Onset of myelination grey matter white matter
Figure 7. Evolution of aspartoacylase activity (specific activity, |jM/ min/mg protein) - mean + S.D.; n=4) in the optic nerve during postnatal development.
A differential distribution of aspartoacylase activity between gray and white matter has been has been demonstrated in previous studies. The study by D'Adamo et al.36 on the occurrence of aspartoacylase activity in the developing rat brain showed an approximately three-fold difference between white and gray matter in the adult cerebral hemispheres, whereas, in another study on adult bovine brain, the specific activity of aspartoacylase in the deepest tissue (white matter) was 15-fold higher than in tissue closest to the pial surface i.e. gray matter.38
The experiments on the cellular distribution of aspartoacylase activity very much reflect the results of our findings on regional brain tissue studies. At the celluar level, the predominate expression of aspartoacylase activity is seen in the O-2A progenitor lineage that gives rise to the myelinating cells of the CNS. In contrast aspartoacylase activity in whole tissue is limited to white matter tracts of the brain, with the largest developmental increases seen in regions of greatest myelination. This is very much in contrast to the expression of its substrate, NAA, where the highest levels are found in gray matter and in all neuronal cell-types studied so far.8, 9 49 Thus the enzyme catalysing NAA catabolism appears to be located in a different cellular compartment from the principle NAA store. However, an interesting exception is the presence of both NAA and aspartoacylase activity in O-2A progenitor lineage with the highest levels of both found in mature oligodendrocytes. Type-2 astrocytes do not synthesise NAA (unpublished data) but do express the highest levels of aspartoacylase. The significance of this finding is discussed later. The development of aspartoacylase in cortical type-1 astrocytes is in contrast to the recent finding of Baslow et a!.39 where the expression of enzyme activity in cultured glial cells was limited to oligodendrocytes alone. In our present study freshly isolated type-1 astrocytes had activities at the lower end of the detection limit. However, there was an increased expression of aspartoacylase activity over time in culture. Another unexpected finding was the presence of the highest cellular aspartoacylase activity in type-2 astrocytes. It is generally accepted that type-1 astrocytes in culture represent cells that in vivo constitute reactive astrocytes, the predominant cell-type of glial scar tissue.90 It may well be that type-2 astrocytes also represent a type of reactive astrocyte that does not appear in normal brain, but may develop in response to injury. These cells form scar tissue that limits the spread of damage,91, 92 and express neurocan, a chondoitin sulphate proteoglycan, which is only seen on type-2 astrocytes in culture.93 These cells may require aspartoacylase for either repair of damaged myelin, or hydrolysis of excess NAA released by cell death that, if remains unchecked, may result in the concomitant water imbalance.
Firstly, one could suggest a simple scavenging role for aspartoacylase, for example preventing potential neurotoxic effect on oligodendrocytes, such as that seen in Canavan's Disease where mutation of aspartoacylase results in accumulation of extracellular NAA in the brain, leading to spongy degeneration of the white matter tracts. However, there is no direct evidence that high levels of extracellular NAA are indeed toxic to oligodendrocytes. Work in our laboratories has shown no detrimental effects of NAA (concentrations of up to 5 mM for three days) on oligodendrocytes in culture (unpublished data). More recently Kitada et a!.94 have demonstrated that the absence-like seizure and spongiform degeneration in the CNS, exhibited in the tremor rat, are due to genomic deletion within a region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of NAA was shown in the mutant brain, in correlation with the severity of the vacuole formation. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4 to 10Hz polyspikes or spikewave-like complexes in cortical and hippocampal EEG. In addition NAA applied to a bath of normal rat brain slice preparations, rapidly induced a long-lasting depolarisation concomitantly with repetitive firings in hippocampal CA3 neurons.95 These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly. A more plausible explanation may be that NAA utilisation is separate from its site of storage. In particular the majority of NAA could be released from the neurones to provide substrate for myelination by oligodendrocytes during development, whereas the NAA in oligodendrocytes is expressed or used in response to white matter damage when neuronal-oligodendrocyte interactions are unstable. Neuronal stores of NAA may be also be involved in the re-myelination process following injury. This would explain the reversible decrease in NAA sometimes observed in MS lesions.96
We note however that at the tissue level, gray matter contains high levels of NAA, but low levels of aspartoacylase. Therefore, a secondary role for NAA in modulating neuronal homeostasis throughout the CNS would not be inconsistent with our data.
Recent studies have confirmed the localization of aspartoacylase to white matter tracts,97 where the authors used double-label immunohistochemistry for aspartoacylase and several cell-specific markers. The aspartoacylase was co-localized throughout the brain with CC1, a marker for oligodendrocytes. Many cells were labeled with aspartoacylase antibodies in white matter, including cells in the corpus callosum and cerebellar white matter. Moreover, only a few cells were labeled in gray matter. 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.
The presence of aspartoacylase in the adult brain demonstrates that it may have a function after myelination is complete that involves repair of damaged myelin. It seems unlikely that such high activities would be required to maintain a repair function. This implies an additional function for aspartoacylase in the adult brain. The presence of aspartoacylase at low levels in the adult olfactory bulb and cortex, which are gray matter tracts, further suggests that its function is not solely for the repair of damaged myelin.
The inverse correlation seen between aspartoacylase and NAA in white and gray matter could be due to a higher turnover of NAA in the white matter during myelination, making the apparent concentration in white matter tracts lower. However, our study have shown that mature oligodendrocytes grown in culture express NAA to similar levels as neurones and may themselves contribute to the overall NAA pool observed in vivo by magnetic resonance.
In conclusion, this study demonstrates the presence of NAA and aspartoacylase in cultured mature oligodendrocytes and may thus present a clearer picture of NAA metabolism in the brain. The developmental and anatomical distribution of aspartoacylase correlates with the maturation of white matter tracts in the rat brain. Moreover, the cellular studies showed that the aspartoacylase activity is limited to glial cells, especially in the O-2A progenitor lineage, with no detectable enzyme activity in neurons. Despite the indications that NAA metabolism correlates closely with myelination, the function/s of NAA nevertheless, still remains obscure, and until this ambiguity is resolved, the utility of NAA as a diagnostic tool in certain brain disorders will require further work.
Was this article helpful?