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

Strategies to rescue the consequences of inducible arginase-1 deficiency in mice

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken beta-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies

Impact of inducible Arg1 knockout on phenotypic presentation.

<p>(A) Typical appearance of Arg1 knockout mouse at humane endpoint (right), with healthy-appearing vehicle-treated control at the same timepoint (left). (B) Percent changes in body weight during the experimental period relative to body weights taken four days following injections are shown on y-axis. (C) Kaplan-Meier survival curve comparison depicts that tamoxifen-treated mice display significantly reduced survival rates when compared to ROSA and vehicle-treated mice. Data are mean ± SEM for n = 5–11 in each group. Statistical significance between groups was determined by Student's <i>t</i>-test (*<i>P<0.05</i>). (D) Quantitative analysis of walking footprint patterns based on measurements of stride length, forepaw base and hindpaw base width, and distance between front and hind footprint placement. (n = 6).</p

Tamoxifen-mediated inducible Arg1 knockout leads to hyperammonemic crisis.

<p>Plasma ammonia assay performed at five different time-points of three age groups. Values are expressed as percentage change in plasma ammonia concentration in tamoxifen-treated mice compared to vehicle-treated control mice. Ammonia concentrations ranged between 305–595 µmol/L (vehicle-treated mice) and 903–1791 µmol/L (tamoxifen-treated mice). Data are mean ± SEM for n = 3–5 in each group. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Down-regulation of Arg 1 expression in tamoxifen-treated Arg-Cre mice.

<p>(A) Transcriptional response of <i>Arg1</i> to tamoxifen induction. Real-time qPCR analysis of <i>Arg 1</i> gene expression was performed in liver, brain and kidney tissues 12 days after tamoxifen administration. Differences in RNA input for reverse transcription were normalized using Ct values obtained in parallel for mouse 18S rRNA. Fold change was calculated relative to the vehicle-treated samples using the comparative threshold method (2^−ΔΔCt). Values are mean ± SEM for n = 3–6 in each group. Statistical significance between groups was determined by Student's <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001. (B) Western blot analysis of total liver extracts (20 µg/well) from four age groups. Arg1 protein expression was evaluated by immunoblotting with an anti-Arg1 antibody (C-terminal) to confirm the level of knockdown. α-tubulin was used as loading control. Representative immunoblot from three independent experiments is shown. (C) Real-time qPCR analysis of <i>Arg 2</i> gene expression in kidney.</p

Plasma amino acid analysis of mice from different age groups (pooled) measured at humane endpoint.

<p>Values are mean ± SEM for n = 11–12 and are expressed as micromoles per liter (µmol/L). BCAA, branched chain amino acid; NS, not significant.</p

Generation of inducible Arg1-deficient mice.

<p>(A) Experimental setup for the gene targeting strategy. <i>Arg1^flox</i> mice were crossbred with CreER^T2 mice to generate Arg1-Cre mice. Four separate groups of mice have been tested for tamoxifen-mediated Cre removal of exons 7 and 8 of <i>Arg1</i>. The arrows depict the locations of primers used for genotyping the resulting mice with approximate sizes of the different PCR products shown. (B) Representative agarose gel of PCR genotyping using genomic DNA from ear punch or tail biopsies of vehicle- and tamoxifen-treated Arg-Cre mice. Arg1-Cre mice exhibited two bands at 1.2 kb and 252 bp, while knockout mice showed a single band at 195 bp.</p

Reduced arginase enzyme activity and urea production in tamoxifen-treated Arg-Cre mice.

<p>(A) Arginase activity in liver tissue extracts from vehicle- and tamoxifen-treated mice. The livers were homogenized and the arginase enzyme activity assay was carried out as mentioned in the “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080001#s2" target="_blank">Materials and Methods</a></i>” section. (B) Functional capacity of hepatocytes assessed by determining urea production. Isolation of primary mouse hepatocytes was performed based on the two-step collagenase perfusion technique. Typical morphology of mouse hepatocytes cultured on a single layer of collagen gel at 24 h (top panel). Hepatocyte urea production was spectrophotometrically determined at 544 nm (bottom panel). Values are mean ± SEM for n = 3–7 in each group. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p