Canavan disease, a rare early-onset human spongiform leukodystrophy: insights into its genesis and possible clinical interventions.

The brain contains high concentrations of the amino acid N-acetyl-l-aspartate (NAA) and its' glutamate adduct N-acetyl-l-aspartylglutamate (NAAG), both synthesized primarily by and stored in neurons. Upon depolarization both are exported to extracellular fluid (ECF) with NAA targeted to oligodendrocytes and NAAG targeted to astrocytes where they are hydrolyzed by specific enzymes. While the functions of these substances are incompletely known, their unique tri-cellular metabolism is apparently vital to normal brain function. Canavan disease (CD) is a globally occurring but rare early-onset human spongiform leukodystrophy associated with inborn genetic errors affecting the activity of aspartoacylase (ASPA), the enzyme highly expressed in oligodendrocytes that hydrolyzes NAA. Several hypotheses attempt to explain how the lack of ASPA activity results in the inability of oligodendrocytes to build or maintain axon-enveloping myelin sheaths, a failure reflected in the CD syndrome by profound neurological disturbances. Based on evidence provided by recent studies, as well as on descriptions of several atypical mild cases of CD and of a singular human case of an inborn error where NAA cannot be synthesized, we provide insights into the possible genesis of the CD syndrome and many of its phenotypic expressions. In this article we also evaluate current hypotheses, and discuss possible clinical interventions that may be of value in treatment of CD.

Differential expression of carnosine, homocarnosine and N-acetyl-L-histidine hydrolytic activities in cultured rat macroglial cells.

N-acetyl-L-histidine (NAH) and N-acetyl-L-aspartate (NAA) are representatives of two series of substances that are synthesized by neurons and other cells in the vertebrate central nervous system (CNS). Histidine containing homologs of NAH are beta-alanyl-L-histidine or carnosine (Carn) and gamma-aminobutyrl-L-histidine or homocarnosine (Hcarn). A homolog of NAA is N-acetylaspartylglutamate (NAAG). These substances belong to a unique group of osmolytes in that they are synthesized in cells that may not to be able to hydrolyze them, and are released in a regulated fashion to a second compartment where they can be rapidly hydrolyzed. In this investigation, the catabolic activities for NAH, Carn, and Hcarn in cultured macroglial cells and neurons have been measured, and the second compartment for NAH and Hcarn has been identified only with astrocytes. In addition, oligodendrocytes can only hydrolyze Carn, although Carn can also be hydrolyzed by astrocytes. Thus, astrocytes express hydrolytic activity against all three substrates, but oligodendrocytes can only act on Carn. The cellular separation of these hydrolytic enzyme activities, and the possible nature of the enzymes involved are discussed.

Effects of ethanol and of alcohol dehydrogenase inhibitors on the reduction of N-acetylaspartate levels of brain in mice in vivo: a search for substances that may have therapeutic value in the treatment of Canavan disease.

N-Acetylaspartate (NAA) is an important osmolyte in the vertebrate brain that participates in an intercompartmental metabolic cycle. It is synthesized primarily in neurons from L-aspartate (Asp) and acetyl-CoA and, after its regulated release, it is hydrolysed by aspartoacylase in an oligodendrocyte compartment to produce Asp and acetate. NAA also gives a strong 1H magnetic resonance spectroscopic signal, which has led to its widespread use as a neuronal marker. Utilizing this noninvasive technique, the NAA concentrations in normal brain and in brains exhibiting a variety of CNS disease syndromes have been studied. In normal individuals, the concentration of NAA has been observed to be relatively stable over long periods. However, in many CNS disease processes there are long-term changes in the level of NAA that have been considered to signal changes in neuron density or function. We report that the concentration of NAA in brain is malleable and that, in addition to normal endogenous variation or changes due to disease processes, it can be modified by a variety of exogenous drugs and other substances. As a result of this investigation, we have also been able to identify a new class of NAA-active compounds--pyrazole and pyrazole derivatives--that have the ability to reduce brain NAA concentrations in normal mice. The importance of these findings in understanding the NAA intercompartmental cycle, and its role in Canavan disease, a genetic aspartoacylase deficiency disease, are discussed.

