Naa Is A Surrogate Marker Of Neuronal Health

NAA is an abundant free amino acid present in the vertebrate brain and is enriched only in neurons and its processes33. This neuronal specificity makes it an ideal marker for monitoring neuronal and axonal health. It has been shown to be consistently reduced in

Figure 4. Wallerian degeneration in normal appearing white matter from a patient diagnosed with relapsing-remitting MS for 9 months. In both cross section (A), and longitudinal section (B), myelin ovoids lacking axons (arrows) were detected. In longitudinal section (B), these myelin ovoids often lay in rows. (From Bjartmar et al,32 with permission).

Figure 4. Wallerian degeneration in normal appearing white matter from a patient diagnosed with relapsing-remitting MS for 9 months. In both cross section (A), and longitudinal section (B), myelin ovoids lacking axons (arrows) were detected. In longitudinal section (B), these myelin ovoids often lay in rows. (From Bjartmar et al,32 with permission).

neurodegenerative diseases, including MS. In vivo it can be reliably measured non-invasively by MRS. In tissues it can easily be detected and quantified by high performance liquid chromatography (HPLC).

3.1 NAA is Specific for Mature White Matter Axons

NAA as measured by MRS, is currently the best and most specific noninvasive marker of axonal pathology in multiple sclerosis. An issue has been raised, however, that NAA is expressed also by oligodendroglial lineage cells34-36. In order to investigate NAA specificity for white matter axons, transected and contralateral non-transected mature and developing rat optic nerves were analyzed by HPLC and immunohistochemistry37. In adult transected optic nerves, axons degenerated rapidly reflected by decreasing NAA levels (Figure 5A) while myelin profiles, oligodendrocytes and NG2+ oligodendrocyte progenitor cells (OPCs) remained abundant. Because NAA became undetectable in these axon-free nerves, the data suggest that neither differentiated oligodendrocytes nor adult OPCs contribute to detectable NAA levels in mature CNS white matter tracts in vivo37.

Interestingly, NAA levels increased significantly over time in contralateral non-transected nerves in operated animals. The mechanism(s) behind this is unclear, but the observation raises several possibilities regarding NAA and neuronal compensation.

Urenjak et al.34 reported that O-2A cells isolated from developing rodent CNS express NAA in vitro. As numerous progenitor cells proliferate and differentiate in transected developing rat optic nerves38, NAA levels were examined following transection of P4 optic nerves. At P14 and P20, average NAA per axon-free nerve was reduced by 80% and 94%, respectively, compared to age- matched control nerves. These data indicate that a minor proportion (<20%) of total NAA in developing nerves is located outside

Figure 5. (A) Axonal degeneration correlates with NAA reduction in transected adult optic nerve and is undetected 24 days post transaction (DPT). In contrast 10-20% of NAA values in developing optic nerve appear to be non-axonal. (B) The percentage of BrdU labeled (proliferating) NG2+oligodendroglial lineage cells in developing nerves are markedly higher than the adult nerves and may account for the non-axonal NAA. (From Bjartmar et al,37 with permission).

Figure 5. (A) Axonal degeneration correlates with NAA reduction in transected adult optic nerve and is undetected 24 days post transaction (DPT). In contrast 10-20% of NAA values in developing optic nerve appear to be non-axonal. (B) The percentage of BrdU labeled (proliferating) NG2+oligodendroglial lineage cells in developing nerves are markedly higher than the adult nerves and may account for the non-axonal NAA. (From Bjartmar et al,37 with permission).

axons. In developing nerves as much as 25 to 33%of NG2+OPCs were proliferating compared to 0.6 to 3.3% in adult nerves. The number of proliferating OPCs is therefore markedly higher in transected developing optic nerves compared to that of adult rats (Figure 5B). Considering the relatively high NAA expression by O-2A OPCs in vitro34, the NAA pool detected in vivo in these developing nerves without axons is likely to be associated with proliferating progenitor cells. Whether OPCs present in some MS lesions can contribute to NAA as measured by MRS remains to be determined39-41. In chronic MS lesions, however, most OPCs appear non-proliferating39, and the density of these cells is generally similar or reduced compared to non-lesion white matter40,41 suggesting that OPCs do not contribute significantly to total NAA in chronic lesions.

