Elsevier

Progress in Neurobiology

Volume 81, Issue 2, February 2007, Pages 89-131
Progress in Neurobiology

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

https://doi.org/10.1016/j.pneurobio.2006.12.003Get rights and content

Abstract

The brain is unique among organs in many respects, including its mechanisms of lipid synthesis and energy production. The nervous system-specific metabolite N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A in neurons, appears to be a key link in these distinct biochemical features of CNS metabolism. During early postnatal central nervous system (CNS) development, the expression of lipogenic enzymes in oligodendrocytes, including the NAA-degrading enzyme aspartoacylase (ASPA), is increased along with increased NAA production in neurons. NAA is transported from neurons to the cytoplasm of oligodendrocytes, where ASPA cleaves the acetate moiety for use in fatty acid and steroid synthesis. The fatty acids and steroids produced then go on to be used as building blocks for myelin lipid synthesis. Mutations in the gene for ASPA result in the fatal leukodystrophy Canavan disease, for which there is currently no effective treatment. Once postnatal myelination is completed, NAA may continue to be involved in myelin lipid turnover in adults, but it also appears to adopt other roles, including a bioenergetic role in neuronal mitochondria. NAA and ATP metabolism appear to be linked indirectly, whereby acetylation of aspartate may facilitate its removal from neuronal mitochondria, thus favoring conversion of glutamate to alpha ketoglutarate which can enter the tricarboxylic acid cycle for energy production. In its role as a mechanism for enhancing mitochondrial energy production from glutamate, NAA is in a key position to act as a magnetic resonance spectroscopy marker for neuronal health, viability and number. Evidence suggests that NAA is a direct precursor for the enzymatic synthesis of the neuron specific dipeptide N-acetylaspartylglutamate, the most concentrated neuropeptide in the human brain. Other proposed roles for NAA include neuronal osmoregulation and axon-glial signaling. We propose that NAA may also be involved in brain nitrogen balance. Further research will be required to more fully understand the biochemical functions served by NAA in CNS development and activity, and additional functions are likely to be discovered.

Introduction

N-Acetylaspartate (NAA, Fig. 1) is an enigmatic molecule present at exceptionally high concentrations in the brain (Tallan et al., 1956, Tallan, 1957). The levels found in various areas of the brain can reach 10 mM or greater (Bluml, 1999, Miyake et al., 1981, Pan and Takahashi, 2005), making it one of the most concentrated molecules in the CNS. After five decades of research by neuroscientists and clinicians into the roles played by NAA, its metabolic and neurochemical functions remain controversial, and its connections to several disease states are uncertain. For three decades after its discovery, NAA research was limited to a few laboratories scattered around the world, and progress was slow. There were two seminal findings that brought NAA to the attention of the general neuroscience and neurology communities, dramatically accelerating the pace of research into the neurochemistry and neurobiology of this unique molecule. First was the prominence of the NAA proton signal in magnetic resonance spectroscopy (MRS), making NAA one of the most reliable markers for brain MRS studies (Barany et al., 1987, Fan et al., 1986, Luyten and den Hollander, 1986). The second was the connection to the rare but fatal hereditary genetic disorder known as Canavan disease (Bartalini et al., 1992, Divry and Mathieu, 1989, Hagenfeldt et al., 1987, Matalon et al., 1988).

In the case of Canavan disease, it was found that a mutation in the gene for the enzyme aspartoacylase (ASPA; EC 3.5.1.15) resulted in an inability to catabolize NAA, leading to a progressive, fatal leukodystrophy in affected infants. In the case of the prominent NAA signal in MRS, it was found that the levels of NAA in various parts of the brain correlate with neuronal health or integrity. Decreased levels of NAA as detected by MRS have been interpreted to indicate neuronal/axonal loss, or compromised neuronal metabolism. In contrast, high levels of NAA were found in the brains of many Canavan disease patients, who lacked the degradative enzyme ASPA, suggesting that excess NAA levels may have detrimental effects in the CNS. These findings provided further impetus for investigators to pursue intensified research into NAA biochemistry and the correlations between altered NAA levels and various neuropathological conditions. Subsequent studies led to important discoveries as diverse as links between NAA catabolism and myelin lipid metabolism, and reversible decreases in brain NAA levels associated with pathologies ranging from hypoxia to multiple sclerosis.

