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.

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-[(3)H]glutamine, whereas PMA-induced differentiation reduced both.

Progress toward acetate supplementation therapy for Canavan disease: glyceryl triacetate administration increases acetate, but not N-acetylaspartate, levels in brain.

Canavan disease (CD) is a fatal genetic neurodegenerative disorder caused by mutations in the gene for aspartoacylase, an enzyme that hydrolyzes N-acetylaspartate (NAA) into L-aspartate and acetate. Because aspartoacylase is localized in oligodendrocytes, and NAA-derived acetate is incorporated into myelin lipids, we hypothesize that an acetate deficiency in oligodendrocytes is responsible for the pathology in CD, and we propose acetate supplementation as a possible therapy. In our preclinical efforts toward this goal, we studied the effectiveness of orally administered glyceryl triacetate (GTA) and calcium acetate for increasing acetate levels in the murine brain.

Rapid sodium cyanide depletion in cell culture media: outgassing of hydrogen cyanide at physiological pH.

During the course of in vitro studies on cyanide exposure with SH-SY5Y human neuroblastoma cells, we found that sodium cyanide (NaCN) up to a concentration of 10 mM had no significant toxic effect under our culture conditions. Further investigation of this apparent cyanide resistance revealed that the sodium cyanide was being rapidly depleted from the cell culture medium. Cyanide was interacting with constituents of the cell culture medium and was somehow being detoxified or removed from solution.

Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease.

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.

SH-SY5Y neuroblastoma cells: a model system for studying biosynthesis of NAAG.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide that is thought to modulate neurotransmitter release through pre-synaptic mGluR3 receptors. Despite years of research into NAAG biochemistry, almost nothing is known about NAAG biosynthesis. To date, NAAG biosynthesis has only been demonstrated conclusively in explanted animal neural tissues, including frog retina, rat dorsal root ganglia and crayfish nerve cord, but not in human cells or tissues.

Immunohistochemical localization of aspartoacylase in the rat central nervous system.

Aspartoacylase (ASPA; EC 3.5.1.

Localization and Synaptic Release of N-acetylaspartylglutamate in the Chick Retina and Optic Tectum.

The neuropeptide, N-acetylaspartylglutamate (NAAG), was identified in the chick retina (1.4 nmol/retina) by HPLC, radioimmunoassay and immunohistochemistry. This acidic dipeptide was found within retinal ganglion cell bodies and their neurites in the optic fibre layer of the retina.