Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase

Abstract

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags^-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags^-/- mice, established the dose of the vector needed to rescue Nags^-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags^-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags^-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

Figure 1

Differing behaviors of Nags^−/− (orange) and Nags^+/+ (teal) mice with respect to cessation of drinking, eating, hanging, rearing up, grooming, and walking (A) during the 24 h before withdrawal of NCG + Cit supplementation and (B) in the 24 h period after withdrawal of NCG + Cit supplementation at 4 PM. The same results are shown in (C) for wild-type mice before (solid) and after (hatched) withdrawal of NCG + Cit supplementation. Shaded rectangles indicate the period of darkness between 6 PM and 6 AM.

Figure 2

Different behaviors of the Nags^−/− (orange) and Nags^+/+ (teal) mice with respect to cessation of drinking, eating, hanging, rearing up, grooming, and walking (A) during 24 h before withdrawal of NCG + Cit supplementation and (B) in the 24 h period after withdrawal of NCG + Cit supplementation at 10 AM. The same results are shown in (C) for wild-type mice before (solid) and after (hatched) withdrawal of NCG + Cit supplementation. Shaded rectangles indicate the period of darkness between 6 PM and 6 AM.

Figure 3

Phenotype of the Nags^−/− mice after expression of the NAGS gene from the AAV2/8TBG.mNAGS vector (left panels), or the AAV2/8NAGS.mNAGS vector (right panels). (A,B) Duration of activity on the voluntary wheel. (C,D) Plasma ammonia concentrations. (E–H) Abundance of the Nags mRNA (E,F) and NAGS protein (G,H) in the liver. (I,J) Changes in the abundance of Nags mRNA and NAGS protein in the liver after a single injection of 10¹¹ viral particles. Each column in panels E–H represents a mean and its associated SEM in n = 8 mice; each data point in panels I and J represents a mean and its associated SEM in n = 6 mice where 100% is set as the mean value for four Nags^+/+ mice in panels E–H. Abundance of the Nags mRNA and NAGS protein in injected and Nags^+/+ mice were normalized to the abundance of 18S rRNA and vinculin, respectively.

Figure 4

NAGS protein in the livers of Nags^−/− mice injected with increasing doses of either AAV2/8TBG.mNAGS (A) or AAV2/8NAGS.mNAGS (B) vector. AAV2/8TBGnull (Null) vector was injected as a control. Vinculin was used as a loading control. Full-length immunoblots are presented in Supplementary Fig. S1.

Figure 5

NAGS protein in the livers of Nags^−/− mice after single injection with 10¹¹ particles of either AAV2/8TBG.mNAGS (A) or AAV2/8NAGS.mNAGS (B) vector. Vinculin was used as a loading control. Full-length immunoblots are presented in Supplementary Fig. S2.

Figure 6

Phenotype of the Nags^−/− mice that received AAV2/8NAGS.mNAGS-E354A gene therapy. Duration of activity on the voluntary wheel (A), plasma ammonia (B) and glutamine (C) concentrations, abundance of the liver NAGS mRNA (D) and protein (E) after withdrawal of NCG. Each column represents a mean and its associated SEM of measurements in n = 8 animals; 100% is the mean value for four Nags^+/+ mice.

Figure 7

NAGS protein in the livers of Nags^−/− mice injected with 10¹¹ viral particles of the AAV2/8NAGS.mNAGS vector (WT NAGS lanes), with either 10¹¹ or 10¹⁰ viral particles of the AAV2/8NAGS.E354A-mNAGS vector (E354A NAGS lanes), or with 10¹¹ viral particles of the AAV2/8TBGnull vector (Null). Vinculin was used as a loading control. Full-length immunoblots are presented in Supplementary Fig. S3.

Figure 8

Behavior of Nags^−/− mice that received the same dose of either AAV2/8NAGS.mNAGS-E354A or AAV2/8NAGS.mNAGS gene therapy. (A) Average running time during 6 h epochs before withdrawal of NCG supplementation. (B) Average running time during 6 h epochs after withdrawal of NCG supplementation. (C) Average running distance during 6 h epochs before withdrawal of NCG supplementation. (D) Average running distance during 6 h epochs after withdrawal of NCG supplementation. Orange—AAV2/8NAGS.mNAGS-E354A. Blue—AAV2/8NAGS.mNAGS. Shaded rectangles indicate nighttime when mice are more active. (NCG + NAG) indicates that both NCG and NAGS are present in the livers of experimental animals. (NAG only) indicates that NCG supplementation has been withdrawn and NAG is produced by the mNAGS expressed from the AAV-vector while NCG is absent from the livers of experimental animals. Shaded rectangles indicate periods of darkness between 6 PM and 6 AM.

Figure 9

Thermal stability of wild-type human (A) and mouse (B) NAGS, E360D human NAGS (C), and E354D mouse NAGS (D) in the presence of either L-arginine (purple) or D-arginine (teal). Different shades of purple and teal represent technical replicates of thermal unfolding.