The most intense research focus on NAA in recent years has involved MRS-based and magnetic resonance imaging (MRI)-based analyses of changing NAA levels under numerous neuropathological and psychopathological conditions. Unfortunately, spectroscopic studies have dramatically outnumbered studies into the basic biochemistry of NAA in the brain, and this disparity has complicated the interpretation of MRS results in various disease states due to the lack of basic knowledge on NAA function and metabolism. Nonetheless, much important information on NAA has been garnered by means of spectroscopic studies.

NAA is unique not only by virtue of its exceptionally high concentration in the brain, but also due to the powerful signal that it gives off in water-suppressed proton MRS spectrograms (Luyten and den Hollander, 1986). The acetate moiety of NAA is coupled through the amine nitrogen of aspartate (Fig. 1), and the three equivalent hydrogen atoms of the acetate group resonate in NMR with a single, sharp peak having a chemical shift of 2.02 ppm relative to the standard tetramethylsilane. While the peak at 2.02 ppm is prominently attributable to NAA, this signal includes smaller contributions from other acetylated compounds, such as from the neuron-specific dipeptide, N-acetylaspartylglutamate (NAAG) (Caramanos et al., 2005), N-acetylneuraminic acid (Varho et al., 1999), and underlying coupled resonances of glutamate and glutamine. NAAG, the most concentrated peptide in the brain, may contribute 15–25% (Pouwels and Frahm, 1997, Pouwels and Frahm, 1998) to the acetate signal that is usually ascribed to NAA (Barker et al., 2006), so the reductions in the NAA peak associated with various brain disorders either involve coincident reductions in NAAG, or they may underestimate the drop in NAA in situations where NAAG levels remain constant.

Despite technical differences in the methods applied to acquire localized proton spectra from the human brain on different MR scanners and at different magnetic field strengths, the resulting spectra demonstrate substantial visual similarities from one MR scanner to another and between different brain regions. MRS reliably quantifies NAA directly from the number of moles per unit brain volume and can therefore provide valuable insights into its variations regionally, between white and gray matter, and in a wide variety of clinical brain disorders. However, magnetic resonance techniques have substantial limitations, including the fact that a relatively large volume of tissue must be sampled to obtain a reliable signal to noise ratio. This lack of spatial resolution often means that the signals acquired tend to average metabolite concentrations over gray matter, white matter, and CSF (Barker et al., 2006). Further, MRS can only detect compounds present at high concentrations, and therefore most metabolites cannot be analyzed by these techniques. Despite these limitations, MRS and MRI provide a great deal of information about several important metabolites of interest to clinicians and neuroscientists.

In MRS spectra of normal human brain the major peaks observed from left to right on the spectrogram include myo-inositol, choline (including glycerophosphocholine, phosphocholine, and free choline), total creatine (creatine and phosphocreatine), and NAA (including NAAG) (Fig. 2). NAA represents the largest peak in spectra of healthy brain tissue. In pathological conditions ranging from hypoxia to brain injury, additional peaks are seen representing lipids and lactate. In practice, while the cerebral concentrations of total creatine (creatine + phosphocreatine) remain relatively constant, changes in NAA, either as an absolute concentration or as a ratio between NAA versus total creatine (NAA/Cr), have proved diagnostically important. Similarly, increased signals for lactate and lipid have substantial diagnostic value.

Comparisons of the various MRS and MRI techniques currently available to neuroscientists and clinicians have been published (Bammer et al., 2005, Barker et al., 2006, Di Costanzo et al., 2003). NAA levels measured by MRS have been shown to be changed in a number of neurological disorders and conditions. Most of these studies have detected decreases in NAA concentrations in the affected brain areas, with the notable exception of Canavan disease which involves accumulation of NAA throughout the brain (Wittsack et al., 1996). In earlier studies, the decreases in NAA associated with various neuropathologies were interpreted to represent irreversible loss of neurons. However, accumulating evidence indicates that these decreases in regional NAA levels can also represent reversible neuronal or mitochondrial dysfunction (Bates et al., 1996, Clark, 1998, De Stefano et al., 1995, Demougeot et al., 2004, Gasparovic et al., 2001, Kalra et al., 1998, Narayanan et al., 2001). This issue will arise later in the discussion in a number of contexts.

