Canavan disease (CD) is a rare autosomal recessive leukodystrophy that is caused by mutations of the aspartoacylase gene (ASPA). ASPA is highly expressed in mature oligodendrocytes (OLs), where it catalyzes the hydrolysis of the most abundant amino acid in the brain, N-acetyl-aspartate (NAA) to acetate and aspartic acid. The mechanism of CD pathogenesis, however, remains unknown. One hypothesis has been that the loss of ASPA results in reduced levels of acetate, a precursor for myelin lipid synthesis. We recently described the identification of the ENU-induced nonsense mutation, Q193X, in the mouse Aspa gene that results in the absence of detectable ASPA protein expression in Aspanur7 homozygous mutant mice, which display severe spongy degeneration (vacuolation) throughout the CNS, strikingly resembling CD. High levels of NAA are found in the CSF and urine of CD patients. Similarly, NAA is increased in the CNS of Aspanur7 mutants. Therefore, another hypothesis implicates elevated NAA levels as the leading cause of myelin degeneration observed in CD. This proposal aims to investigate the molecular mechanisms that are responsible for CD pathogenesis by taking advantage of the Aspanur7 mutant. Our previous studies and recent preliminary data on the Aspanur7 mouse indicate that myelin degeneration in CD is not primarily due to a limited supply of NAA- derived acetate for the myelin lipid synthesis. Thus, a role of ASPA outside myelination is also possible. Here, we favor the hypothesis that CD pathogenesis is caused by ASPA deficiency in mediating NAA clearance in the CNS and thereby protecting myelin and/or OLs from NAA damage. We plan to address this hypothesis by establishing cocultures of purified retinal ganglion neurons (RGCs) with oligodendrocyte progenitor cells (OPCs) that result in myelination (Specific Aim 1). By comparing myelination levels and structure between cocultures of OPCs derived from the ASPA-deficient mice and wild-type ones we will be able to determine whether ASPA is required or not for myelination. These cocultures will also be used to assess the possibility that ASPA has an NAA-scavenger activity in the CNS that protects myelin and/or OLs from NAA damage (Specific Aim 1). The proposed role of ASPA as NAA-scavenger in the CNS will be further investigated in vivo by generating ASPA-deficient animals that synthesize significantly reduced NAA levels due to the deficiency of the solute carrier family 25 member 12 (Slc25a12) gene (Specific Aim 2). We anticipate that the accumulation of NAA observed in the CNS of the Aspanur7 mutants will be significantly reduced in the Aspanur7/nur7;Slc25a12-/- animals, which might lead to amelioration of their CD symptoms as compared to the Aspanur7/nur7 mice, supporting that ASPA's activity in OLs protects OLs and/or myelin from the potential detrimental effects of NAA accumulation observed in CD. Overall, the results produced by the proposed Aims could promote our understanding on the mechanism of the CD pathogenesis and eventually help us develop therapeutic targets to prevent disease progression and thereby potentially enhance myelin repair in the CNS of the CD patients.
Determining the role of ASPA activity in mature oligodendrocytes is the main focus of this research. More specifically, we will check the hypothesis that ASPA has a primary role outside myelination, which is related to NAA clearance for myelin and/or OL protection in the CNS. We anticipate that the new knowledge produced by our experimental approaches will help us dissect the molecular mechanisms responsible for Canavan disease pathogenesis and develop therapeutic targets that promote myelin repair for this leukodystrophy.