Functions of N-acetyl-L-aspartate and N-acetyl-L-aspartylglutamate in the vertebrate brain: role in glial cell-specific signaling.

N-Acetyl-L-aspartate (NAA) and its derivative N-acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and L-glutamate, hydrolyzed to NAA and L-glutamate by astrocytes, and further hydrolyzed to L-aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell-specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.

Canavan's spongiform leukodystrophy: a clinical anatomy of a genetic metabolic CNS disease.

Canavan disease (CD) is a globally distributed early-onset leukodystrophy. It is genetic in nature, and results from an autosomally inherited recessive trait that is characterized by loss of the axon's myelin sheath while leaving the axons intact, and spongiform degeneration especially in white matter. There is also a buildup of N-acetyl-L-aspartate (NAA) in brain, as well as NAA acidemia and NAA aciduria. The cause of the altered NAA metabolism has been traced to several mutations in the gene for the production of aspartoacylase, located on chromosome 17, which is the primary enzyme involved in the catabolic metabolism of NAA. In this review, an attempt is made to correlate the change in NAA metabolism that results from the genetic defects in CD with the processes involved in the development of the CD syndrome. In addition, present efforts to counter the results of the genetic defects in this disease are also considered.

The existence of molecular water pumps in the nervous system: a review of the evidence.

Recently, the presence if both influx and efflux molecular water pumps (MWP's) in vertebrate cells has been reported. These appear to use a common mechanism; the intercompartmental cotransport of water uphill against a gradient as a hydrophylic osmolyte is transported down its own gradient, in a regulated fashion, by a membrane spanning cotransporter protein. In each case, the dwell time of the transported osmolyte is short in that it is metabolically converted and its products either eliminated or recycled, thereby maintaining the required high intercompartmental gradient. An influx water pump osmolyte has been identified as a sodium-glucose complex, and an efflux water pump osmolyte as N-acetylhistidine. These osmolytes may also be archetypal representatives of many other osmolytes with similar functions in a variety of cells. When recycled, the osmolyte metabolites appear to be dewatered during high affinity binding that is associated with their active transport back across the membrane prior to intracellular resynthesis of the osmolyte. Since these cyclical systems result in the pumping of water, they also appear to create a previously unrecognized motive force which results in the establishment of unidirectional transcellular water flows between apical and basolateral cell membranes. As neurons represent highly specialized forms of animal cells, and cells which are also extremely sensitive to changes in osmotic pressure, the presence of these water pumps in the CNS could be significant. There would be connotations with regard to how neurons regulate water balance and transaxonal flow as well as to how these factors affect the integrated function of the nervous system. In this article, evidence of the presence of MWP's in the nervous system, and how they might relate to aspects of both normal and abnormal brain function is reviewed.

Expression of aspartoacylase activity in cultured rat macroglial cells is limited to oligodendrocytes.

N-acetyl-L-aspartate (NAA) is an important osmolyte in the vertebrate brain and eye, and its cyclical metabolism is accomplished in two separate compartments. In the brain, NAA is synthesized primarily in neurons, and after its regulated release, NAA is hydrolyzed by aspartoacylase, which is present in a glial-associated compartment. However, the precise nature of this hydrolytic compartment has remained obscure. It has been proposed that one role of aspartoacylase in the central nervous system (CNS) is as part of a molecular water pump (MWP) that uses the NAA intercompartmental cycle to remove nerve cell metabolic water against a water gradient and that oligodendrocytes comprise the second compartment in this metabolic sequence. The absence of aspartoacylase activity in Canavan disease (CD), a rare early onset genetic spongiform leukodystrophy, is associated with CNS edema, intramyelinic swelling and a progressive loss of oligdendrocytes. In order to evaluate the MWP hypothesis and its possible relationship to the etiology of CD further, both oligodendrocytes and astrocytes obtained from neonatal rat brain were grown in culture and tested for the presence of aspartoacylase activity. The results of this study show for the first time that aspartoacylase activity is expressed only in oligodendrocytes. The meaning of this observation in understanding the function of the NAA metabolic cycle is discussed.