Reports on conditions other than Canavan disease that result in raised NAA levels are relatively few. An early investigation reported that anesthetics and GABA administration caused NAA levels to increase in rodent brains (Buniatian et al., 1965), but to our knowledge, these findings have not been confirmed elsewhere. Two recent reports have shown that NAA levels in the brain are increased under certain circumstances other than Canavan disease. First, striatal NAA levels as measured by MRS were reported to be elevated between 7% and 12% in children with sickle cell disease (Steen and Ogg, 2005). Second, administration of the anti-psychotic drug haloperidol has been reported to elevate NAA levels in the striatum of rats by 23% as measured by high performance liquid chromatography (HPLC) (Harte et al., 2005). No changes were observed in other brain areas. Reports from another group did not support these results, and instead these researchers detected no changes in NAA levels in frozen rat brain micro-punches after haloperidol treatment using HPLC (Bustillo et al., 2004) or high field MRS (Bustillo et al., 2005). Further studies are required to determine the effects of haloperidol on brain NAA levels.

The diseases and disorders in which brain NAA levels are decreased include brain ischemia, brain injury, brain cancer, multiple sclerosis and Alzheimer disease, among others (Danielsen and Ross, 1999). Several chemical treatments have been reported to reduce brain NAA levels, including a report that 4 day treatment with ethanol or alcohol dehydrogenase inhibitors reduced whole brain NAA levels by between 5% and 20% (Baslow et al., 2000). In a subsequent study, 5 day administration of lithium chloride was reported to reduce whole brain NAA levels by approximately 13% in the rat model of Canavan disease (Baslow et al., 2002). Selected MRS studies detailing changes in brain NAA levels associated with various conditions are discussed below.

The current state of knowledge on the use of MRS to analyze changing NAA levels in experimental ischemia in animals has been reviewed (Demougeot et al., 2004, Hoehn et al., 2001). The use of MRS in medical diagnosis of human neurological disease has also been described (Danielsen and Ross, 1999, Gillard et al., 2005, Lin et al., 2005). In human and animal studies, proton MRS and MRI have consistently demonstrated large increases in lactate levels, and significant decreases in NAA levels in ischemic brain tissue several hours after onset (Bruhn et al., 1989b, Di Costanzo et al., 2003, Fenstermacher and Narayana, 1990, Franke et al., 2000, Graham et al., 1994). However, there is a paucity of published information concerning the early response of neuronal NAA metabolism in human stroke patients. Localized decreases in NAA are seen within a few hours of ischemic onset in patients with clinical evidence of stroke, and the levels as measured by MRS are typically low or absent in chronic infarcts (Gideon et al., 1994, Gillard et al., 2005). Several key issues concerning spectroscopic examination of NAA remain unresolved. These include the basis of the relatively slow observed decreases in NAA signal after the onset of stroke, the clinical and functional correlates of NAA loss, and the issue of whether NAA levels can recover over time under certain circumstances. To the extent that satisfactory answers to these issues can be obtained from animal research, MRS of NAA can profoundly alter the way we diagnose, monitor and treat human stroke. First we will examine MRS studies of stroke and hypoxia in humans, and then we will explore findings from animal studies of ischemia to focus on possible mechanisms underlying the neurochemical changes observed by spectroscopy.

Parsons and coworkers have shown that the ratios of both NAA to creatine (NAA/Cr) and lactate to creatine (Lac/Cr) are diagnostic of the long-term outcome in stroke patients (Parsons et al., 2000). High Lac/Cr ratios (indicating anaerobic glycolysis) and low NAA/Cr ratios are indicative of poor outcome after stroke. However, animal and human studies have shown that the loss of NAA is relatively slow such that NAA levels may be only partially reduced by the time lactate levels begin to return to normal (Graham et al., 1993, Petroff et al., 1988). The Lac/Cr ratio during the subacute phase of stroke in humans has been particularly useful in determining long-term outcome, and is more predictive than diffusion weighted imaging of infarct size alone (Parsons et al., 2000, Walker et al., 2004). Kreis and colleagues provide evidence from studies after near-drowning that NAA levels in the human brain respond to hypoxia in a similar way to that described for animals (Kreis et al., 1996). In children examined repeatedly with quantitative MRS, T1/2 for NAA was estimated at about 48 h after near-drowning. Mean NAA concentrations were reduced by 25% after 1–2 days, and by 35–50% after 3–4 days. Five or more days after the hypoxic insult, NAA levels reached a minimum of about 25% of control. The degree and the rate of decline of NAA occurred considerably slower in a predominantly white matter region of the brain than that described above for a predominantly gray matter region. The reason for the slower reductions in the concentration of NAA in white matter remains unknown.

