Liver-specific knockout of arginase-1 leads to a profound phenotype similar to inducible whole body arginase-1 deficiency

Abstract

Arginase-1 (Arg1) converts arginine to urea and ornithine in the distal step of the urea cycle in liver. We previously generated a tamoxifen-inducible Arg1 deficient mouse model (Arg1-Cre) that disrupts Arg1 expression throughout the whole body and leads to lethality ≈ 2 weeks after gene disruption. Here, we evaluate if liver-selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of gene delivery or hepatocyte transplantation to rescue the phenotype. Liver-selective Arg1 deletion was induced by using an adeno-associated viral (AAV)-thyroxine binding globulin (TBG) promoter-Cre recombinase vector administered to Arg1 "floxed" mice; Arg1^fl/fl ). An AAV vector expressing an Arg1-enhanced green fluorescent protein (Arg1-eGFP) transgene was used for gene delivery, while intrasplenic injection of wild-type (WT) C57BL/6 hepatocytes after partial hepatectomy was used for cell delivery to "rescue" tamoxifen-treated Arg1-Cre mice. The results indicate that liver-selective loss of Arg1 (> 90% deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality ≈ 3 weeks after Cre-induced gene disruption. Delivery of Arg1-eGFP AAV rescues more than half of Arg1 global knockout male mice (survival > 4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels observed in WT animals. Significant Arg1 enzyme activity from engrafted WT hepatocytes into knockout livers can be achieved but not sufficient for rescuing the lethal phenotype. This raises a conundrum relating to liver-specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, gene and cell-directed therapies suggest that rescue of extra-hepatic Arg1 expression may also be necessary for disease correction. Further studies are needed in order to illuminate the detailed mechanisms for pathogenesis of Arg1-deficiency.

Keywords: AAV, adeno-associated virus; Arg1, arginase-1; Arg1-eGFP, arginase-1 enhanced green fluorescent protein; Arginase; Gene therapy; Hepatocyte; Inducible knockout mice; Liver; TBG, thyroxine-binding globulin; Urea cycle; WT, wild-type; gc, genome copies; ip, intraperitoneal.

Fig. 1

AAV-TBG-Cre delivery results in loss of liver Arg1 expression. A. PCR genotyping of 11 tissues from a representative mouse injected with high-dose AAV-TBG-Cre. Lanes: 1, brain; 2, heart; 3, lung; 4, liver; 5, kidney; 6, spleen; 7, small intestine; 8, esophagus; 9, tongue; 10, skeletal muscle; 11, tail. The 195 bp band is diagnostic for the Arg1^∆ allele, while the 252 bp and 1.2 kb bands represent the Arg1^fl allele. B. Western blot analysis of Arg1. Liver extracts from three low-dose Cre-injected mice express variable amounts of Arg1, whereas extracts from three mid-dose Cre-injected mice express almost no Arg1 protein. Tubulin is used as a loading control. C. Arg1 enzyme activity assay in the three groups of AAV-TBG-Cre-injected mice euthanized at day 21–22 post-injection (n = 7 high-, 6 mid-, 3 low-dose, respectively). None of the low-dose Cre-injected mice (n = 6) displayed symptoms requiring euthanization at this time point and three in this group were not sacrificed nor assayed for enzymatic activity. Liver Arg1 enzymatic activity from a cohort of untreated animals is shown for comparison on the right. D. Immunostaining for Arg1 (FITC, green) and glutamine synthetase (Alexa Fluor 568, red) in liver sections from a low-dose Cre-injected mouse (left panel) and a WT mouse (right panel).

Fig. 2

Liver-specific Arg1 KO leads to weight loss and humane euthanization endpoint. A. Weight loss traces. B. Kaplan-Meier survival curves. Data for high- (n = 7), mid- (n = 6) and low-dose (n = 6) AAV-TBG-Cre-injected mice are shown.

Fig. 3

Blood arginine levels at two time points post-AAV-TBG-Cre injection (n = 7 high-, 6 mid-, 3 low-dose, respectively). Samples were analyzed from dried blood spots by mass spectrometry as described in the Methods section. The two dotted lines represent arginine levels in two cohorts of untreated Arg1^fl mice (n = 15 total) measured on two separate occasions.

Fig. 4

AAV transgene delivery of Arg1-eGFP restores Arg1 hepatic enzyme activity and prolongs lifespan relative to control eGFP-treated mice. A. Arg1 hepatic enzymatic activity measured at endpoints in two groups (Arg1-eGFP treated, n = 10 and eGFP-treated, n = 3), as well as from a cohort of untreated animals (same as in Fig.1C) for comparison. B. Corresponding Kaplan-Meier survival curves for two groups of mice. C. Immunostaining for Arg1 (FITC, green) and glutamine synthetase (Alexa Fluor 568, red) in liver sections from representative mice; mouse R10 (euthanized after 3 months of transgene expression from unrelated skin lesions; top panel) and mouse R4 (euthanized after 7 months of transgene expression; bottom panel). See Table 1 for details on individual mice.

Fig. 5

WT Arg1-expressing hepatocyte delivery only modestly extends lifespan of induced Arg1 deficiency despite significant re-population of donor livers. A. Kaplan-Meier survival curves of mice (n = 9, retrorsine-treated; n = 3, non-retrorsine-treated). B. Arg1 enzyme activity in livers of hepatocyte-repopulated mice at endpoint, as well as from a cohort of untreated animals (same as in Fig.1C) for comparison. C. Arg1 immunostaining (FITC, green) in a liver section from a representative hepatocyte-transplanted Arg1-deficient recipient mouse 15 weeks post-transplantation (middle panel) and 20 days post-tamoxifen-induced Arg1 knockout, along with corresponding low-magnification brightfield image (left panel) and a section from a non-treated C57BL/6 WT control mouse (right panel). D. Blood arginine levels at three time points post-tamoxifen (T) in retrorsine-treated mice. The two dotted lines represent arginine levels in two cohorts of untreated Arg1^fl mice (n = 15 total) measured on two separate occasions, as in Fig.3.