¹⁴C-NAA/¹⁴C-aspartate (product/substrate) ratio obtained with ¹⁴C-malate suggests greater NAA biosynthetic activity. We conclude that NAA biosynthesis occurs in both the cytoplasm and mitochondria of SH-SY5Y cells, and that the contribution from the mitochondrial compartment is quantitatively larger than that in the extramitochondrial compartment.
N-acetylaspartate synthesis in the brain: Mitochondria vs. microsomes
2008, Brain Research
Citation Excerpt :
Protein standards also were chromatographed under similar conditions to ascertain the approximate molecular weight of the eluted enzyme. A radiometric assay with quantitation of the product by TLC-phosphor imaging technique as described earlier (Madhavarao et al., 2002, 2003) was followed with minor modifications. In brief, the assay was performed in same buffer {sorbitol phosphate buffer (pH 7.0) or PBS glycerol (pH 7.2)} containing [14C] l-Asp and unlabelled l-Asp at 1.06 mM with specific activity of ~ 6.7 mCi/mmol (13.5 × 106 CPM) and 1.0 mM acetyl CoA.
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
2007, Brain Research
Citation Excerpt :
Most mutations in this report yielded undetectable ASPA activity using a TLC-based assay and a fixed concentration of NAA near the published Km value (Namboodiri et al., 2000). Previous reports using different ASPA enzyme assays sometimes detected residual activity for various CD-associated ASPA mutations, likely as a result of longer enzyme–substrate incubations, higher substrate concentrations, and inherent background activity that is absent from the TLC-based assay (Madhavarao et al., 2002). With a partial homology model and a hypothetical catalytic mechanism for ASPA-mediated NAA hydrolysis, we can offer explanations for how several CD-associated mutations result in a loss of activity in vivo.
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.

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A radiometric assay for aspartoacylase activity in cultured oligodendrocytes

Abstract

Section snippets

Materials

Results and discussion

Acknowledgements

J. Biol. Chem.

J. Pediatr.

Mol. Brain. Res.

Mol. Brain Res.

Prog. Neurobiol.

A radiometric assay for aspartoacylase activity in human fibroblasts: application for the diagnosis of Canavan’s disease

Clin. Chim. Acta

Expression of aspartoacylase activity in cultured rat macroglial cells is limited to oligodendrocytes

J. Mol. Neurosci.

Prenatal diagnosis of Canavan disease-problems and dilemmas

J. Inherit. Metab. Dis.

Developmental and regional distribution of aspartoacylase in rat brain tissue

J. Neurochem.

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase

J. Neurochem.