A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia

Abstract

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 ^-/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1^+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Keywords: carbamoyl phosphate synthetase (CPS1) deficiency; citrulline; glutamine; hyperammonemia; murine model.

Figure 1.. Strategy to generate the Cps1KO (tm1b) murine model.

The genomic outline for this mouse is shown (left). In the top right image, 5’ LRPCR shows confirms the targeting in the N1 (tm1a) mice. With this approach, we were able to successfully introduce the gene specific forward primer 5’ in the homology arm. We also included a reverse primer in lacZ at en2/Frt junction. 3’ loxP image (middle right): In the middle image (right), loxP confirmation of the targeted N1 mice is demonstrated. This demonstrates germline transmission and that this is conditionally ready (tm1a). In the bottom right image, the demonstration of the Cre transgene positive mice confirms that successful recombination at the loxP sites has taken place. With this step a constitutive knockout is generated. With the LacZ cassette having a forward primer and a reverse in the downstream distal LoxP homology arm, determination of the Cps1 and Cre positive animals successfully recombined at the loxP sites and generated the mutant (tm1b). (Abbreviations: LRPCR refers to long range polymerase chain reaction and DTA refers to the Diphtheria Toxin A gene. The black rectangles represent exon regions of Cps1).

Figure 2.. Characteristics of Neonatal CPS1-Deficient Mice.

A) The genotypes are distributed among 104 progeny of CPS1 matings of heterozygote mice; B) Western blot showing hepatic CPS1 protein in wild type (+/+), heterozygote (+/−) and knockouts (−/−); representative immunostaining for CPS1 shows hepatic CPS1 expression in liver in Cps1^+/+ (C) and Cps1^+/− (D) mice with absent expression in knockout mouse liver (E); (F) CPS1 activity in hepatic extracts from Cps1^+/+, Cps1^+/− and knockout mice correspond with that of CPS1-specific mRNA (G); plasma ammonia levels of neonatal Cps1^+/+, Cps1^+/− and knockout mice (n=5 per group; except in the CPS1 enzyme assay [n=3 Cps1^+/−, n=4 Cps1^+/+ and n=5 Cps1^−/−]). (Data are presented as mean ± standard deviation.) (CP = Carbamoyl Phosphate).

Figure 3.. Plasma Amino Acids.

Amino acids were analyzed from plasma from constitutive CPS1 knockout mice and littermate controls within 12 hours of birth (n=5 per group; data presented as mean ± standard deviation in μmol/L). (ASP = aspartic acid, GLU = glutamic acid, GLN = glutamine, ALA = alanine, CIT = citrulline, ARG = arginine, ORN = ornithine; LYS = lysine, TAU = taurine, GLY = glycine, PHE = phenylalanine, TYR = tyrosine)

Figure 4.. Hepatic Amino Acids.

Amino acids were analyzed from liver lysates from constitutive CPS1 knockout mice and littermate controls within 12 hours of birth (n=5 per group; data presented as mean ± standard deviation in nmol/mg protein). (ASP = aspartic acid, GLU = glutamic acid, GLN = glutamine, ALA = alanine, CIT = citrulline, ARG = arginine, ORN = ornithine; LYS = lysine, TAU = taurine, GLY = glycine, PHE = phenylalanine, TYR = tyrosine)