Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase Deficiency by Neonatal Gene Therapy.

UNLABELLED: Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT: These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

l-Citrulline Protects from Kidney Damage in Type 1 Diabetic Mice.

RATIONALE: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of l-arginine (l-arg), the substrate for endothelial nitric oxide synthase (eNOS), failed to improve vascular function. l-Citrulline (l-cit) supplementation not only increases l-arg synthesis, but also inhibits cytosolic arginase I, a competitor of eNOS for the use of l-arg, in the vasculature. AIMS: To investigate whether l-cit treatment reduces DN in streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice and rats and to study its effects on arginase II (ArgII) function, the main renal isoform. METHODS: STZ-C57BL6 mice received l-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and l-cit-treated STZ-rats were evaluated. RESULTS: l-Citrulline exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis, and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, l-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 weeks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater blood urea nitrogen levels, hypertrophy, and dilated tubules than diabetic wild type (WT) mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic WT animals. l-Cit also restored nitric oxide/reactive oxygen species balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, l-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1ß and IL-12(p70) generation in the human proximal tubular cells. CONCLUSION: l-Citrulline supplementation established an anti-inflammatory profile and significantly preserved the nephron function during T1D.

Clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy.

A patient diagnosed at 9 months with a milder form of propionic acidemia was functioning at a near normal intellectual level and a normal neurological level at age 8. After 2-week history of feeling "poorly" but functioning normally, she became acutely ill and succumbed to heart failure and ventricular fibrillation in 12 h. At post-mortem the heart was hypertrophied and had low carnitine levels, despite carnitine supplementation and repeatedly normal plasma carnitine levels. The findings in this patient provide a possible mechanism for the cardiac complications that are becoming more apparent in propionic acidemia.

Expression of the liver form of arginase in erythrocytes.

Arginase I (AI) has a critical function in mammalian liver as the final enzyme in the urea cycle responsible for the disposal of ammonia from protein catabolism. AI is also expressed in various extrahepatic tissues and may play a role in regulating arginine levels and in providing ornithine for biosynthetic reactions that generate various critical intermediary metabolites such as glutamate, glutamine, GABA, agmatine, polyamines, creatine, proline, and nitric oxide. AI is expressed in red blood cells (RBCs) only in humans and certain higher primates. Macaca fascicularis has been identified as an evolutionary transition species in which RBC-AI expression is co-dominantly regulated. The M. fascicularis AI gene was analyzed to understand AI expression in erythrocytes. Erythroid progenitor cells [nucleated red blood cells (nRBCs)] isolated from cord blood were utilized to demonstrate AI expression by immunocytochemical staining using anti-AI antibody. Introduction of EGFP reporter vectors into nRBC showed that the proximal 1.2 kbp upstream of the AI gene is sufficient for AI expression. Expression of a second arginase isoform, AII, in nRBCs was discovered by cDNA profiling. This contrasts with mature fetal or adult RBCs which contain only the AI protein. In addition, an alternatively spliced AI (AI(')) variant was observed from erythroid mRNA analysis with an alternative splice acceptor site located within intron 2, causing the insertion of eight additional amino acids yet retaining significant enzymatic activity.