Metabolic acetate therapy improves phenotype in the tremor rat model of Canavan disease

Once the genetic basis of Canavan disease was discovered (Matalon et al. 1988), the primary question centered on whether the etiology involved toxic buildup of NAA, or impaired brain metabolism associated with the inability to catabolize NAA. Under the assumption that excess NAA was toxic, ASPA gene transfer appeared to be the most logical method of reversing the effects of ASPA deficiency in Canavan patients. This method has been attempted in animal models of Canavan disease (Klugmann et al. 2005; McPhee et al. 2005) and in a group of over 20 children with the disease (Janson et al. 2002; Leone et al. 2000). No positive long-term outcomes on motor function have been reported using ASPA gene transfer to ameliorate ASPA deficiency. Taking the approach that failure to metabolize NAA leads to a brain acetate deficiency during postnatal development and myelination, the current investigation is the first in a series of ongoing studies into the potential use of GTA supplementation for the treatment of Canavan disease.

We found that GTA is effective in significantly reducing the severe phenotypic sequelae of ASPA enzymatic deficiency in the tremor rat model of Canavan disease over the course of a 4-month study. In tremor rats treated with GTA, motor performance was significantly improved, with the greatest improvements occurring later in the longitudinal study (Fig. 2). Further, galactocerebroside content in myelin was modestly (∼20%) but significantly increased (see Table 3). Galactocerebrosides are one of the functionally important classes of myelin-associated lipids that are significantly reduced in Canavan disease patients and ASPA-deficient animal models (Madhavarao et al. 2005; Wang et al. 2009). Additionally, vacuolation in the brain and spinal cord was found to be modestly reduced with GTA treatment (Fig. 8), and the decreased vacuolation was positively correlated with improved performance on the Rotarod treadmill test (Fig. 4). These results support the acetate deficiency hypothesis of Canavan disease. Ongoing studies with ASPA -/- knockout mice (Matalon et al. 2000) are underway to confirm these results in another model system.

In previous studies, we found that administration of GTA to mice at a dose of 5.8 g/kg increases acetate levels in the brain over 15-fold within an hour, and the levels remain elevated for several hours after administration (Mathew et al. 2005). Further, short and long term toxicity studies have shown that GTA is well tolerated (Bailey et al. 1991, 1992; Madhavarao et al. 2009). GTA does not require specific transport mechanisms to enter the cytoplasm of cells. Once internalized in cells, GTA is broken down to acetate and glycerol by the action of non-specific esterases. These factors make GTA especially useful for acetate delivery to the brain. Plasma acetate derived from GTA breakdown in the intestine, liver and bloodstream can also be taken up by the brain.

The current GTA treatment partially reversed the severe motor dysfunction of tremor rats. Rotarod treadmill motor performance scores in treated female tremor rats reached approximately 50% of those achieved by age-matched wild-type rats. This compares favorably with performance scores for untreated female tremor rats, which were only about 15% of those seen with wild-type rats. So, despite significant improvement in motor performance, the treated rats still had substantial leeway for further improvement. Additionally, GTA treatment improved the spongiform vacuolation in brain only partially, and substantial vacuolation was still observed in GTA-treated tremor rats. Finally, myelin lipid content was improved modestly but did not reach wild-type levels, indicating that further optimization of the treatment regimen is necessary to increase effectiveness. Male and female tremor rats performed differently in treadmill balance time and locomotion testing. Female tremor rats consistently performed better than males. Additionally, female tremor rats responded more favorably to GTA treatment. The reason for the poor performance of the males is uncertain, but may relate to the genetic deletion in tremor rats, which spans three genes in addition to the gene for ASPA (Kitada et al. 2000).

Several possible improvements could be made to the current GTA supplementation regimen that may provide increased efficacy. GTA was only administered twice daily during the treatment period, and it may be found that increasing the frequency of administration provides greater recovery of function. In addition, GTA was administered in water, whereas it is likely that administration in a complete infant formula would provide increased efficacy due to improved nutrition. Further, tremor rat pups were not treated for the first postnatal week due to the difficulty in administering GTA orally to such young rats. It may be possible to increase acetate delivery to prenatal tremor pups and newborns by administering high levels of GTA to the pregnant and nursing maternal rats. Because esterase activity is high in gut and liver, it may be possible to increase acetate delivery to the brain by combining GTA with mild esterase inhibitors, such as the flavenoids found in grapefruit juice (Li et al. 2007). Future studies comparing intravenous administration of GTA to oral administration will be important to determine if efficacy can be improved by bypassing the digestive system.

