A rare leukodystrophy, or spongy degenerative disease involving brain white matter was described in an infant by Myrtelle May Moore Canavan in 1931, which she tentatively identified as Schilder’s disease, or diffuse cerebral sclerosis of Schilder (Canavan, 1931
). This fatal infantile condition was later determined to be a unique autosomal inheritable white matter degenerative disease by van Bogaert and Bertrand in 1949 (van Bogaert and Bertrand, 1949
). Despite the fact that these clinicians were the first to correctly identify Canavan disease as a distinct disorder from Schilder’s sclerosis, the disease is nonetheless most often referred to as Canavan disease. The genetic disorder has been designated by other names in the literature, including “Canavan-van Bogaert-Bertrand disease” and “spongy degeneration of the brain, van Bogaert-Bertrand type”. A number of early reviews on Canavan disease are available (Adachi et al., 1973
; Hogan and Richardson, Jr., 1965
; Pratt, 1972
). Adachi et al.
recognized three forms of Canavan disease based on time of onset; congenital, infantile and juvenile (Adachi et al., 1973
), however the vast majority of confirmed cases fit into the infantile category (Matalon et al., 1995
Infants with Canavan disease appear normal at birth, but usually show signs of delayed development and decreased muscle tone (hypotonia), including head lag between 2 and 6 months of age. By one year, macrocephaly is often evident, and motor development is severely impaired. Infants later develop optic atrophy, and hypotonicity converts to limb stiffness and spasticity. The affected children become increasingly debilitated with age, often including seizures and an inability to move voluntarily or swallow. Death typically occurs before adolescence, but some Canavan patients with milder forms survive into their 20’s or beyond.
It was not until the 1980’s that the connection between elevated NAA levels and a progressive demyelinating cerebral atrophy was discovered. Kvittingen and coworkers first described a condition of “N
-acetylaspartic aciduria” in a child with progressive cerebral atrophy, wherein the excretion of NAA was dramatically elevated in the urine (Kvittingen et al., 1986
). They observed demyelination in cerebral CT scans from the child, and as the child aged they noted cerebral atrophy and enlarged ventricles. Based on the dramatic increase in urinary NAA excretion, these authors surmised that this infantile leukodystrophy might be due to either increased synthesis or reduced degradation of NAA. Shortly thereafter, N
-acetylaspartic aciduria was identified as being due to a deficiency of ASPA activity by Hagenfeldt and colleagues, who found reduced ASPA activity in skin fibroblasts from a child with severe leukodystrophy. They proposed that the observed dysmyelination in the CNS was due to a failure of NAA to serve as an acetate carrier of acetyl groups from mitochondria to cytoplasm for lipogenesis (Hagenfeldt et al., 1987
). Matalon and colleagues were the first to connect N
-acetylaspartic aciduria and ASPA deficiency specifically to Canavan disease by showing high NAA levels in urine, and a lack of ASPA activity in skin fibroblasts in 3 children with Canavan disease (Matalon et al., 1988
). They proposed three possible mechanisms whereby a lack of ASPA activity could lead to the spongy degeneration and dysmyelination observed in Canavan disease patients. First, because NAA had been shown to be involved in the production of cerebronic acid (Shigematsu et al., 1983
), Matalon et al.
proposed that a defect in myelination could be responsible for the progression of the leukodystrophy. Second, they proposed that because aspartate can act as a neurotransmitter (Bradford and Thomas, 1969
), the lack of deacetylase activity might lead to a disruption of aspartate neurotransmission, resulting in neurological disturbances. Finally, they suggested that high levels of brain NAA might directly lead to myelin damage and spongy degeneration. Determining which of these mechanisms were involved in Canavan disease pathogenesis would require the cloning of the ASPA gene, and further basic research.