The silent nature of early axonal loss and the lack of reliable measures of disease progression make biologic markers that reflect early axonal damage and treatment efficiency in patients with MS essential. In this respect, the present data support NAA, as measured by MRS, as a specific in vivo marker for adult myelinated axons.

3.2 Magnetic Resonance Spectroscopy Detects NAA in Vivo

Clinical parameters such as relapses and progression underestimate the actual damage to tissue that occurs in MS. Hence, conventional magnetic resonance imaging (cMRI) has been an important tool for following disease activity and evolution in MS. However despite the correlation between axon loss and hypointensity in T1-weighted images of MS brain42,43 cMRI generally lacks pathological specificity as many factors can influence image contrast10,11. Though sensitive in detecting lesions, it cannot distinguish inflammation, gliosis, demyelination, and more importantly axon loss—the pathologic substrate of irreversible disability44. To adequately understand disease progression and monitor efficacy of newer therapeutic strategies in clinical trials, it would be useful to be able to assess the accumulated load of irreversible damage. MRS determination of NAA may provide such an index.

MRS is one of the modern quantitative MR techniques that have the potential to overcome some of the limitations of cMRI. Other modern MR techniques like magnetization transfer and diffusion weighted MRI enable one to quantify the extent of structural changes occurring within and outside the MS lesion with increase specificity45. MRS can add information on the biochemical nature of such changes, with the potential to improve significantly our ability to monitor inflammatory demyelination and axonal injury. It facilitates noninvasive assessment of neurons, axons, and membrane integrity through the quantification of W-acetylaspartate (NAA), total choline (Cho), and creatine (Cr) levels. Decreased NAA, coupled with Cho and Cr variations, have been reported in MS lesions, normal-appearing white matter (NAWM), and cortical gray matter10,44.

Figure 6. Proton MR spectra from a normal brain (A), NAWM of a patient with MS (B), and chronic periventricular plaque of the same patient with MS. (From Arnold, 1999,8 with permission).

That MRS detection of NAA concentration is an accurate measure of axonal density has been confirmed by histology of biopsied samples46. To determine NAA levels in MS spinal cords, HPLC analysis of whole cord cross sections was performed postmortem. At cervical and lumbar levels, average NAA levels were significantly decreased by 53% and 55% respectively. Since these patients were severely disabled (Expanded Disability Status Scale >7.5) the data indicates that reduced NAA levels in chronic MS as detected by MRS, can reflect irreversible functional impairment24.

Falini et al.47 tested the utility of MRS in defining the extent of metabolic changes in benign versus secondary progressive MS and found significant differences in NAA pattern according to the phase (acute vs. chronic) and the clinical form (benign vs. progressive) of the disease. MRS also detected reduced NAA levels in NAWM, substan- tiating the histopathologic findings of axonal loss or Wallerian degeneration outside MS le-sions32. The extent of NAWM changes varies between different patient groups48, but the changes are invariably present in all major MS phenotypes49 and correlate with the level of physical disability48 and cognitive impairment50.

Gray matter involvement at cortical and subcortical levels in MS is gaining increased recognition12, 51. MRS was able to document substantial NAA reduction in this area as well52, 53 corroborating reports of diffuse axonal pathology starting at the early stage of the disease54.

The concept that MS has a diffuse pathology poses questions for the current localized MRS detection of NAA55. It assumes that relevant metabolic changes occur only at sites of imaging abnormality. This problem of partial coverage was addressed by recent studies utilizing Whole Brain NAA (WBNAA). One study showed that the interindividual WBNAA variation in a cohort of normal, middle-aged women (range 42+/-5 years of age) is small, 3% (p<0.01), and the intra-individual temporal variation is smaller still56. Comparison of the WBNAA of 12 rapidly remitting MS patients and age matched controls showed that the MS group had markedly reduced WBNAA. This difference was noted to be greater among older than younger patients. The linear prediction of equations of WBNAA with age indicate a faster decline in the patients, ~0.8% per year of age (p=0.22)55. In a study of WBNAA dynamics in RR-MS patients, measurement defined 3 subgroups: stable, exhibiting an insignificant change in NAA level per year of clinically definite disease duration (p=0.54); moderate decline, -2.8% per year (<0.01); and rapid decline, -27.9 % per year (p<0.01). Thus, WBNAA offers a quick, highly reproducible measure of disease progression and may help stratify patients for active therapeutic intervention in MS57. This is particularly compelling now, in the light of the observation that axonal injury starts early in the course of the disease and that partially effective treatment for MS is available for certain group of patients.

