Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease

Abstract

Genetic mutations that severely diminish the activity of aspartoacylase (ASPA) result in the fatal brain dysmyelinating disorder, Canavan disease. There is no effective treatment. ASPA produces free acetate from the concentrated brain metabolite, N-acetylaspartate (NAA). Because acetyl coenzyme A is a key building block for lipid synthesis, we postulated that the inability to catabolize NAA leads to a brain acetate deficiency during a critical period of CNS development, impairing myelination and possibly other aspects of brain development. We tested the hypothesis that acetate supplementation during postnatal myelination would ameliorate the severe phenotype associated with ASPA deficiency using the tremor rat model of Canavan disease. Glyceryltriacetate (GTA) was administered orally to tremor rats starting 7 days after birth, and was continued in food and water after weaning. Motor function, myelin lipids, and brain vacuolation were analyzed in GTA-treated and untreated tremor rats. Significant improvements were observed in motor performance and myelin galactocerebroside content in tremor rats treated with GTA. Further, brain vacuolation was modestly reduced, and these reductions were positively correlated with improved motor performance. We also examined the expression of the acetyl coenzyme A synthesizing enzyme acetyl coenzyme A synthase 1 and found upregulation of expression in tremor rats, with a return to near normal expression levels in GTA-treated tremor rats. These results confirm the critical role played by NAA-derived acetate in brain myelination and development, and demonstrate the potential usefulness of acetate therapy for the treatment of Canavan disease.

Fig. 1

Immunohistochemistry and western blots were used to confirm the ASPA null phenotype in tremor rats. ASPA expression is shown for wild-type (a), and tremor rats (b). ASPA-immunoreactive cells in (a) are predominantly oligodendrocytes in the internal capsule, and surrounding forebrain fiber tracts. Immunoreactivity is absent in brain tissue from tremor rats (b). Inset in (b) is a western blot of brain protein extracts from wild-type (WT) and tremor rats (Tr) showing a single immunoreactive band at approximately 37 kD in the wild-type rat extract that is absent in the tremor rat extract. VL Lateral ventricle, int internal capsule, CP caudate putamen

Fig. 2

Longitudinal study of Rotarod performance in GTA-treated and untreated tremor rats. The differences between the GTA-treated and untreated groups were significant by ANOVA (p < 0.001) and significant by post-hoc analysis with Tukey’s HSD (p < 0.05). Rotarod performance of female tremor rats is shown in (a), performance of male tremor rats is shown in (b). Animal numbers per test session were variable; n = 11–16 (av. 12) for treated tremor females (TR-F), 4–18 (av. 12) for untreated tremor females (UT-F), 9–14 (av. 10) for treated tremor males (TR-M), and 7–16 (av. 11) for untreated tremor males (UT-M). See Table 2 for statistical analyses of individual testing sessions

Fig. 3

Average values of total horizontal activity, distance moved (cm) and move time (s) were recorded for the treated and untreated tremor rats at 4 time points at approximately 70, 85, 98, and 110 (±3) days of age. The data were collected for 1 h in an open-field apparatus. Data are shown for testing at 98 and 110 days of age; the testing days when treated female and treated male tremor rats showed the greatest improvements respectively. The number of rats in the different groups tested were as follows: treated female (Tr Fem, 12); untreated female (Ut Fem, 15); treated male (Tr Male, 12), and untreated male (Ut Male, 14)

Fig. 4

Regression analysis of the severity of vacuolation in the cerebellum/brain stem (a) and spinal cord (b) with the average Rotarod performance of individual rats (n = 7). The randomly selected sample consisted of 2 untreated female tremor rats, 2 treated female tremor rats, one untreated male tremor rat, one treated male tremor rat, and one untreated female wild-type rat of the same strain. The correlation coefficients, r, determined for plots (a) and (b) were 0.72 and 0.61, and were found to be significant with p values of 0.01 and 0.04 for the F values of 13.15 and 7.79 (by ANOVA), respectively. The vacuolation scores were determined by a veterinary pathologist blinded to the study. The values for the treated tremor rats tended to fall intermediate between values found for the untreated tremor rats and the wild-type rat

Fig. 5

AceCS1-immunoreactivity in oligodendrocytes in white matter from an 18-day-old wild-type rat. The fimbria of the hippocampus is shown in (a) and (b), demonstrating numerous immunoreactive oligodendrocytes. AceCS1 expression in the corpus callosum of an 18-day-old rat is shown in (c) and (d). Immunoreactive oligodendrocytes in the internal capsule are shown in (e) and (f). AceCS1 expression was generally stronger in oligodendrocyte nuclei than in their cytoplasm. CA3 Pyramidal cell layer of hippocampal Ammon’s horn CA3 region, cc corpus callosum, CP caudate/putamen, fim fimbria of the hippocampus, ic internal capsule, LV lateral ventricle, st stria terminalis. Bar (in f): 120 µm (a,c,e), 30 µm (b,d,f)

Fig. 6

AceCS1-immunoreactivity comparison between adult wild,type and GTA treated and untreated tremor rats: low magnification images (left column), higher magnification images (right column). AceCS1 immunoreactivity in the cortex of an adult control rat (a,b) shows expression predominantly in cell nuclei in all layers of cortex. In a representative untreated adult tremor rat (c,d), AceCS1 expression was moderately upregulated, including in oligodendrocytes in the corpus callosum (cc). In a representative adult GTA-treated tremor rat (e,f), AceCS1 expression was reduced relative to untreated tremor rats, and was similar to the expression levels in control animals. Bar (in f): 300 µm (a,c,e), 120 µm (b,d,f)

Fig. 7

AceCS1 expression in layer V of cortex. In wild-type adult rats, AceCS1 was expressed predominantly in scattered cell nuclei in layer V of cortex (a). In adult tremor rats, the number of immunoreactive cell nuclei in layer V was increased, and tended to occur in clusters not seen in wild-type rats (b). In GTA-treated tremor rats, the number of AceCS1 immunoreactive cell nuclei was reduced relative to untreated tremor rats, and tended to be more scattered as in the case of wild-type rats (c). Bar 60 µm

Fig. 8

AceCS1 expression and vacuole reduction in the lateral hypothalamus with GTA treatment. AceCS1-immunoreactivity in an adult wild-type rat (control) is shown in (a), with the supraoptic nucleus denoted by the arrow. The lateral hypothalamus of an adult tremor rat is shown in (b), exhibiting extensive vacuolation throughout the area, and upregulation of AceCS1 expression. The lateral hypothalamus of a GTA-treated adult tremor rat is shown in (c), with reduced AceCS1 expression and reduced vacuolation as compared with the untreated tremor rat. Bar 300 µm