With regard to the parallels between changes in brain NAA levels as measured by MRS and the clinical outcome in stroke patients, much anecdotal evidence and a few controlled studies have equated water suppressed proton MRS measurements of NAA with neurological outcome. Neurological status on admission and neurological outcome a few weeks or months later do indeed correlate with NAA concentration and the ratio of NAA to creatine/phosphocreatine (NAA/Cr) in gray matter after near-drowning. The use of NAA as a surrogate for neuronal cell number and viability, and thus for diagnosis and prognosis in these clinical settings has been supported by clinical research (Danielsen and Ross, 1999). Finally, the question of whether NAA concentrations ever recover is crucial from the clinical management perspective, since the interpretation of NAA loss as representing neuronal cell death rests strongly on this premise. Several reports have documented NAA concentration recovery in gray matter after stroke, suggesting that some degree of reversible neuronal injury accompanies ischemia (Walker et al., 2004). Reports of recovery of NAA in white matter regions are more numerous, and in MELAS, a metabolically induced regional stroke characterized by accumulation of lactate and loss of NAA, recovery of NAA is regarded as the norm (Lin et al., 2003b). In a recent study of focal ischemia (1 h middle cerebral artery occlusion) in baboons, Coon et al. showed that lactate was significantly elevated 3 days and 10 days post-injury, and NAA was reduced significantly at both time points (Coon et al., 2006). In less severe injuries encompassing less than 30% of the affected hemisphere, NAA levels partially normalized by day 10. High lactate levels correlated negatively with functional outcome, whereas high NAA correlated positively with functional outcome. In a large study of 71 stroke patients, a substantial recovery of NAA levels was seen in patients with small strokes 100 days after the ischemic event (Walker et al., 2004).

A recent investigation on patients with ischemic injuries in the region of the middle cerebral artery examined risk factors associated with stroke, such as age, hypertension and diabetes to determine if metabolite ratios were affected in contralateral structures (Walker et al., 2006). These investigators found that of all the assessed risk factors, only hypertension correlated significantly with reduced NAA/Choline ratios in the basal ganglia and periventricular white matter in normal appearing regions contralateral to the injury. They concluded that hypertension affects metabolite ratios in normal brain tissue making MRS measurements after stroke problematic in hypertensive patients when using contralateral tissue as a control. These findings highlight the sensitivity of brain NAA to perturbations, and the ability of MRS to detect these changes, but also emphasize the many caveats associated with the use of these techniques to monitor ischemia and other brain injuries.

Animal studies provide a much more detailed picture of the progression of the pathophysiology associated with stroke and ischemia, but there are reasons for considering animal models as imperfect surrogates for studying stroke and hypoxia in humans. For example, animal models of stroke are done under highly controlled conditions, and the injuries are relatively uniform in extent and severity. Further, experimental ischemia in animals is induced under anesthesia, rather than in unanesthetized animals, which may affect the outcomes significantly. In contrast, human stroke is highly heterogeneous in nature and extent, and occurs under extremely varied conditions. Nonetheless, experimental models of ischemia have been very useful in following the evolution of ischemic injury using MRS, and in confirming the results by independent methods.

Barker and Gillard have recently summarized evidence from numerous animal studies showing that the decreases in NAA occur more slowly than increases in lactate after focal ischemia, and that larger drops in NAA are seen in the core of the infarct than in peripheral areas (Barker and Gillard, 2005). There is an approximate 10% decline in NAA over the first 6–8 h after occlusive stroke, with a T1/2 of about 15 h for the remaining 50%. The lowest in vivo levels of NAA are observed only after 50–70 h in animal studies (Higuchi et al., 1996, Sager et al., 1995). Animal studies into the early response to experimental ischemia have shown that increases in lactate can be observed by MRS within 40 min of onset, and small decreases in NAA/Cr were observed at 1 h (Yi et al., 2002). Studies in animals have demonstrated that the concentration of NAA, as measured by HPLC, may not always be a good surrogate for neuronal viability after experimental stroke (Demougeot et al., 2003). In these studies, NAA was dramatically reduced in the core infarct at 3 days (approximately 90%), but was partly recovered after 8 days (approximately 60% of control), and returned to near normal by 30 days post-ischemia (approximately 90% of control). Similar findings have been reported in rats subjected to a 15 min middle cerebral artery occlusion, where NAA/Cr was decreased significantly at 1 day post-injury, but recovered to near normal levels 2 weeks after injury (Wang et al., 2006). Histology showed that the partial recovery of NAA concentrations correlated with strong microglial activation and proliferation in the core of the infarct, suggesting that activated microglia may be capable of synthesizing NAA (Demougeot et al., 2003). Large declines in NAA levels associated with brain ischemia parallel the onset of histopathological events including dramatically reduced neuronal numbers, reduced cell size, nuclear pyknosis and infiltration first of polymorphonuclear cells, followed by infiltration of mononuclear cells, which are most evident between 24 and 48 h in the case of photochemically induced focal lesions in rats (Lee et al., 1996).

