Creatine is synthesized from arginine by L-arginine:glycine amidinotransferase (AGAT) and S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) and can be taken up by cells by creatine transporters (CRT). While creatine is mainly synthesized by the liver and the kidney, most of other tissues, including the brain, also express AGAT and GAMT. There is evidence that the permeability of the blood-brain barrier (BBB) for creatine is limited, suggesting that the brain is dependent on its own creatine synthesis. In order to better understand creatine synthesis and transport in the central nervous system (CNS), we studied the regional distribution of cells expressing AGAT, GAMT and the creatine transporter CRT1 in the adult rat brain by non-radioisotopic in situ hybridization. AGAT and GAMT presented an ubiquitous neuronal and glial expression, whereas CRT1 was present in neurons and oligodendrocytes throughout the brain, but not in astrocytes. This indicates that all cells in the CNS can synthesize creatine from arginine. The absence of expression of CRT1 in astrocytes and particularly in those contacting capillary endothelial cells (BBB) reinforces the idea that under normal conditions the creatine used by the brain is synthesized mainly in the CNS. Furthermore, the expression of CRT1 by neurons and oligodendrocytes indicates that creatine trafficking is possible in those brain areas of main creatine consumption.

Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH 4 Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.

2Nitric oxide (NO) is synthesized from a unique precursor, arginine, by nitric oxide synthase (NOS). In brain cells, arginine is supplied by protein breakdown or extracted from the blood through cationic amino acid transporters (CATs). Arginine can also be recycled from the citrulline produced by NOS activity, through argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) activities, and metabolized by arginase. NOS, AS and AL constitute the so-called citrulline-NO cycle. In order to better understand arginine transport, recycling and degradation, we studied the regional distribution of cells expressing CAT1, CAT3, AS, AL, neuronal NOS (nNOS) and arginase II (AII) in the adult rat brain by nonradioisotopic in situ hybridization. CAT1, AL and AII presented an ubiquitous neuronal and glial expression, whereas CAT3 and AS were confined to neurons. nNOS was restricted to scattered neurons and a few brain nuclei and layers. We demonstrate by this study that cells expressing nNOS all appear to express the entire citrulline-NO cycle, whereas numerous cells expressing AL do not express AS. The differential expression of these genes within the same anatomical structure could indicate that intercellular exchanges of citrulline-NO cycle metabolites are relevant. Thus vicinal interactions should be taken into account to study their regulatory mechanisms.

Hyperammonemia in the brain leads to poorly understood alterations of nitric oxide (NO) synthesis. Arginine, the substrate of nitric oxide synthases, might be recycled from the citrulline produced with NO by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). The regulation of AS and AL genes during hyperammonemia is unknown in the brain. We used brain cell aggregates cultured from dissociated telencephalic cortex of rat embryos to analyze the regulation of AS and AL genes in hyperammonemia. Using RNase protection assay and non-radioactive in situ hybridization on aggregate cryosections, we show that both AS and AL genes are induced in astrocytes but not in neurons of aggregates exposed to 5 mM NH4Cl. Our work suggests that the hyperammonemic brain might increase its recycling of citrulline to arginine.

No abstract

L-Arginine is a substrate for the synthesis of proteins, nitric oxide (NO), creatine, urea, proline, glutamate, polyamines and agmatine. In the central nervous system (CNS), arginine is extracted from the blood and exchanged by cells through carriers called cationic amino acid transporters (CAT) and belonging to the so-called system y+. In order to better understand the arginine transport in the CNS, we studied in detail the regional distribution of the cells expressing the CAT2(B) transcript in the adult rat brain by non-radioisotopic in situ hybridization. We show that CAT2(B) is expressed in neurons and oligodendrocytes throughout the brain, but is not detected in astrocytes. The pattern of localization of CAT2(B) in the normal adult rat brain fits closely that of CRT1, a specific creatine transporter. Our study demonstrates that the in vivo expression of CAT2(B) differs from that reported in vitro, implying that local cellular interactions should be taken into account in studies of gene regulation of the CAT2(B) gene. Our work suggests that CAT2(B) may play a role in case of increased NO production as well as arginine or creatine deficiency in the brain.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from lifethreatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed threedimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency.Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable. K E Y W O R D S 3D organotypic brain cell cultures, argininosuccinate lyase (ASL) deficiency, argininosuccinic acid, astrocytes, creatine, guanidino compounds, guanidinosuccinic acid, neurons, neurotoxicity, urea cycle disorders Johannes Häberle and Olivier Braissant contributed equally to this study.