The human ASPA gene was cloned by Kaul and colleagues in 1993, as discussed above (section 2.2.) (Kaul et al., 1993
). Once Canavan disease was determined to be a specific autosomal recessive genetic disorder, it was assigned a Mendelian Inheritance in Man (MIM) number (Hamosh et al., 2005
) with the designation of #271900. Early genetic studies found that two mutations were prevalent among Ashkenazi Jewish patients with Canavan disease (Kaul et al., 1994a
). A missense mutation at codon 285 resulted in a substitution of glutamate to alanine, and was found to account for 83.6 % mutations identified in 104 alleles from 52 unrelated Ashkenazi Jewish patients. A nonsense mutation was found in 13.4% of the alleles from Jewish patients at codon 231, which converts tyrosine to a stop codon. The incidence of mutations in the ASPA gene are far less common in non-Jewish patients, and the mutations are distinct, and more diverse (Kaul et al., 1996
; Sistermans et al., 2000
). The most prevalent non-Jewish mutation was identified in codon 305, a missense mutation substituting alanine for glutamic acid. This mutation was observed in 35.7% of the 70 alleles from 35 unrelated non-Jewish patients (Kaul et al., 1994
). Fifteen other mutations were detected in 24 other Canavan disease patients. Recently additional mutations have been reported, in some cases with the children dying immediately after birth (Zeng et al., 2002
). The diverse number of mutations associated with Canavan disease limits the usefulness of DNA analyses for prenatal diagnosis in non-Jewish parents. However, preimplantation genetic diagnosis has recently been successfully employed to screen in vitro
embryos from Ashkenazi Jewish parents prior to implantation (Yaron et al., 2005
Two animal models of Canavan disease exist (see Section 3.3. below); the so-called Tremor rat
(Serikawa et al., 1987
) and the ASPA knockout mouse (Matalon et al., 2000
). The Tremor rat
model is a stable line developed from a naturally occurring mutant with a genomic deletion on chromosome 11 spanning 4 genes, including the aspartoacylase gene, olfactory receptor gene, vanilloid receptor subtype I gene, and the calcium/calmodulin-dependent protein kinase IV gene (Kitada et al., 2000
). The Tremor rat
shows no ASPA activity in brain, and greatly reduced activity in kidney, and also exhibits increased brain NAA levels (Kitada et al., 2000
). Tremor rats
exhibit muscular tremors starting at about 2 weeks of age, which give way to absence-like seizure activity in later development. Pathology in the CNS involves white matter spongiform degeneration and hypomyelination (Kondo et al., 1991
). Similarly, homozygous ASPA −/− knockout mice exhibit subcortical and white matter vacuolation. In addition, they fail to thrive, exhibit macrocephaly, display tremors, and some develop seizures by 6 months of age (Matalon et al., 2000
). Homozygous ASPA −/− mice lack detectable ASPA activity, and show impaired motor activity as well as a nine fold increase in NAA levels in urine as compared with control mice. Because ASPA is strongly expressed in kidney proximal tubule cells it might be expected that ASPA deficiency would result in kidney pathologies, but this does not appear to be the case, as reports of Canavan disease patients or ASPA-deficient animals do not make note of observable kidney damage or disease.
Despite the great deal of work done over the last 5 decades to understand this fatal inheritable CNS leukodystrophy, there is still controversy over the specific etiology resulting in progressive white matter disease and subcortical vacuolation. Experimental and clinical attempts to halt or reverse the pathogenesis of Canavan disease will be discussed below (see sections 3.3. and 3.4.).
3.1. NAA neurotoxicity
An unresolved issue concerning NAA involves its potential toxicity to neurons or oligodendrocytes when the concentration is substantially elevated in the brain, as in the case in Canavan disease (Kitada et al., 2000
; Leone et al., 1999
). Despite the established connection between mutations in the gene for ASPA in Canavan disease, and the lost capacity to deacetylate NAA, the specific connection between ASPA deficiency and the failure of proper CNS development remains controversial (Matalon et al., 1995
; Matalon and Michals-Matalon, 1998
). It has been proposed that lack of deacetylase activity against NAA leads to toxic increases in the concentration of NAA in the brain (Akimitsu et al., 2000
; Kitada et al., 2000
; Leone et al., 1999
; Leone et al., 2000
). The level of extracellular NAA may be a critical factor in determining if it has toxic effects. Pliss and colleagues reported that injection of 0.25 micromoles of NAA into the lateral cerebral ventricles of rats did not induce any detectable neuronal death in the hippocampus, (Pliss et al., 2003
) whereas Akimitsu et al.