Function of the N-acetyl-L-histidine system in the vertebrate eye. Evidence in support of a role as a molecular water pump.

N-acetyl-L-histidine (NAH) is a major constituent of poikilotherm brain, eye, heart, and muscle, but for which there is no known function. NAH is characterized by high tissue concentrations, a high tissue/extracellular fluid (ECF) gradient, and by a continuous selective and regulated efflux into ECF. In the eye, there is a complete compartmentalization of the synthetic and hydrolytic enzymes, with synthesis of NAH from AcCoA and L-histidine (His) occurring in the lens, and its hydrolysis to acetate and His restricted to surrounding ocular fluids. Using 14C-isotopes, the cycling of NAH between lens and ocular fluids in a simple support medium consisting of NaCl (0.9%), Ca2+ (4 mEq/L) and D-glucose (5 mM) at pH 7.4 has previously been observed. In the present study, using the isolated lens of the goldfish eye, each of the components of that support medium has been individually varied in order to determine its effect on NAH release down its intercompartmental gradient. As a result of these and related studies, it is suggested that NAH may function as a metabolically recyclable gradient-driven molecular water pump. It is proposed that water influx or generation of metabolic water serves as the trigger mechanism to open a Ca-dependent gate for the release of NAH down its gradient, along with its associated water. Preliminary analyses suggest that in addition to its potential for multiple daily cycles, a strongly ionized hydrophilic molecule, such as NAH, may include a large power function as a result of its attraction to water, and it has been calculated that an aqua complex of each NAH molecule may have 33 dipole-dipole-associated water molecules as it passes into ECF. It is this unique combination of a capacity for multiple cycles per day, coupled with a large power function, that may allow for such an intracellular osmolyte to be present in relatively low concentration in comparison to total cellular osmolality, and yet to perform a large and important task with little expenditure of energy. With each NAH molecule recycled up to 10 times/d, and a power factor of 33, there could be 330 mmol of water transported/mmol of NAH each day. With typical NAH concentrations in brains of poikilothermic vertebrates of 5-10 mmol/kg, there is the potential for up to 3.3 mol (60 mL) of water to be removed each day/kg of brain, a value that represents about 8% of total brain water content. Dewatering of the released osmolyte would occur in two additional steps, consisting of its hydrolysis and the subsequent active uptake of its metabolites. It is also suggested that NAH is the archetype of several metabolically and structurally related cellular osmolytes found in both poikilotherms and homeotherms, for which there is similarly no known function, and these may form a family of cycling hydrophilic osmolytes that serve as molecular water pumps in a variety of tissues. These include the basic His containing derivatives: NAH, carnosine, anserine, ophidine, and homocarnosine, and the acidic aspartate derivatives: N-acetyl-L-aspartate (NAA) and N-acetyl-L-aspartylglutamate (NAAG). In each of these cases, the high intracellular/extracellular osmolyte gradient appears to be maintained by combining a hydrophilic protein amino acid with a nonprotein moiety to block its use in other intracellular metabolic pathways, and by blocking catabolism of the derivative by maintaining its hydrolytic enzyme in an extracytosolic membrane or extracellular compartment. Unlike other known water-regulating mechanisms, the proposed cellular system is unique in that as a water pump, it can function as a water regulator independently of extracellular solute composition or osmolality. Finally, based on the hypothesis developed, the NAH system would represent the first cellular water pump to be identified.

A review of phylogenetic and metabolic relationships between the acylamino acids, N-acetyl-L-aspartic acid and N-acetyl-L-histidine, in the vertebrate nervous system.

