Immunohistochemical localization of aspartoacylase in the rat central nervous system.
Aspartoacylase (ASPA; EC 3.5.1.15) catalyzes deacetylation of N-acetylaspartate (NAA) to generate free acetate in the central nervous system (CNS). Mutations in the gene coding ASPA cause Canavan disease (CD), an autosomal recessive neurodegenerative disease that results in death before 10 years of age. The pathogenesis of CD remains unclear. Our working hypothesis is that deficiency in the supply of the NAA-derived acetate leads to inadequate lipid/myelin synthesis during development, resulting in CD. To explore the localization of ASPA in the CNS, we used double-label immunohistochemistry for ASPA and several cell-specific markers. A polyclonal antibody was generated in rabbit against mouse recombinant ASPA, which reacted with a single band (approximately 37 kD) on Western blots of rat brain homogenate. ASPA colocalized throughout the brain with CC1, a marker for oligodendrocytes, with 92-98% of CC1-positive cells also reactive with the ASPA antibody. Many cells were labeled with ASPA antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Relatively fewer cells were labeled in gray matter, including cerebral cortex. No astrocytes were labeled for ASPA. Neurons were unstained in the forebrain, although small numbers of large reticular and motor neurons were faintly to moderately stained in the brainstem and spinal cord. Many ascending and descending neuronal fibers were moderately stained for ASPA in the medulla and spinal cord. Microglial-like cells showed faint to moderate staining with the ASPA antibodies throughout the brain by the avidin/biotin-peroxidase detection method, and colocalization studies with labeled lectins confirmed their identity as microglia. The predominant immunoreactivity in oligodendrocytes is consistent with the proposed role of ASPA in myelination, supporting the case for acetate supplementation as an immediate and inexpensive therapy for infants diagnosed with CD.
Madhavarao
Chikkathur N
CN
Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Moffett
John R
JR
Moore
Roger A
RA
Viola
Ronald E
RE
Namboodiri
M A Aryan
MA
Jacobowitz
David M
DM
D025601:Adenomatous Polyposis Coli Protein / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002452:Cell Count; D002490:Central Nervous System / Q000166:cytology / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D007124:Immunoenzyme Techniques / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D037102:Lectins / Q000378:metabolism; D008297:Male; D051379:Mice; D017628:Microglia / Q000378:metabolism; D009836:Oligodendroglia / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014148:Tranexamic Acid / Q000378:metabolism; D053675:Versicans
D025601:Adenomatous Polyposis Coli Protein; D005904:Glial Fibrillary Acidic Protein; D006023:Glycoproteins; D037102:Lectins; C508324:Vcan protein, rat; D053675:Versicans; D001224:Aspartic Acid; D014148:Tranexamic Acid; C000179:N-acetylaspartate; D000581:Amidohydrolases; C019751:aspartoacylase