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.

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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.

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Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in corneaspecific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.5q31-linked corneal dystrophies are autosomal dominant disorders characterized by age-dependent progressive accumulation of protein deposits in the cornea followed by visual impairment. The pathological inclusions display two different types of ultrastructure: amyloid fibrils in lattice type I dystrophy (CDLI) and non-amyloid amorphous aggregates in Groenouw type I (CDGGI) and Reis-Bü cklers (CDRB). The fourth, Avellino corneal dystrophy (CDA), is a mixed form, which is characterized by amyloid and non-amyloid lesions in the same cornea. Mutations in the BIGH3 gene encoding for kerato-epithelin (KE) 1 were found to be responsible for this entire group of conditions (1). BIGH3 is expressed in many tissues, including cornea, and is up-regulated by trnasforming growth factor-␤ (2). The function of KE is not yet understood, although it is thought to be an extracellular matrix protein mediating cell adhesion (3).Population analysis revealed two hot spots of mutations in KE, Arg-124 and Arg-555 (4). There is a strong correlation between each particular mutation and the affected phenotype. Changes of Arg-555, R555W and R555Q, result in the conditions of non-amyloid nature, CDGGI and CDRB, respectively. Changes of Arg-124 result in a more complex pattern of deposition: R124C causes CDLI, a localized amyloidosis; R124H causes a mixed form, CDA. The most recently described mutation, R124L, results in non-amyloid deposition (5).Recently, by using immunohistology we demonstrated that KE is specifically co-localized with pathologic inclusions in all analyzed forms of the disease (6). This supports the idea that the mutated protein aggregates in pathologic deposits (simil...