www.elsevier.com/locate/brainresBrain Research 1016 (2004) 268–271

Short communication

Aspartoacylase deficiency does not affect N-acetylaspartylglutamate level

or glutamate carboxypeptidase II activity in the knockout mouse brain

Sankar Surendrana, Edward L. Ezellb, Michael J. Quastc, Jingna Weic, Stephen K. Tyringd,Kimberlee Michals-Matalona, Reuben Matalona,*

aDepartment of Pediatrics, Childrens Hospital, The University of Texas Medical Branch, Rm# 3.350, Galveston, TX 77555-0359, USAbDepartment of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, UTMB, Galveston, TX, USA

cDepartment of Neurosciences and Cell Biology, UTMB, Galveston, TX, USAdDepartment of Dermatology, University of Texas Health Science Center, Houston, TX, USA

Accepted 10 May 2004

Available online 19 June 2004

Abstract

Aspartoacylase (ASPA)-deficient patients [Canavan disease (CD)] reportedly have increased urinary excretion of N-acetylaspartylglu-

tamate (NAAG), a neuropeptide abundant in the brain. Whether elevated excretion of urinary NAAG is due to ASPA deficiency, resulting in

an abnormal level of brain NAAG, is examined using ASPA-deficient mouse brain. The level of NAAG in the knockout mouse brain was

similar to that in the wild type. The NAAG hydrolyzing enzyme, glutamate carboxypeptidase II (GCP II), activity was normal in the

knockout mouse brain. These data suggest that ASPA deficiency does not affect the NAAG or GCP II level in the knockout mouse brain, if

documented also in patients with CD.

D 2004 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: other

Keywords: Canavan disease; N-Acetylaspartylglutamate; Glutamate carboxypeptidase II; ASPA gene knockout mouse

Canavan disease (CD) is an autosomal recessive disorder in patients with CD [18]. The knockout mouse brain has

caused by aspartoacylase (ASPA) deficiency [16]. This

enzyme hydrolyzes N-acetylaspartic acid (NAA) to acetate

and aspartate [7]. While ASPA is localized in oligodendro-

cytes [4], the substrate, NAA, is restricted to neurons in the

gray matter of the brain [14,26]. Deficiency of the enzyme

results in the accumulation of NAA in the brain and elevated

NAA excretion in the urine of patients with CD [16,17].

Abnormalities in the brain of patients with CD include

spongy degeneration with swollen astrocytes and elongated

mitochondria [1–3,10,13]. Patients with CD show mental

retardation, macrocephaly, hypotonia and head lag [29].

The knockout mouse for CD shows neurological impair-

ment and neuropathology similar to what has been observed

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.05.035

* Corresponding author. Tel.: +1-409-772-3466; fax: +1-409-772-

9595.

E-mail address: rmatalon@utmb.edu (R. Matalon).

vacuolation throughout the white matter in the deep cortex

and the white matter bundles in the corpus striatum [18].

The hippocampus has vacuolation in the pyramidal area and

sparing of the dentate gyrus. The Purkinje cell layer,

granular layer and white matter of the cerebellum are also

affected in the knockout mouse [18].

Deficiency of ASPA results in the accumulation of NAA

in the brain of knockout mice as observed in patients with

CD [18]. The levels of glutamate and glutamate-GABA

pathway-associated enzymes aspartate aminotransferase,

glutamate dehydrogenase and a-ketoglutarate dehydroge-

nase [21–23,25] are lower in the knockout mouse brain. In

neurons, NAA and glutamate are ligated into N-acetylas-

partylglutamate (NAAG) in the presence of N-acetylaspar-

tate-L-glutamate ligase [28]. The acidic peptide NAAG is

most prevalent in the brain, with a high affinity for metab-

otropic glutamate receptors, mGluR3 [12,15]. NAAG is

cleaved into NAA and glutamate by glutamate carboxypep-

Fig. 1. A representative 400 MHz proton NMR spectrum from a knockout

mouse brain showing the spectral region where the NAA and NAAG

methyl protons resonate. The NAAG methyl peak is resolved from the

NAA methyl.

Fig. 2. Measurement of glutamate carboxypeptidase II 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. (n= 10F S.E).

S. Surendran et al. / Brain Research 1016 (2004) 268–271 269

tidase II (GCPII)/N-acetylated-a-linked acidic dipeptidase

(NAALADase) [6,11,20]. While GCP II is located in

astrocytes, NAAG is restricted to neurons [15,19].

Patients with CD showed elevated excretion of urinary

NAAG [8,9]. Because ASPA deficiency is the basic cause of

CD [16], it is unclear whether the enzyme defect results in

the accumulation or the depletion of brain NAAG to cause

elevated excretion of urinary NAAG. To investigate the

possibility, the NAAG level was estimated in the brain of

ASPA-deficient mouse. In addition, the NAAG hydrolyzing

enzyme, GCPII, activity was also measured.