Animal studies of ischemia provide the opportunity to compare results from MRS with results from other more direct techniques for measuring NAA levels. Sager and colleagues have used multiple techniques to study NAA levels and the levels of other metabolites in experimental ischemic injury in rodents. HPLC analyses of focal and global ischemia in rats indicated a slightly more rapid decline in NAA levels in focal ischemia, reaching approximately 50% of controls in 8 h (Sager et al., 1995). The most conspicuous difference between focal and global ischemia was that aspartate levels decreased in focal ischemia, but were increased significantly over the course of 24 h after global ischemia, possibly indicating that global ischemia brings NAA and its degratory enzyme ASPA, normally segregated in distinct compartments, into contact. Using MRS, HPLC and microdialysis, Sager and colleagues showed that MRS and HPLC gave comparable results with respect to reductions in NAA levels, but that MRS tended to overestimate NAA concentrations, and underestimate NAA reductions during ischemia (Sager et al., 1999a). Clearance of NAA from the extracellular space, as measured by microdialysis, was observed after focal ischemia, but not after global ischemia. NAA export mechanisms may explain the differences in NAA loss between focal and global ischemia, where surrounding intact tissue in focal ischemia is able to remove NAA to the circulation and maintain a concentration gradient, whereas globally damaged brain may suffer from compromised glial clearance mechanisms.

In a subsequent study, Sager and colleagues used standard histopathological techniques in conjunction with HPLC analysis of NAA levels and NAA immunohistochemistry to examine ischemic injury (middle cerebral artery occlusion) in mice (Sager et al., 2000). NAA declined to 50% and 20% of control levels in infarcted tissue after 6 and 24 h, respectively, and no further decrease was observed during the following 6 days. Six hours after ischemic injury, the number of normal-appearing neurons in infarct-damaged cortical tissue decreased to 70% of control, and the majority of neurons were eosinophilic, indicating damage. After 24 h almost no normal-appearing neurons were seen. The number of eosinophilic neurons decreased steadily to virtually zero after 7 days. The number of immunopositive cells staining for standard neuronal markers within the infarct was progressively reduced, and after 3–7 days the staining was confined to discrete granulomatous material in the center of the infarct, which was infiltrated with macrophages. The central granulomatous material also stained positively for NAA. NAA levels as measured by HPLC 7 days after ischemia were still detectable, remaining between 10% and 20% of controls. Astrocyte markers progressively increased at the circumference of infarcted areas, and these areas also showed increased immunoreactivity against NAA. The authors concluded that NAA becomes trapped in dying neurons and in cell debris, thus restricting its use as a marker of neuronal density. There are reasons for questioning the “NAA trapping hypothesis” as an adequate explanation for the remaining NAA levels at 7 days post-ischemic injury. First, it is possible that low levels of NAA production could be associated with macrophages or reactive microglia in the infarct and peri-infarct areas under pathological conditions. Further, other acetylated compounds may contribute 10% or more to the so-called “NAA signal” in MRS, and some of these compounds may be more resistant to decline after injury than NAA (Pouwels and Frahm, 1997, Sager et al., 2001). Finally, we have observed that the method used to perform NAA immunohistochemistry (Moffett et al., 1993, Moffett and Namboodiri, 1995) causes strong, non-specific antibody binding to damaged brain tissue (unpublished observations), as was observed by Sager and colleagues in areas bordering the ischemic injury (Sager et al., 2000).