3.3 Dynamics of NAA in Multiple Sclerosis

It was initially thought that NAA reduction was always secondary to axonal loss. However, as demonstrated by in vivo MRS, reduced NAA in acute lesions is at least partly reversible, indicating that early axonal damage due to inflammatory demyelination can be reversible10. This view is compatible with clinical recovery during remissions in MS, which is attributed to axonal recovery due to mechanisms such as resolution of inflammation, remyelination or redistribution of axolemmal ion-channels. Given the limited levels of neuronal regeneration within the CNS, reversible levels of NAA in MS

white matter also suggest that mechanisms other than axonal loss contribute to overall NAA reductions in MS.

To address whether axonal loss is the only contributor to decreased levels of NAA in chronic MS spinal cords, NAA per axonal volume was calculated (Figure 7. A-G)24. Compared to myelinated axons in control white matter, average NAA per axonal volume was reduced in myelinated axons in MS non-lesion white matter by 30% (p = 0.05), and in demyelinated axons in MS lesions by 42% (p = 0.01). These data raise several interesting interpretations of MRS data. One possibility is that myelin related molecules and/or oligodendrocytes influence axonal levels of NAA. When NAA was expressed per axonal volume, demyelinated axons contained 42% less NAA than myelinated axons in control spinal cords. It is well established that myelination and demyelination dynamically influence axonal neurofilament phosphorylation by regulating kinase and phosphatase activity locally58. NAA metabolism appears to be related to neuronal activity in a tract as well. Since these MS patients were paralyzed, the 30% reduction of NAA in normal appearing

Figure 7. Correlation between axonal loss and levels of N-acetyl-aspartate (NAA) in MS spinal cord white matter. Axonal density was determined in neurofilament-stained cryostat sections, and NAA levels were determined by HPLC in adjacent sections. Top panel: representative micrographs showing axons in control white matter (A), MS white matter without lesion (B), and in an MS lesion (C). Scale bars = 40-^m. Lower panels: HPLC chromatograms (D, E, and F) from tissue sections adjacent to those immunostained in (A), (B) and (C) respectively. Levels of NAA per axonal volume in myelinated and demyelinated axons in control and MS samples (G). Average NAA per axonal volume was significantly reduced in MS non-lesion white matter samples (30%, p = 0.05) and in samples from chronic MS lesions (42%, p = 0.01). (From Bjartmar et al.,24 with permission).

Figure 7. Correlation between axonal loss and levels of N-acetyl-aspartate (NAA) in MS spinal cord white matter. Axonal density was determined in neurofilament-stained cryostat sections, and NAA levels were determined by HPLC in adjacent sections. Top panel: representative micrographs showing axons in control white matter (A), MS white matter without lesion (B), and in an MS lesion (C). Scale bars = 40-^m. Lower panels: HPLC chromatograms (D, E, and F) from tissue sections adjacent to those immunostained in (A), (B) and (C) respectively. Levels of NAA per axonal volume in myelinated and demyelinated axons in control and MS samples (G). Average NAA per axonal volume was significantly reduced in MS non-lesion white matter samples (30%, p = 0.05) and in samples from chronic MS lesions (42%, p = 0.01). (From Bjartmar et al.,24 with permission).

