Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism.

Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.

Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism.

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing "floxed" Arg1 mice with CreER(T2) mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.

Five novel mutations in ARG1 gene in Chinese patients of argininemia.

BACKGROUND: Argininemia is an autosomal recessive genetic disorder caused by hepatocyte arginase deficiency. It could be detected by blood amino acids analysis (high arginine) and confirmed by molecular diagnosis. The clinical manifestations in patients are similar to cerebral palsy so the diagnosis is usually much delayed. Reports of argininemia from mainland China are few, and genetic analyses have not been reported. PATIENTS AND METHODS: Five Chinese patients with argininemia were investigated. They had progressive spastic tetraplegia, poor physical growth from 1 month to 4 years. When argininemia was found at the ages of 4 to 12 years, four of patients had mental retardation, and three had seizures. RESULTS: Elevated blood arginine and significantly decreased erythrocyte arginase activity in five patients confirmed the diagnosis of arginase deficiency. Liver dysfunction was found in four patients, two of whom had mildly elevated blood ammonia levels. Cranial magnetic resonance imaging showed progressive cerebral atrophy in three patients. Six mutations in the ARG1 gene were identified, of which only one (c.703 G>A, p.G235R) in exon 7 has been reported before; c.34 G>T (p.G12X) in exon 1, c.67delG (p.G23fsX31) in exon 2, c.539G>C (p.R180 T) in exon 5, c.374C>T (p.A125 V) in exon 4, and c.646-649del CTCA (p.T215fsX219) in exon 6 were novel mutations. CONCLUSIONS: Argininemia is one of the few treatable causes of pediatric spastic paraparesis. Early metabolic investigation is very important to reach a diagnosis and better outcome. Five Chinese patients with late-diagnosed argininemia were reported. The mutation spectrum of ARG1 gene should be different from other populations.

Analysis of novel ARG1 mutations causing hyperargininemia and correlation with arginase I activity in erythrocytes.

Hyperargininemia (HA) is an autosomal recessive disease that typically has a clinical presentation that is distinct from other urea cycle disorders. It is caused by the deficient activity of the enzyme arginase I, encoded by the gene ARG1. We screened for ARG1 mutations and measured erythrocyte enzyme activity in a series of 16 Brazilian HA patients. Novel mutations, in addition to previously described missense mutations, were analysed for their effect on the structure, stability and/or function of arginase I (ARG1) using bioinformatics tools. Three previously reported mutations were found (p.R21X; p.I11T and p.W122X), and five novel mutations were identified (p.G27D; p.G74V; p.T134I; p.R308Q; p.I174fs179). The p.T134I mutation was the most frequent in the Brazilian population. Patients carrying the p.R308Q mutation had higher residual ARG1 decreased activity, but presented no distinguishable phenotype compared to the other patients. Bioinformatics analyses revealed that missense mutations (1) affect the ARG1 active site, (2) interfere with the stability of the ARG1 folded conformation or (3) alter the quaternary structure of the ARG1. Our study reinforced the role of Arg308 residue for assembly of the ARG1 homotrimer. The panel of heterogeneous ARG1 mutations that cause HA was expanded, nevertheless a clear genotype-phenotype correlation was not observed in our series.

Neonatal cholestasis: an uncommon presentation of hyperargininemia.

Hyperargininemia is a rare inborn error of metabolism due to arginase deficiency, which is inherited in an autossomal recessive manner. Arginase is the final enzyme of the urea cycle and catalyzes the conversion of arginine to urea and ornithine. This condition typically presents in early childhood (between 2 and 4 years of age) with developmental delay associated with progressive spastic paraparesis. Neonatal presentation is very uncommon with a poorly described outcome. Here, we discuss two cases of neonatal cholestasis as initial clinical presentation of hyperargininemia. In case 1, diagnosis was established at 2 months of age upon investigation of the etiology of cholestatic injury pattern and hepatosplenomegaly, and treatment was then initiated at when the patient was 3 months old. Unfortunately, the patient had progressive biliary cirrhosis to end-stage liver disease complicated with portal hypertension for which she underwent successful orthotopic liver transplant at 7 years of age. In case 2, hyperargininemia was identified through newborn screening and treatment was started when patient was 21 days old. Cholestasis was only identified in the patient's further evaluation and it resolved 2 weeks into treatment. The patient is currently 18 months old and her development and neurological examination remain unremarkable. Neonatal cholestasis as first presentation of hyperargininemia is rare, but this disorder should be included in the differential diagnosis of unexplained cholestasis in the neonate. In fact, these two cases suggest that arginase deficiency may be the cause of cholestatic liver disease.

Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis.

The balance between the products of L-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. L-arginine can be oxidized by host inducible NO synthase to produce NO, which contributes to parasite killing. In contrast, L-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considerable potential to modulate infectivity and disease pathogenesis. In this study, we compared the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg(-)) L. major. We found that arg(-) L. major are impaired in their macrophage infectivity in vitro independent of host inducible NO synthase activities. As with in vitro results, the proliferation of arg(-) L. major in animal infections was also significantly impaired in vivo, resulting in delayed onset of lesion development, attenuated pathology, and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg(-) L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg(-) than in WT-infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity.

NTPDase and 5'-nucleotidase activities of synaptosomes from hippocampus of rats subjected to hyperargininemia.

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5'-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5'-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 microL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1-1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.

Clinical, biochemical, and molecular spectrum of hyperargininemia due to arginase I deficiency.

The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Urea cycle disorders are a group of inborn errors of hepatic metabolism that often result in life threatening hyperammonemia and hyperglutaminemia. Deficiencies of all of the enzymes of the cycle have been described and although each specific disorder results in the accumulation of different precursors, hyperammonemia and hyperglutaminemia are common biochemical hallmarks of these disorders. Arginase is the enzyme involved in the last step of the urea cycle. It catalyzes the conversion of arginine to urea and ornithine. The latter reenters the mitochondrion to continue the cycle. Hyperargininemia is an autosomal recessive disorder caused by a defect in the arginase I enzyme. Unlike other urea cycle disorders, this condition is not generally associated with a hyperammonemic encephalopathy in the neonatal period. It typically presents later in childhood between 2 and 4 years of age with predominantly neurological features. If untreated, it progresses with gradual developmental regression. A favorable outcome can be achieved if dietary treatment and alternative pathway therapy are instituted early in the disease course. With this approach, further neurological deterioration is prevented and partial recovery of skills ensues. Early diagnosis of this disorder through newborn screening programs may lead to a better outcome. This review article summarizes the clinical characterization of this disorder; as well as its biochemical, enzymatic, and molecular features. Treatment, prenatal diagnosis and diagnosis through newborn screening are also discussed.

Arginase expression in mouse embryonic development.

We are using the model of the developing mouse embryo to elucidate the pattern of arginase expression in mammalian cells in normal animals and in arginase I (AI) deficiency during development by digoxigenin-labeled RNA in situ hybridization. Our goal is to understand the regulation of these isozymes, with the expectation that this knowledge will help patients suffering from AI deficiency. We found that AI mRNA was widely and strongly expressed in the normal developing mouse embryo; in contrast, a relatively strong AII mRNA signal was found only in the intestine. In the AI knockout mouse embryo, no AII overexpression was found. These results indicated that arginases are needed in mouse embryonic development and AI is the principal form required. The strong AI expression in the peripheral nervous system suggests that the pathogenesis of the neurological retardation in AI deficiency may be conditioned by AI deficiency in the nervous system during embryonic development.

Functional consequences of the G235R mutation in liver arginase leading to hyperargininemia.

Hyperargininemia is a rare autosomal disorder that results from a deficiency in hepatic type I arginase. This deficiency is the consequence of random point mutations that occur throughout the gene. The G235R patient mutation has been proposed to affect the catalytic activity and structural integrity of the protein [D. E. Ash, L. R. Scolnick, Z. F. Kanyo, J. G. Vockley, S. D. Cederbaum, and D. W. Christianson (1998) Mol. Genet. Metab. 64, 243-249]. The G235R (patient) and G235A (control) arginase mutants of rat liver arginase have been generated to probe the effects of these point mutations on the structure and function of hepatic type I arginase. Both mutant arginases were trimeric by gel filtration, but the control G235A mutant had 56% of wild-type activity and the G235R mutant had less than 0.03% activity compared to the wild-type enzyme. The G235R mutant contained undetectable levels of tightly bound manganese as determined by electron paramagnetic resonance, while the G235A mutant had a Mn(II) stoichiometry of 2 Mn/subunit. Molecular modeling indicates that the introduction of an arginine residue at position 235 results in a major rearrangement of the metal ligands that compromise Mn(II) binding.