Aspartoacylase Deficiency In Cd

Aspartoacylase is one of two amino acylases, aminoacylase I hydrolyses acetate from other aminoacids. Aspartoacylase (aminoacylase II or ASPA) is specific for N-acetylaspartic acid (NAA), which is hydrolyzed to aspartate and acetate (4). Aspartoacylase is localized in the oligodendrocytes, with little or no enzyme activity in astrocytes for the oligodendrocyte participation in the formation of a myelin sheath (31). Deficiency of the enzyme results in accumulation of NAA in the brain, causing spongy degeneration. Aspartoacylase deficiency can also be determined in peripheral organs such as lungs and kidneys (14). Aspartoacylase activity in the brain increases with age as mylination progresses (32).

The substrate for ASPA, NAA, is synthesized from acetyl-coenzyme A and aspartate by L-aspartate N-acetyltransferase (33). N-Acetylaspartic acid may be involved in osmotic regulation, neuromodulation and lipogenesis during myelination (34). The rapid increase of the NAA in the brain during the active myelination, and the localization of ASPA in oligodendrocytes support a roll for NAA in the myelination process (35,36). Deficiency of ASPA with severe myelin disease further indicates the importance of NAA in promoting intake myelin metabolism.

Canavan Naa Metabolism

Figure 1. Targeted disruption of the murine aspartoacylase gene. (A). Map of the murine ASPA gene, the targeting vector (pKOS/ASPA/62) restriction enzyme cutting sites. The position of a 400 bp sized probe 5' from the targeting vector and the position of 600 bp sized probe 3' from the targeting vector are shown. The probes were generated by PCR using 12/13 and 29/21 primer pairs. The numbered boxes denote exons, however exon six is not shown in the figure. A targeting vector for deleting 10 bp from exon 4 was engineered. B. Southern blot analysis of Bgll digested genomic DNA from F1 intercross progeny using the PCR generated 5' probe. C. Southern blot analysis of EcoRI digested genomic DNA from F1 intercross progeny using the PCR generated 3' probe. The homozygous knockout mouse (Fig 2) showed pathophysiology similar observed in patients with CD including ASPA deficiency, NAA accumulation in the brain, excessive urinary NAA excretion (Table 1) and spongy degeneration of the brain.

Figure 1. Targeted disruption of the murine aspartoacylase gene. (A). Map of the murine ASPA gene, the targeting vector (pKOS/ASPA/62) restriction enzyme cutting sites. The position of a 400 bp sized probe 5' from the targeting vector and the position of 600 bp sized probe 3' from the targeting vector are shown. The probes were generated by PCR using 12/13 and 29/21 primer pairs. The numbered boxes denote exons, however exon six is not shown in the figure. A targeting vector for deleting 10 bp from exon 4 was engineered. B. Southern blot analysis of Bgll digested genomic DNA from F1 intercross progeny using the PCR generated 5' probe. C. Southern blot analysis of EcoRI digested genomic DNA from F1 intercross progeny using the PCR generated 3' probe. The homozygous knockout mouse (Fig 2) showed pathophysiology similar observed in patients with CD including ASPA deficiency, NAA accumulation in the brain, excessive urinary NAA excretion (Table 1) and spongy degeneration of the brain.

The central nervous system contains high level of NAA (36). The concentration of NAA in the human fetus brain in utero is approximately 2.5^ol/g (36), while in the normal adult human brain the level varies 8-10 ^ol/g (36-39). The accumulation in the brain of NAA due to ASPA deficiency, and the efflux of this compound form the brain, leads to elevated urinary NAA. Thus, in Canavan disease, patients had urine NAA 1440.5 ± 873.3 ^mol/mmol Cr in contrast to 23.5 ± 16.1 ^ol/mmol Cr in normal subjects (3,14). Therefore, urinary NAA is one of the markers to determine CD. However, mild elevation of NAA with macrocephaly must not be predicted as Canavan disease, unless spongy degeneration of the brain is confirmed (30).

Figure 2. The knockout mouse reflects Canavan's phenotype

Table 1. Aspartoacylase activity and NAA levels in the homozygous knockout mouse and wild type.

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