The brain content of NAAG was determined using

nuclear magnetic resonance (NMR) spectral analysis as

followed earlier [21,25]. All animal procedures were ap-

proved by the Institution’s Animal Care and Use Commit-

tee. Brain samples from wild type and knockout mice were

frozen in liquid nitrogen, crushed and then were placed in a

12% solution of perchloric acid for overnight extraction at 4

jC. Following perchloric extraction, samples were centri-

fuged at 10,000� g for 10 min at 4 jC. Supernatants wereneutralized, lyophilized and redissolved in D2O. High

resolution, 400 or 750 MHz proton NMR spectra were

run on the supernatants. Measurements were performed on

Varian Unity-plus spectrometers using water-suppressed

proton NMR spectroscopy. The NMR parameters for the

single pulse experiments were: TR = 10 s; acquisition

time = 3 s; saturation delay = 2 s; and signal averages = 128.

The residual water peak was set to 4.70 ppm. The peak

integral for the NAAG methyl protons (singlet, 2.04 ppm)

was compared to the creatine methyl (singlet, 3.04 ppm) to

calculate relative metabolite concentrations.

To perform the GCP II assay, wild type and knockout

mice were sacrificed, the brain was removed and brain parts

(cerebrum, hypothalamus, cerebellum and brainstem) were

separated as followed earlier [24,25]. The brain was soni-

cated in ice-cold 50 mM Tris–HCl buffer (pH 7.4) and

centrifuged at 30,000� g for 30 min at 4 jC. The superna-

tant was discarded and the pellet was resuspended in 50 mM

Tris–HCl containing 0.5% Triton X100. After centrifuga-

tion at 30,000� g for 30 min, the supernatant containing

membrane was collected and the protein in the supernatant

was assayed using a BCA protein assay reagent (Pierce,

Illinois). Glutamate carboxypeptidase II activity was deter-

mined by analyzing the hydrolysis of [3H]NAAG radio-

labelled at the glutamate residue.

Glutamate carboxypeptidase II activity was measured as

described earlier [5,19]. The reaction mixture for the assay

contained 50 mM Tris–Hcl (pH 7.4), 0.05% Triton X100,

0.3 Aci N-acetyl-L-aspartyl-L-[3,4-3H]glutamate (Perkin

Elmer, MA), 119.17 nM non-radiolabelled NAAG (Sigma,

Michigan), 1 mM cobalt chloride (Sigma, MI) and 25 Agmembrane protein in a total volume of 250 Al. Assay

reactions were incubated at 37 jC water bath and were

quenched after 60 min with 250 Al of ice-cold sodium

phosphate buffer (0.1 M, pH 7.4). The assay mixture was

then applied to a distilled water-prewashed mini column of

AG 1-X8 anion-exchange resin [200–400 mesh, formate

form (Bio-Rad Laboratories, California)], and glutamate

was selectively eluted with 1 ml of scintillation flour

(Sigma). Radioactivity was determined by LS 6000 IC

scintillation spectrometry (Beckman, California).

Glutamate carboxypeptidase II activity is expressed as

nmol of formed glutamate/mg protein/h, and the calcula-

tions were performed as followed earlier [5]. Data were

analyzed using ANOVA.

To examine whether the ASPA gene mutation affects the

NAAG content of the brain, the level of NAAG was mea-

sured. NMR spectral integral analysis indicated that NAAG

was not affected in the knockout mouse brain compared to the

wild type (Fig. 1). The ratio of NAAG/Cr. in the knockout

mouse brain was 0.032F 0.003 (n = 5F S.E.) compared to

that in the wild type, 0.028F 0.004 (n = 4F S.E.).

S. Surendran et al. / Brain Research 1016 (2004) 268–271270

The hydrolyzing enzyme, GCP II, activity was normal in

the whole brain of the knockout mouse as observed in the

wild type (Fig. 2). The activity of GCP II in different parts

of the brain (cerebrum, hypothalamus, cerebellum and

brainstem) of the knockout mouse was similar to that

observed in the wild type (Fig. 2).

ASPA deficiency is the basic cause of CD [16]. Elevated

excretion of urinary NAAG was reported in ASPA-deficient

patients [8,9]. Because the neuropeptide NAAG is abundant

in the brain [15,19], whether ASPA deficiency affects brain

NAAG to result in an increased excretion of urinary NAAG

is not known. Brain NAAG may contribute to lead elevated

urinary NAAG by two possible ways. Either depletion of

NAAG by nerve cell rupture or accumulation of NAAG in

the brain due to a defect in the hydrolysis of the enzyme,

GCP II. Subsequently, the resulting NAAG will be removed

through urine as waste. Therefore, brain NAAG was deter-

mined in the mouse model for CD using NMR spectroscopy.

The amount of NAAG in the knockout mouse brain was

found to be similar to that in the wild type. This data

suggests that ASPA deficiency does not affect the normal

level of NAAG in the mouse brain.

The amount of NAAG in the brain can also be interpreted

by measuring NAAG hydrolyzing enzyme, GCP II, activity

[11,20,27]. Elevated or reduced levels of NAAG in the brain

affect the normal activity of GCP II [11,20,27]. Therefore,

GCP II activity is also to be studied in the mouse brain. The

normal activity of GCP II in the ASPA-deficient mouse

brain observed in the present study suggests that ASPA

deficiency does not affect the hydrolyzing enzyme activity

and this is likely due to the normal level of brain NAAG

seen in the mouse.

These observations in the knockout mouse brain suggest

that ASPA deficiency does not affect the level of brain

NAAG or the hydrolyzing enzyme, GCP II. Presumably,

brain NAAG is not likely involved to result in an increased

excretion of urinary NAAG in the knockout mouse, if

documented also in patients with CD.

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