The connection between NAA metabolism and lipid synthesis in the brain was first noted in the 1960s. D’Adamo and colleagues found that the acetate moiety of NAA is incorporated into brain lipids, and proposed that NAA acts to provide acetate for brain lipid synthesis (D’Adamo et al. 1968; D’Adamo and Yatsu 1966). Injecting carbon-14 and tritium-labeled NAA into the brains of developing and adult rats, they found that the acetate moiety was readily incorporated into brain lipids during postnatal myelination. Approximately 1/3 of the carbon incorporated into lipids during myelination was derived from the acetate group of NAA. This finding is consistent with the recent findings from our laboratory and the Ledeen laboratory that lipid synthesis is reduced by about 1/3 in the brains of developing ASPA -/- mice (Madhavarao et al. 2005) and tremor rats (Wang et al. 2009).

The present studies confirm earlier investigations indicating a role for NAA as an acetyl source in brain lipid synthesis, and show that NAA is likely to serve multiple developmental and neurochemical roles in the nervous system. We propose that the high concentration of NAA is a nervous system-specific mechanism for storing and transporting acetate to be used for acetyl coenzyme A synthesis, and subsequent acetylation reactions critical for brain development and function. A number of studies have indicated that production of NAA in neuronal mitochondria is somehow linked to energy metabolism in the brain (Patel and Clark 1979), and this role could involve the use of NAA as a storage and transport form of acetate. Importantly, NAA is probably a major source of acetate that is transported from the site of synthesis in neurons to the site of catalysis in oligodendrocytes where the free acetate can be reconverted to acetyl coenzyme A for lipid synthesis (Patel and Clark 1980) and protein acetylation reactions. This would allow neurons to regulate the rate at which acetate is supplied to their ensheathing oligodendrocytes.

The ASPA deficiency in the mouse knock-out model of Canavan disease (Aspa -/- mice) results in an 80% reduction in total brain acetate levels, as well as significant decreases in the levels of certain polar and non-polar lipids including cerebrosides during the period of postnatal myelination (Madhavarao et al. 2005). The decrease in brain lipid content has been verified in the tremor rat model of Canavan disease (Wang et al. 2009), and in the Nur7 ASPA-deficient mouse (Traka et al., 2008). However, further work with the Nur7 mouse suggested that disrupted myelin synthesis was not the only pathogenic mechanism involved in the etiology of the spongy degeneration associated with Canavan disease. Specifically, Nur7 ASPA-deficient mice were generated that were heterozygous for a null allele of the galactolipid-synthesizing enzyme UDP-galactose:ceramide galactosyltransferase (Cgt), which further reduced brain cerebroside content. Despite this further reduction in brain cerebroside content, the mice were not more severely affected than those harboring only homozygous Nur7 mutations in the Aspa gene.

These findings implicate other potential mechanisms in the pathophysiology of ASPA deficiency in addition to defective myelin synthesis. One aspect of the neuropathologies associated with ASPA deficiency that does not comport directly with disrupted myelination is the substantial brain and spinal cord vacuolation observed in gray matter, and sparing of many white matter areas (Surendran et al. 2005; Traka et al. 2008). The vacuolation appears between postnatal days 14 and 21 in the Nur7 ASPA-deficient mouse, which coincides with the peak of postnatal myelination. We have also observed vacuolation in many gray matter areas and sparing of specific white matter tracts in the adult tremor rat brain (unpublished observations). Recent studies on oligodendrocyte maturation have highlighted the important role of histone acetylation and deacetylation in the epigenetic control of cellular differentiation from oligodendrocyte precursor cells to mature oligodendrocytes (Copray et al. 2009; MacDonald and Roskams 2009; Ye et al. 2009). Because neurons transfer NAA to oligodendrocytes (Chakraborty et al. 2001), and oligodendrocytes strongly express ASPA (Klugmann et al. 2003; Madhavarao et al. 2004), it is likely that NAA-derived acetate is an important source of acetyl coenzyme A in oligodendrocytes for histone acetylation reactions that regulate chromatin structure and gene transcription. The finding that ASPA is expressed in the nuclei of oligodendrocytes, as well as in their cytoplasm (Hershfield et al. 2006), is consistent with this role for NAA-derived acetate. The dramatic reduction in acetate availability in oligodendrocytes resulting from ASPA deficiency could impact histone acetyltransferase reactions required for epigenetic gene regulation. The resultant disruption of oligodendrocyte differentiation would explain the observed loss of mature oligodendrocytes in the cerebellum and brainstem of Nur7 ASPA-deficient mice (Traka et al. 2008).

