Immunohistochemistry and Biosynthesis of N‐Acetylaspartylglutamate in Spinal Sensory Ganglia

Abstract

N-Acetylaspartylglutamate (NAAG) is a nervous system-specific dipeptide which has been implicated in chemical neurotransmission. Antisera were prepared against NAAG in order to study its cellular distribution. When these antisera were applied to tissue sections of rat spinal sensory ganglia, NAAG-like immunoreactivity was detected within a subpopulation of relatively large neuronal cell bodies in cervical, lumbar, and thoracic ganglia. In order to confirm the presence of NAAG within these neurons, the dipeptide was extracted and purified from spinal ganglia using high-performance liquid chromatography and its composition confirmed by amino acid analysis. Further, the biosynthesis of NAAG was studied in vitro by following the incorporation of either [3H]glutamine or [3H]glutamate into the glutamate residue of the purified dipeptide. [3H]Aspartate was not incorporated efficiently into NAAG under these conditions, suggesting a precursor role for the large N-acetylaspartate pool. The incorporation of radiolabeled amino acids into newly synthesized NAAG by spinal sensory ganglia was not inhibited by incubation of the cells with anisomycin or cycloheximide at concentrations which significantly inhibited protein synthesis. These data suggest that NAAG is present in a subpopulation of primary afferent spinal neurons and that its biosynthesis is mediated by a dipeptide synthetase.

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

“…The first reliable report of NAAG biosynthesis in tissue explants demonstrated that incubation of excised rat dorsal root ganglia with cycloheximide or ansinomycin at 200 μM did not inhibit incorporation of radiolabel into the dipeptide (Cangro et al, 1987). In this report, the addition of protein synthesis inhibitors actually increased NAAG biosynthesis, and the authors attributed this to the increased availability of radiolabeled glutamate for NAAG biosynthesis, due to the reduced incorporation of glutamine and glutamate into proteins.…”

“…It is synthesized enzymatically from NAA and glutamate (Arun et al, 2006;Boltshauser et al, 2004;Cangro et al, 1987;Gehl et al, 2004) and is localized in specific types of neurons throughout the CNS (Anderson et al, 1987;Moffett et al, 1993;Moffett and Namboodiri, 1995;Tieman et al, 1991;Tieman and Tieman, 1996). NAAG is released from synapses in a calcium dependent manner (Williamson et al, 1991), and acts through presynaptic metabotropic glutamate receptors to modulate the release of classical neurotransmitters (Xi et al, 2002;Zhao et al, 2001).…”

“…The presence of a "NAAG synthetase" enzyme has been postulated based on indirect means in several studies (Arun et al, 2006;Cangro et al, 1987;Gehl et al, 2004;Urazaev et al, 2001;Williamson and Neale, 1988). Most recent studies which have successfully demonstrated NAAG biosynthesis were done in neural explants, including excised rat dorsal root ganglia (Cangro et al, 1987), excised frog retinas (Williamson and Neale, 1988), crayfish nerve cord (Urazaev et al, 2001) and hemisected rat spinal cord (Gehl et al, 2004). In several recent reports, NAAG biosynthesis has been observed in cell culture, including primary rat astrocyte cell culture (Gehl et al, 2004), and in a continuous human neuroblastoma cell line (Arun et al, 2004;Arun et al, 2006).…”

“…The first convincing studies on NAAG biosynthesis were conducted by Cangro and colleagues using excised rat dorsal root ganglia (DRG) in short-term culture (Cangro et al, 1987). They incubated DRG in tissue culture for 3 hr with radiolabeled glutamine, and found very low levels of incorporation into NAAG during that time frame.…”

“…synthesis have been reported to have no effect on the incorporation of radiolabeled precursors into NAAG in neural explants (Cangro et al, 1987;Gehl et al, 2004), suggesting that NAAG is not synthesized on ribosomes as a portion of a protein, and then cleaved to generate the active molecule as is the case with many other bioactive peptides. The first reliable report of NAAG biosynthesis in tissue explants demonstrated that incubation of excised rat dorsal root ganglia with cycloheximide or ansinomycin at 200 μM did not inhibit incorporation of radiolabel into the dipeptide (Cangro et al, 1987).…”