reported that injection of 8 micromoles of NAA into the lateral ventricle of rats induced strong seizures (Akimitsu et al., 2000
). However, these same investigators observed no effects after injecting 2 micromoles of NAA into the lateral ventricles of normal rats (Kitada et al., 2000
). Seizures are one of the symptoms of late stage Canavan disease, but it has not been conclusively shown that elevated NAA levels are responsible for the seizure activity. Akimitsu and colleagues used exceptionally high concentrations of NAA (~0.5M to 1M) to elicit seizure activity, so there is some question about physiological relevance to Canavan disease, where the concentration may be elevated no more than 2 fold (Kitada et al., 2000
). This translates to approximately 15 to 25mM, which is far below the 500mM to 1M concentrations injected by Akimitsu and colleagues to induce seizures in rats.
Using organotypic hippocampal slice preparations, Tranberg and colleagues showed that the addition of 10 mM NAA to the culture medium had no effect on increasing cell death after 24 hours, indicating that NAA is not toxic to neurons or glia at extracellular concentrations far higher than normal (Tranberg et al., 2004
). The potential toxic or excitotoxic actions of NAA have not been fully explored, but the evidence suggests that high levels of NAA in the brains of Canavan disease patients may be involved in some aspects of the pathogenesis, possibly by inducing seizure activity. However, it is also possible that the observed hypomyelination could also lead to seizure activity by way of disrupting normal neurotransmission.
3.2. NAA-related osmotic/hydrostatic pressure in Canavan Disease
A well-cited hypothesis concerning the etiology of Canavan disease is that the lack of deacetylase activity against NAA leads to osmotic dysregulation in the brain, which then results in a buildup of NAA-associated water in the extracellular fluid, resulting in the dysmyelination and subcortical vacuolation observed in Canavan patients (Baslow, 2000a
; Baslow, 2002
; Baslow, 2003a
; Baslow, 2003b
; Baslow and Guilfoyle, 2006
). Currently to our knowledge, there is no direct evidence that has shown that excess NAA in the brain causes dysmyelination through osmotic damage, but nonetheless, this hypothesis is often invoked in discussions of the pathogenesis of Canavan disease. The osmotic damage hypothesis of Canavan disease may have had its origins in work on osmoregulation in ocular tissues of fish (Baslow and Yamada, 1997
-acetylhistidine is present at high concentrations in the lens of poikilotherms including fish, and Baslow proposed that it could be involved in tissue fluid balance (Baslow, 1997
). It was also proposed that what appeared to be an inverse phylogenetic relationship between the concentrations of NAA and N
-acetylhistidine in brain, eye and other tissues might indicate that they served similar membrane transport or fluid balance functions in different physiological contexts. Baslow proposed that the synthetic and hydrolytic compartments for NAA and N
-acetylhistidine were distinct and involved a cellular or fluid boundary, and therefore the two compounds served similar transport or fluid balance functions in different organisms.
