Aspa Gene Knockout In The Mouse

In order to study the pathophysiology of CD, a knockout mouse for CD has been generated. The knockout mouse was created by deleting 10 base pair of exon four of the murine ASPA gene (Fig. 1) (40). 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.

5.1. NAAG Levels in CD Mouse

N-acetylaspartylglutamate is an NAA-glutamate adduct that is synthesized primarily in neurons, is affected in patients with CD. The enzyme, glutamate carboxypeptidase II that cleaves NAAG into NAA and glutamate. In neurons, NAA and glutamate are converted into NAAG in the presence of ^-acetylaspartate-L-glutamate ligase (41,42). Patients with CD showed approximately twenty-one fold higher NAAG level in the urinary excretion (43,44). However, the knockout mouse brain for CD did not show any difference in the levels of NAAG or the hydrolyzing enzyme, NAALADase (Fig. 3). The ratio of NAAG/Cr. In the knockout mouse brain was 0.032 ± 0.003 compared to that in the wild type, 0.028 ± 0.004 (45). These studies in the knockout mouse brain suggest that ASPA deficiency does not affect NAAG. Therefore it is likely that NAAG seen in the urine of patients with CD may be due to some other factors and not by ASPA deficiency (CD). The activity of NAALADase has also been described in renal tissue (41,46). However, NAAG is located only in the central nervous system suggesting that NAALDase in peripheral tissue may function as house keeping enzyme (47).

5.2. Low Level of Glutamate in the CD Brain and Gene Expression Studies

In the central nervous system, the termination of chemical neurotransmission involves the rapid removal of neurotransmitter, such as glutamate, y-aminobutyric acid (GABA) from synapse by specific transport systems. Glutamate is generated from the hydrolysis of N-acetylaspartylglutamate (NAAG) by the enzyme, glutamate carboxy-peptidase II (NAALADase). The levels of NAAG and glutamate carboxy-peptidase II were found to be normal in the knockout mouse brain (45). Glutamate transporter, EAAT4 may have combined function of transporter and inhibitor glutamate receptor (48), is mainly located in the cerebellum and its concentrations increase with development (48,49) suggesting an important role of EAAT4 in the developing brain. Abnormal gene expression in the CD mouse brain is shown in Table 2. There is down-regulation of glutamate transporter, EAAT4, which may be associated with the cause for low glutamate level in the CD mouse brain. There is up-regulation of genes involved in glial activation, apoptosis, and inflammatory response and cell death (50).

5.3. Low GABA Levels in CD

Transport of glutamine from astrocytes to neurons is important for the synthesis of y-aminobutyric acid (GABA). The converted glutamate from glutamine, serves as a precursor for GABA (50). The glutamate product, GABA is lower in the CD mouse brain (40). GABA is a major inhibitory neurotransmitter. The low level of GABA seen in the CD mouse brain is possibly associated with the down-regulation of GABA receptor, GABRA6 (Table 1) to result abnormal cortical excitation. The enzyme associated with the glutamate-GABA pathway, succinyl semiladehyde dehydrogenase (SSADH) was reported with the mental retardation, hypotonia and seizures (52), symptoms also seen in patients with CD. However, the knockout mouse brain did not show any difference from the wild type (Table 3) suggesting that SSADH is not involved in the phenotype seen in CD (53).

Figure 3. Measurement of glutamate carboxypeptidase II (NAALADase) activity in the brain of knockout mouse for CD. Glutamate carboxypeptidase II activity in the whole brain, cerebrum, hypothalamus, cerebellum and brainstem of knockout mouse did not have significant difference compared to that in the wild type.

Figure 3. Measurement of glutamate carboxypeptidase II (NAALADase) activity in the brain of knockout mouse for CD. Glutamate carboxypeptidase II activity in the whole brain, cerebrum, hypothalamus, cerebellum and brainstem of knockout mouse did not have significant difference compared to that in the wild type.

Table 2: Aspartoacylase gene knockout leading to abnormal gene expression in the brain of Canavan mouse.

Gene Bank

Genes

Expression

accession no.

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