N-Acetyl-L-histidine (NAH) and N-acetyl-L-aspartic acid (NAA) are major constituents of vertebrate brain and eye with distinct phylogenetic distributions. They are characterized by high tissue concentrations, high tissue/extracellular fluid gradients, and a continuous regulated efflux into the extracellular fluid. As a result of parallel investigations over the past three decades, evidence has accumulated that suggests that the metabolism of NAA in the CNS of both homeothermic and poikilothermic vertebrates and the metabolism of NAH in the CNS of poikilothermic vertebrates are related. Tissue distribution and concentrations are similar, as well as timing of appearance during embryological development and their synthetic and degradative biochemistry. Both amino acids appear to be involved in a rapid tissue-to-fluid-space cycling phenomenon across a membrane. Evidence accumulating for each amino acid suggests a dynamic and important role in the CNS and the eye of vertebrates. A genetic disease in humans, Canavan's disease, is associated with NAA aciduria and aspartoacylase deficiency with concomitant accumulation of NAA and a spongy degeneration of the brain. In this article, evidence linking NAA and NAH metabolism is reviewed, and the hypothesis that NAA and NAH complement each other and are metabolic analogues involved with membrane transport is developed. Their enzyme systems also appear to exhibit plasticity in relation to osmoregulatory forces on an evolutionary time scale, with an apparent interface at the fish-tetrapod boundary.

Canavan disease. Analysis of the nature of the metabolic lesions responsible for development of the observed clinical symptoms.

Canavan disease (CD), a rare recessive autosomal genetic disorder, is characterized by early onset and a progressive spongy degeneration of the brain involving loss of the axon's myelin sheath. After a relatively normal birth, homozygous individuals generally develop clinical symptoms within months, and usually die within several years of the onset of the disease. A biochemical defect associated with this disease results in reduced activity of the enzyme N-acetyl-L-aspartate amidohydrolase (aspartoacylase) and affected individuals have less ability to hydrolyze N-acetyl-L-asparate (NAA) in brain and other tissues. As a result of aspartoacylase deficiency, NAA builds up in extracellular fluids (ECF) and is excreted in urine. From an analysis of the NAA biochemical cycle in various tissues of many vertebrate species, evidence is presented that there may be two distinct NAA circulation patterns related to aspartoacylase activity. These include near-field circulations in the brain and the eye, and a far-field systemic circulation involving the liver and kidney, the purpose of which in each case is apparently to regenerate aspartate (Asp) in order for it to be recycled into NAA as part of the still unknown function of the NAA cycle. Based on the authors' analysis, they have also identified several metabolic outcomes of the genetic biochemical aspartoacylase lesion. First, there is a daily induced Asp deficit in the central nervous system (CNS) that is at least six times the static level of available free Asp. Second, there is up to a 50-fold drop in the intercompartmental NAA gradient, and third, the ability of the brain to perform its normal intercompartmental cycling of NAA to Asp is terminated, and as a result, the only remaining long-term source of Asp for NAA synthesis is via nutritional supplementation of Asp or its metabolic precursors. Finally, the authors identify a potential maternal-fetal interaction that may be responsible for observed normal fetal development in utero, and that provides a rationale for, and suggests how, CD might respond to far-field nutritional, transplantation, or genetic engineering techniques to alter the course of the disease.

Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase.

1. The brain and ocular fluid of skipjack tuna (Katsuwonus pelamis) contained high levels of N-acetylhistidine deacetylase. 2. This enzyme had a molecular weight of about 120,000 and was activated by zinc or cobaltous ions. 3. Cod (Gadus callarias) brain, ocular fluid and muscle contained a similar metal-activated thiol hydrolase, the muscle enzyme being known as anserinase. 4. The purified enzymes hydrolyzed N-acetylhistidine, carnosine, homocarnosine, anserine and certain other dipeptides. 5. Their specificity resembled that of hog kidney homocarnosinase. 6. In both fish, brain and ocular fluid were rich sources of this hydrolase, whereas muscle contained only trace amounts.