Based on subsequent studies with 14
C-isotopes and excised lens from goldfish, Baslow proposed that N
-acetylhistidine acted as a “molecular water pump” in which the solute flowed down its concentration gradient through calcium channels, and removed 33 millimoles of water per millimole of N
-acetylhistidine against a water gradient (Baslow, 1998
). Further, he proposed that the N
-acetylhistidine molecular water pump was an archetype for similar molecular water pump systems in derived vertebrates, including NAA synthesis, transport and breakdown in the brains of birds and mammals. This molecular water pump hypothesis was elaborated in later publications, wherein Baslow proposed that NAA is a significant neuronal osmolyte and that the inability to deacetylate NAA in oligodendrocytes leads to increased osmotic pressure in the intracellular space between axons and their myelin ensheathment, leading to the intramyelinic splitting and intralamellar edema observed in Canavan disease (Baslow, 1999
; Baslow, 2000a
). Baslow and Guilfoyle used magnetic resonance spectroscopy of samples of NAA in water to estimate the degree of hydration of NAA in order to determine the degree to which NAA could transport water across neuronal membranes (Baslow and Guilfoyle, 2002
). They calculated a hydration factor of 32:1 for NAA, and suggested that NAA was a good candidate for a neuronal osmolyte which acted in concert with a transport protein as a molecular water pump.
Baslow outlined the criteria for meeting the status of a so-called molecular water pump. “In theory many solutes could be used for this purpose as long as certain conditions were met. These conditions include the requirement for a suitable cotransporter protein with associated gate and trigger mechanism for regulation of solute transport, a method for cycling the solute by means of which its intercompartmental gradient can be continuously reestablished, and a coupled energy source”
) (page 89). Currently, NAA does not meet all of these criteria for molecular water pump status, including the fact that no cotransport protein has been identified, and there is only scant evidence on possible gating mechanisms that specifically regulate NAA release from neurons. Results with organotypic hippocampal slices have been instrumental in demonstrating that NAA is not released under hyper-osmotic conditions, but that it is released in a calcium-dependent manner upon stimulation of neuronal NMDA receptors (Tranberg et al., 2004
). NAA release in this system was not stimulated by potassium depolarization, but was prolonged for 20 minutes after a 5 minute application of 60 μM NMDA. The authors noted that the efflux was specific to selected organic ions, including NAA, taurine, glutathione and phosphoethanolamine, but they did not propose a physiological role for the regulated release. Receptor-meditated, calcium-dependent release of NAA from neurons has not been reported by other research groups, but if confirmed, these findings suggest that glutamatergic neuronal activity is linked to NAA efflux from neurons, and that this may be a regulated physiological process (see section 7.1.1. below).
Many different mechanisms are involved in brain water regulation, and cotransport associated with the active or passive movements of various solutes into or out of neurons is one key factor. As such, the solutes that are most actively redistributed in response to osmotic stress must be the same hydrated solutes involved in moving water into and out of neurons. As mentioned above (section 2.3.) the major solutes that are redistributed in response to osmotic stress in the brain include glutamine, glutamate, taurine, choline and myo-inositol (Verbalis, 2006
). For this reason, it seems likely that NAA is only a minor contributor to the osmotically active pool of solutes present in neurons that can respond to changes in osmolarity. Finally, until an osmotically-sensitive NAA export protein is found in neurons, the NAA molecular water pump hypothesis remains speculative.
3.3. Gene Transfer Therapy for Canavan Disease
Based on the assumption that the primary etiology of Canavan disease involves toxic NAA concentration increases in the brain (Leone et al., 1999
), possibly causing osmotic dysregulation and intramyelinic water accumulation, adenoviral transfer of the ASPA gene to the brains of humans has been performed in an to attempt to reverse brain edema and vacuolation (Janson et al., 2002
; Leone et al., 2000
). No follow up studies showing significant myelination or motor improvements in these children have been published to date, so it is difficult to assess to what extent any pathologies were ameliorated. However, a number of studies have been done using the ASPA-null mutant Tremor rat
(see section 3. above), and the ASPA −/− knockout mouse model of Canavan disease, and these studies have yielded somewhat conflicting results with regard to the efficacy of this approach.
