We detected high concentrations of NAAG in the CSF of 22 patients with PMD and mutations in the PLP1 gene. Furthermore, we found high levels of NAAG in the CSF of 7 patients with a dysmyelinating disorder with a clinical presentation of PMD, but without mutations in the PLP1 gene (PMLD). Conversely, the CSF NAA was in the normal range in PMD and PMLD patients (except for two PMD samples and one PMLD sample with increased NAA content). These results confirm our preliminary data (Burlina et al. J. Inherit. Metab. Dis. 2000; 23 (suppl 1: 213). Moreover, NAAG was not increased in other leukodystrophies that were analyzed such as VWMD, Alexander disease and other leukodystrophy of known origin.
We demonstrated that NAAG is significantly and consistently increased in CSF of patients with genetically confirmed PLP disorders but also in patients with an undefined genetically dysmyelinating disorder (PMLD). Thus we have evidence that increased NAAG is a common marker in dysmyelinating leukodystrophies with or without confirmed PLP mutations.
An involvement of the NAA/NAAG cycle has been described only in few disorders. Indeed, in 1990 Rothstein reported elevated levels of both NAA and NAAG in the CSF of patients with amyotrophic lateral sclerosis.11 High concentrations of NAA and NAAG have been detected in urines of CD patients and in the CSF of one patient with Canavan disease, a leukodystrophy due to aspartoacylase deficiency leading to accumulation of NAA.10 We suppose that in Canavan disease the accumulation of NAA lowers the threshold of NAAG synthesis and may inhibit NAAG hydrolysis, leading to a paralleled increased of NAAG and later on a decrease of both NAA and NAAG in the long-term of the disease, as we have shown.12 Recently, Wolf and co-workers described two unrelated girls with severe brain hypomyelination and increased levels of NAAG in the CSF.13
NAAG is the most abundant dipeptide neurotransmitter in the mammalian CNS.14 It exhibits mixed agonist/antagonist properties at the W-Methyl-D-Aspartate (NMDA) receptor. Indeed, NAAG renders NMDA less effective in promoting cerebellar granule cells survival during differentiation, and also raises the threshold of NMDA toxicity to the differentiated granule cells.15 Moreover, NAAG acts as an agonist at metabotropic glutamate receptors group 2 (mGluR3).14
Recently, it has been shown that metabotropic glutamate receptors (mGluRs) are involved in regulating oligodendrocyte excitotoxicity. Deng and co-workers demonstrated that group 1 mGluRs mitigates excitotoxicity damage to oligodendrocyte precursor cells.16 Oligodendrocyte precursor cells share with neurons a high vulnerability to glutamatergic excitotoxicity. Furthermore, a secondary role for NAAG could be the release of glutamate after NAAG hydrolysis.
But NAAG is a "complex" molecule with multiple actions. NAAG can act as a neurotoxin, and also as a neuroprotective agent, depending on the activity of its peptidase.17 Ghadge et al. have demonstrated that the inhibition of GCPII, the dipeptidase which hydrolyzes NAAG to NAA and glutamate, prevented motor neurons death in a cell culture model of amyotrophic lateral sclerosis.18 The authors suggested that increasing NAAG levels, by blocking NAAG hydrolysis may be neuroprotective by activating mGluR3 receptors.18
It has also been shown that in PMD patients axonal dysfunction and/or injury can be detected by proton MRS and in the mouse model of PMD axonal swelling and degeneration are present.3,19 Therefore, the increase levels of NAAG in CSF may be expression of an augmented release of W-acetylaspartylglutamate after neuronal damage.
Because of its prominent signal, always detectable in human healthy brain by in vivo proton MRS, NAA is generally considered an important marker of viable functions in neurons. Indeed, many neurodegenerative disorders with either loss of neural cells or neuronal dysfunction exhibit a decreased signal of NAA in the proton MR spectra.20 In three PMD patients we detected elevated levels of NAA in the CSF. Therefore, we cannot confirm that NAA was increased in all our PMD patients and this does not fully correlate with the increased concentrations of total NAA in the brain of five patients with PLP duplications, as detected by proton magnetic resonance spectroscopy.4 The involvement of the metabolic pathway of NAA and NAAG has been recently confirmed by the report of a new severe neurological disorder. Indeed, Martin and colleagues (2001) reported a child with neurodevelopmental retardation and moderately delayed myelination. In vivo proton MRS of the brain did not detect an NAA signal (hypoacetylaspartia).21 We analysed the CSF of the patient and NAAG was not detectable, indicating that in the case of hypoacetylaspartia both NAA and NAAG can be decreased (see Burlina, Schmitt et al, this volume).22
In conclusion, our findings show that NAAG may be a useful biochemical marker in the diagnosis of Pelizaeus-Merzbacher disease and may help to understand the myelination process.
This study was supported by a grant from the European Union's Biomed 2 Programme (PL 95 1405), European Network on Brain Dys-myelinating Disease: Diagnostic, Molecular and Therapeutic Approach (ENBDD).
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