In order for free acetate derived from NAA hydrolysis to be utilized, it must be enzymatically converted to acetyl coenzyme A. We found that acetyl coenzyme A synthase-1 (AceCS1), which converts free acetate to acetyl coenzyme A, is present in the nuclei and cytoplasm of many oligodendrocytes in 18-day-old rats (Fig. 5). Further, AceCS1 protein expression was upregulated in neurons and oligodendrocytes in adult tremor rats as compared with wild-type controls (Figs. 6 and 7). Expression of AceCS1 in GTA-treated tremor rats was reduced relative to untreated tremor rats and was returned to near wild-type levels. AceCS1 is known to be involved in lipid synthesis (Luong et al. 2000) and has recently been shown to be involved in histone acetylation reactions necessary for cell differentiation (Wellen et al. 2009). The reduced substrate availability for AceCS1 could negatively impact histone acetylation critical for proper oligodendrocyte maturation (Copray et al. 2009; Kumar et al. 2009). It seems likely that ASPA deficiency leads to improper regulation of histone acetylation in developing oligodendrocytes, preventing normal differentiation and leading to oligodendrocyte cell death, dysmyelination, neuronal injury, and inflammation, possibly contributing to vacuole formation. It is noteworthy that GTA treatment also modestly reduced vacuole formation (see Fig. 8). One important conclusion that can be drawn from these observations is that NAA-derived acetate is not only involved in brain myelination but it may also be involved in gene regulation associated with cellular differentiation and brain development.

Another possible mechanism linking reduced acetyl coenzyme A availability and the neuropathologies in Canavan disease involves posttranslational acetylation of proteins in the endoplasmic reticulum, particularly in oligodendrocytes. Cells which have active protein secretory pathways through the endoplasmic reticulum, such as oligodendrocytes, are sensitive to disorders of protein mis-folding. Acetylation and deacetylation of nascent polypeptide chains in the endoplasmic reticulum secretory pathway of cells is required for stabilization and correct folding (Costantini et al. 2007; Spange et al. 2009). Acetyl coenzyme A is required for the acetyltransferase reactions involved in acetylation at lysine sites on newly synthesized proteins. The substantial drop in brain acetate levels that occur in ASPA deficiency could have a negative impact on protein folding and stabilization, thus targeting proteins for endoplasmic reticulum associated degradation (ERAD). In the ASPA knockout mouse, a severe loss of myelin basic protein and PLP/DM20 proteolipid proteins has been observed, combined with a decrease in myelinated fibers and perinuclear retention of myelin protein staining. These are clear indicators of an impairment in protein trafficking in oligodendrocytes (Kumar et al. 2009). Oligodendrocytes are highly susceptible to endoplasmic reticulum stress (ERS) associated with disruptions in protein synthesis and trafficking (Lin and Popko 2009). It is likely that the dramatic drop in NAA-derived acetate in Canavan disease has a negative impact on the protein secretory pathway in oligodendrocytes. Because all of the potential pathogenic mechanisms involve reductions in acetate availability associated with the inability to deacetylate NAA, it is possible that GTA administration may provide needed substrate for acetyl coenzyme A synthesis required for all three cellular functions; fatty acid synthesis, nuclear histone acetylation, and endoplasmic reticulum protein acetylation.

In conclusion, the acetate precursor GTA improved motor performance and increased myelin galactocerebroside content in the tremor rat model of Canavan disease, without overt toxicity. GTA treatment also decreased brain vacuolation, and the improvements in motor performance were positively correlated with the reduced vacuolation. Additionally, GTA treatment returned the elevated AceCS1 expression levels in tremor rats to near control levels. None of the pathological consequences of ASPA deficiency were completely reversed by GTA administration, indicating that additional investigations and modifications to the treatment regimen will be necessary to further improve long-term outcomes. Nonetheless, acetate supplementation is a safe and promising treatment strategy for Canavan disease that is inexpensive and easy to administer. GTA-based acetate supplementation is a unique method for addressing dysmyelination in Canavan disease in particular, and possibly other disorders of myelination, or brain injury (Arun et al. 2010). Finally, the important role of NAA-derived acetate in brain development and myelination is further confirmed. Additional research will be required to determine the role of compromised nuclear histone acetylation and endoplasmic reticulum protein acetylation in the etiology of Canavan disease.