The Tremor rat
has been proposed to be an animal model for petit mal seizures due to the fact that the rats exhibit so-called absence-like seizures, which are characterized by sudden immobility, with the coincident appearance of 5–7 Hz spike–wave-like complexes in cortical and hippocampal electroencephalograms (Seki et al., 2002
; Serikawa et al., 1987
). The Tremor rat
ASPA gene transfer studies that have been done in the last several years have made use of adenoviral gene vectors to deliver the ASPA gene to the brain. The investigations by Seki and colleagues involved delivery of replication-deficient recombinant adenoviral gene vectors containing the ASPA gene into the lateral cerebral ventricles, which was reported to primarily induce expression in ependymal cells of the ventricle lining (Seki et al., 2002
; Seki et al., 2004
). These two studies were done on different mutant rat strains, both of which lacked the gene for ASPA, and which display absence-like seizures. In both studies, a very transient reduction in seizure activity was noted at 1 week after gene transfer, but this effect was not maintained by 10 days after transfer, and no improvement in animal survival rates was observed. The authors noted that the viral vector they employed induced immune system responses which may have interfered with the efficacy of the treatment.
The most recent adenoviral gene transfer studies employed improved gene transfer technology using the recombinant adeno-associated virus serotype 2 vector (AAV-2) (Klugmann et al., 2005
; McPhee et al., 2005
). In one of these gene transfer studies in Tremor
rats, NAA levels were reduced, and seizure activity was diminished, but brain vacuolation and dysmyelination were unaffected, suggesting that these pathological features of the disease are not mediated by excessive NAA concentrations (Klugmann et al., 2005
). More importantly, no motor improvements were observed in the Tremor rats
after gene transfer. In the other study, reduced NAA levels and somewhat improved motor functions were reported (McPhee et al., 2005
). However, motor improvements were modest, and statistical significance in motor performance improvement was determined between Tremor rats
which had received adenovirally transferred green fluorescent protein gene, versus Tremor rats
which had received adenovirally transferred ASPA gene. Statistical significance between naïve (no gene transfer) Tremor rats
and those that received adenovirally transferred ASPA was not reported. Because Tremor rats
with green fluorescent protein gene transfer performed more poorly on the motor tests than the naïve Tremor rats
, the significance of the reported improvements in motor function may be questionable.
An ASPA −/− knockout mouse Canavan disease model has been developed (Matalon et al., 2000
), but somewhat less work has been done with this model to date. A single reported trial of recombinant AAV-ASPA treatment has been attempted with this model (Matalon et al., 2003
). In that study, recombinant AAV-ASPA gene transfer provided excellent ASPA expression and activity in and around vector injection sites, and reduced NAA levels as detected by magnetic resonance spectroscopy. Further, histopathological examinations revealed locally reduced brain vacuolation, particularly in thalamus, in animals that received AAV-ASPA. In spite of the histopathological improvements, no improvements in motor performance, brain myelination or development were reported after ASPA gene transfer in ASPA −/− mice.
A potential problem with gene transfer therapy for ASPA deficiency is that current technology is limited by the available vectors for introducing genes into different brain cell populations. ASPA is present in the brain primarily in oligodendrocytes, but NAA is produced primarily in neurons, so the anabolic and catabolic compartments are distinct. Adenoviral gene transfer (AAV-2) involves the use of a neurotrophic viral vector to deliver a specific expression cassette to target cells, and it was shown that the majority of cells which expressed delivered genes were neuronal (McPhee et al., 2005
). As such, the segregation of the NAA synthetic and degradative compartments was not restored by gene therapy. This inability to direct the vector to oligodendrocytes may limit the usefulness of ASPA gene therapy until vectors which target other cell types in the brain are developed. While gene therapy is a very promising technique, the technologies necessary for proper integration of selected genes into the correct cell populations in the brain remain to be developed, and additional basic research is required.