myelinated spinal cord axons provides direct support for reduced NAA in the absence of demyelination in functionally impaired axons. It is also possible that denervation of a neuron due to damage of afferent axons, for example in one or several remote lesions, causes altered NAA levels. Indeed, acute deafferentiation in the CNS causes trans-synaptic decreases of NAA levels without ultrastructural abnormalities, indicating that denervation and/or impaired function reduces neuronal NAA59. It is also possible that myelination and demyelination may modulate activity of enzymes involved in NAA me tabolism, thus influencing axonal NAA levels locally. Since NAA is synthesized in mitochondria60, 61, changes in NAA could reflect mitochondrial dysfunction62. In patients with acute mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), a primary mitochondrial disorder, NAA levels in the lesions recover partly after the acute illness63. These lesions, and acute demyelinating lesions, exhibit an inverse relationship between reduced NAA and lactate production, suggesting that reduced NAA production due to mitochondrial dysfunction might be associated with increased anaerobic glycolysis62, 63. Local inflammation could indirectly influence mitochondrial function. In acute EAE, an animal model of MS characterized by inflammation and demyelina-tion, mitochondrial dysfunction and reduced NAA has been reported in the absence of neuronal loss62, 64, 65. Finally, in theory, edema (dilution effects) or inflammation-induced metabolic effects may also cause reduction in NAA levels. For example, decrease in NAA levels of up to 50% have been observed in NAWM5 and up to 80% in lesions66, 67. However the observed reductions in NAA levels in these regions are greater in proportion than the volume changes, indicating that the reversible NAA change is unlikely to result merely from edematous dilution effects68.

In summary, reduced NAA levels in MS could reflect several mechanisms such as reversible neuronal/axonal damage due to inflammatory demyelination, altered neu-ronal/axonal metabolism related to activity, axonal atrophy, or axonal loss10, 63.

3.4. Correlation of NAA with other Measures of Neurodegeneration

Brain atrophy reflects the net result of irreversible and destructive pathological processes in MS. Axonal damage and loss, chronic demyelination, and gliosis all contribute to a reduction in brain parenchymal tissue volume and a corresponding expansion of cerebrospinal fluid (CSF) spaces. Hence, measures of brain atrophy (e.g. brain parenchymal volume, index of brain atrophy, whole brain ratio) are a reliable, and meaningful global surrogate for the destructive pathologic process in MS patients 69' 70. In studies correlating NAA and brain atrophy, consistent direct correlations have been observed. The observed correlations between atrophy and decreased NAA/creatine levels suggest that axonal loss is a substantial contributor to atrophy and that MRS can assess that component, even in the very early stages of the disease71.

Efforts to correlate NAA levels to disability status like Expanded disability Status Scale (EDSS) however, have been contradictory. While most studies found a correlation5, 24 other studies did not57, 72. While disappointing, it is not surprising that despite the specificity of NAA as an index of neuronal health, correlation between NAA levels and EDSS has been inconsistent. There are several reasons for this discrepancy. First, lesion location was not considered in these correlations. Understandably, disability or lack thereof resulting from any CNS lesion, do vary with lesion location. Second, it reflects the inherent weakness of EDSS as a clinical instrument72, 73. While it is a useful clinical measure of neurologic impairment, EDSS score consistently fails to reflect the full burden of disease because it is weighted toward cerebellar and spinal cord deficits73, 74. Hence, spinal cord NAA level is well correlated with EDSS score24 while WBNAA is inconsistent. Third, this disparity reflects the brains ability to compensate for accumulating injury and to conceal its extent. Cortical reorganization is known to occur in MS75, 76 and underlies the difficulty in using clinical criteria such as EDSS to predict disease course. In contrast, NAA dynamics yield a direct measure of the brain's pathologic structure load without the distorting overlay of function, thereby more objectively predicting the course of organic pathology which may be more appropriate for monitoring disease progression in clinical trials.

4. CONCLUSION

It is well established that axonal pathology is the basis of permanent disability in MS from its earliest stage. The promise of NAA measurement is that, it provides a specific and readily quantifiable index of neuronal and axonal dysfunction or loss. Therefore, monitoring NAA dynamics provides an index of change associated with irreversible stages in the evolution of diffuse MS lesions central to determining disability. Use in the proper context and together with other modern MR techniques, understanding NAA concentration dynamics may enable early forecast of disease course, reflect disease load and so influence treatment decision; improve clinical trial efficiency, and enhance further understanding of this complex disease.

5. ACKNOWLEDGMENTS

This work was supported by NIH grants NS35058, NS38667 (B.D.T.). The authors thank Dr. Grahame Kidd for the figures and Susan De Stefano for editorial assistance.

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