Short-term correction of arginase deficiency in a neonatal murine model with a helper-dependent adenoviral vector.

Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 x 10(9) viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg(-/-) mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg(-/-) livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg(-/-) mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV.

Disruption of arginase II alters prostate tumor formation in TRAMP mice.

BACKGROUND: Arginase II (AII) is involved in the polyamine synthetic pathway, and elevated levels of expression have been found in a high proportion of prostate cancer samples and patients. However, the biological function of arginase II in prostate cancer still remains to be elucidated. In this study, we utilized the TRAMP mouse prostate cancer model to better understand the contribution of AII on tumor development. METHODS: AII expression was determined in prostates from TRAMP mice at 23 weeks of age by real-time RT-PCR and Western blot analysis. Additionally, AII expression was disrupted in the TRAMP model by crossbreeding arginase II knockout (AII KO) mice with TRAMP mice in order to generate the TRAMP/AII KO line. In each group, genito-urinary (GU) tract weights were determined and a pathological evaluation of the tumors was completed. RESULTS: AII expression was only detectable in those mice without the presence of macroscopic tumors; it was also absent in the TRAMP-C2 cell line, which is characteristic of an advanced prostate tumor. Assessment of the GU weights revealed larger average GU weights in the TRAMP/AII KO mice compared to TRAMP mice. Additionally, a greater percentage of more advanced pathology was found in the TRAMP/AII KO group compared to the TRAMP cohort. CONCLUSIONS: Based on these results, AII deficiency in the TRAMP model seems to accelerate prostate tumor progression, leading to an overall more advanced cancer stage in these mice. These findings support the possibility that prostatic arginase II could be a potentially useful marker of disease progression.

Increased plasma and tissue guanidino compounds in a mouse model of hyperargininemia.

In humans, arginase I (AI)-deficiency results in hyperargininemia, a metabolic disorder with symptoms of progressive neurological and intellectual impairment, spasticity, persistent growth retardation, and episodic hyperammonemia. A deficiency of arginase II (AII) has never been detected and the clinical disorder, if any, associated with its deficiency has not been defined. Since the spasticity and paucity of hyperammonemic crises seen in human AI-deficient patients are not features of the other urea cycle disorders, the likelihood of ammonia as the main neuropathogenic agent becomes extremely low, and the modest elevations of arginine seen in the brains of our mouse model of hyperargininemia make it an unlikely candidate as well. Specific guanidino compounds, direct or indirect metabolites of arginine, are elevated in the blood of patients with uremia. Other guanidino compounds are also increased in plasma and cerebrospinal fluid of hyperargininemic patients making them plausible as neurotoxins in these disorders. We analyzed several guanidino compounds in our arginase single and double knockout animals and found that alpha-keto-delta-guanidinovaleric acid, alpha-N-acetylarginine, and argininic acid were increased in the brain tissue from the AI knockout and double knockout animals. Several compounds were also increased in the plasma, liver, and kidneys. This is the first time that several of the guanidino compounds have been shown to be elevated in the brain tissue of an arginase-deficient mammal, and it further supports their possible role as the neuropathogenic agents responsible for the complications seen in arginase deficiency.

Polyamine homeostasis in arginase knockout mice.

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N(1)-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues.

Ornithine deficiency in the arginase double knockout mouse.

Knockout mouse models have been created to study the consequences of deficiencies in arginase AI and AII, both individually and combined. The AI knockout animals die by 14 days of age from hyperammonemia, while the AII knockout has no obvious phenotype. The double knockout (AI(-/-)/AII(-/-)) exhibits the phenotype of the AI-deficient mice, with the additional absence of AII not exacerbating the observed phenotype of the AI knockout animals. Plasma amino acid measurements in the double knockout have shown arginine levels increased roughly 100-fold and ornithine decreased roughly 10-fold as compared to wildtype. Liver ornithine levels were reduced to 2% of normal in the double knockout with arginine very highly elevated. Arginine and ornithine were also altered in other tissues in the double knockout mice, such as kidney, brain, and small intestine. This is the first demonstration that the fatal hyperammonemia in the AI knockout mouse is almost certainly due to ornithine deficiency, the amino acid needed to drive the urea cycle. Others have shown that the expression of ornithine aminotransferase (OAT) rapidly decreases in the intestine at the same age when the AI-deficient animals die, indicating that this enzyme is critical to the maintenance of ornithine homeostasis, at least at this early stage of mouse development. Although most human AI-deficient patients have no symptomatic hyperammonemia at birth, it is possible that clinically significant ornithine deficiency is already present.