3.4. Potential Acetate Supplementation Therapy for Canavan Disease
Based on the idea that the loss of ability to metabolize NAA in the catabolic oligodendrocyte compartment of NAA metabolism results in intramyelinic osmotic damage, Baslow concluded that the etiology of Canavan disease does not involve an inability of oligodendrocytes to produce myelin (Baslow, 2000a
). He stated his conclusion; “The demyelination in CD [Canavan disease] is probably not owing to the inability of oligodendrocytes to produce myelin, but to the continuous destruction of myelinating oligodendrocytes themselves by the NAA osmotic pressure generated in the sealed paranodal and the internodal regions”
(page 67). However, based upon a growing body of evidence connecting NAA-derived acetate to aspects of myelin lipid formation (Chakraborty et al., 2001
; D’Adamo, Jr. et al., 1968
; D’Adamo, Jr. and Yatsu, 1966
), we and others have proposed that the primary etiological mechanism in the pathogenesis of Canavan disease is reduced lipid synthesis due to a reduced supply of NAA-derived acetate in the brain during myelination (Hagenfeldt et al., 1987
; Madhavarao et al., 2005
; Mehta and Namboodiri, 1995
) (see section 5. below).
ASPA acts to remove acetate from NAA in oligodendrocytes, and therefore ASPA activity against NAA would be expected to increase free acetate levels in these cells. A loss of ASPA enzyme function during postnatal myelination could then lead to an acetate deficiency in oligodendrocytes. Because acetate in the form of acetyl coenzyme A is a key building block of lipids, a lack of ASPA activity in oligodendrocytes could theoretically limit lipid synthesis in the myelinating cells of the brain when axonal myelination is maximal during early infancy. In this regard, we have observed an approximately 80% reduction in free acetate levels in the brains of ASPA −/− mice at the time of peak myelination (Madhavarao et al., 2005
). For these reasons we have proposed dietary acetate supplementation therapy, for example with glyceryl triacetate (GTA), as a possible treatment for Canavan disease (Madhavarao et al., 2005
; Mathew et al., 2005
; Mehta and Namboodiri, 1995
; Namboodiri et al., 2006b
GTA is a triester with 3 acetate groups coupled to glycerol. Esterases present in most tissues of the body can cleave the acetate moieties, generating free acetate. Dosing experiments in mice demonstrated that GTA was 10 to 20 times more effective at delivering acetate to the brain than calcium acetate, and that it was well tolerated by developing mice (Mathew et al., 2005
). If substantially elevated levels of acetate can be maintained in oligodendrocytes through dietary acetate supplementation during postnatal myelination, this should theoretically correct the acetate deficiency that occurs in Canavan disease. It is likely that acetate supplementation of Canavan patients would have to be initiated within the first few months after birth in order to ensure that critical neurodevelopmental stages requiring myelination are accomplished on schedule, including visual system development. Other potential problems would not be addressed by acetate supplementation including excess NAA concentrations in the brain, reduced aspartate levels in oligodendrocytes, and possibly reduced neuronal respiration on glutamate due to product inhibition of the mitochondrial enzymes Asp-NAT and aspartate aminotransferase (see section 6.3. below). If these turn out to contribute significantly to the pathogenesis of Canavan disease, then acetate supplementation therapy may only be partially effective as a treatment.
In a collaborative study between the Namboodiri and Matalon laboratories, preliminary results have been obtained with a relatively small number of ASPA −/− mice supplemented with GTA in the diet. The results indicate reduced vacuolization and reduced water content in the brain, improved motor function, increased lipid content and reduced mortality in homozygous ASPA −/− mice treated with GTA (unpublished observations). Because of the small numbers of treated mice, these results should be considered preliminary, and more conclusive data with a larger number of mice will be needed in order to find out whether GTA supplementation can prevent the severe phenotype associated with ASPA deficiency.
Breeding ASPA −/− mice and Tremor rats is difficult because heterozygote breeding pairs are required, which produce litters averaging only 25% ASPA −/− pups, and because the affected animals do not thrive and often die soon after birth. As such, many breeding cycles are required to obtain sufficient numbers of ASPA deficient pups. We are planning studies with GTA supplementation on a greater number of homozygous animals so the efficacy of acetate can be determined more conclusively as a therapeutic agent for the treatment of Canavan disease.