In order to determine whether NAA levels more closely parallel neuronal loss in transient global ischemia, where overt tissue necrosis is avoided, Sager and coworkers studied the loss of CA1 hippocampal neurons in the two vessel occlusion model of global ischemia in gerbils (Sager et al., 2001). NAA levels as measured by single-voxel proton MRS and HPLC correlated with neuronal loss as determined by histological examination, with a reported 25% reduction in NAA and complete loss of CA1 pyramidal neurons 4 days after ischemic injury. The death of CA1 neurons and the loss of NAA were comparably slow in the global ischemia model as compared with the focal model. NAA could be released from dying neurons and taken up and excreted to the circulation by remaining, ischemic-resistant glia. In global ischemia all brain tissue is damaged to some extent, rather than a specific locus, thus increasing the efflux of NAA from neurons throughout the brain, while simultaneously impairing glial clearance.

In summary, despite complicating issues, reduced NAA levels as detected by MRS can be an extremely valuable marker of brain injury after stroke or hypoxia. In vivo MRS studies support the hypothesis of NAA as a surrogate for neuronal loss and dysfunction, and the clinically associated neurological deficits observed in patients after local or global hypoxia-ischemic incidents. Outcome predictions based upon residual NAA, and increased lactate levels in localized proton spectra have proved rather accurate in such varied ischemia settings as stroke (Barker and Gillard, 2005, Parsons et al., 2000, Wild et al., 2000), neonatal hypoxia (Barkovich et al., 1999, Cappellini et al., 2002), and near-drowning in children (Kreis et al., 1996).

A reasonable working hypothesis, subsequently confirmed in myriad studies, suggests that regional reductions in NAA concentration and NAA-creatine ratio are to be found in individual patients and in patient groups who test positive for clinical dementia. Patients with Alzheimer disease consistently show a 15–20% reduction in NAA/Cr (or NAA concentration) in posterior cingulate gyrus gray matter (Kantarci and Jack, 2003, Moats et al., 1994, Shonk et al., 1995, Waldman et al., 2002). The gray matter in the region of the posterior cingulate gyrus has been found to be the most reliable for the diagnosis of this and other dementias, rather than hippocampus, the region selected for PET diagnosis, and postmortem histological examination of Alzheimer disease. In this regard, NAA levels may reveal something about Alzheimer disease and the metabolic basis of memory and attention. The finding that the posterior cingulate gyrus loses NAA early in the disease supports the notion that this brain region takes part in attention and memory (Raichle et al., 2001). It has proved technically more difficult to define significant loss of NAA from the hippocampus by MRS, the region most implicated in memory, and involved earliest in plaque deposition as demonstrated by histopathological studies of Alzheimer disease brain. This does not argue against existing theory; rather it indicates the technological limitations of in vivo MRS measurements in the temporal lobe. In the hippocampus, a relatively small structure in the magnetically inhomogeneous environs of the petrous temporal bone, localized short echo time MRS has measurement errors of 20% or more, sufficient to mask any biological differences in NAA associated with Alzheimer disease. On the other hand, use of the more robust MRS technique, long echo time chemical shift imaging confirms a loss of NAA from the hippocampus in Alzheimer disease which is directly proportional to the volume loss of that structure (Schuff et al., 2002). Schuff and colleagues have reported that NAA reductions and volume loss made independent contributions to the correct discrimination of Alzheimer disease patients from controls, with a better than 80% correct diagnosis using NAA declines alone, which improved to 90% when NAA loss and volume reductions were combined (Schuff et al., 2006).

In the familial form of Alzheimer disease NAA/Cr is markedly reduced from the posterior cingulate gyrus (Lin et al., 2003a), and pre-symptomatic carriers have significantly reduced NAA/Cr and NAA-myo-inositol (NAA/MI) metabolite ratios (Godbolt et al., 2006). A precursor of Alzheimer disease is the recently defined syndrome of mild cognitive impairment. Evolution to true Alzheimer disease occurs in a sizeable proportion of these patients and is also accompanied by a reduction in brain NAA/Cr (Kantarci et al., 2000, Kantarci et al., 2002, Kantarci et al., 2003). Perhaps most instructive in this exploration of a role for NAA in a disease for which histopathological confirmation is notoriously lacking, is the recent MRS data from the well defined knock-out models of Alzheimer disease in mice (Jack et al., 2004, Jack et al., 2005). In mice that coexpress mutated forms of human presinilin-1 and amyloid-β precursor protein, accelerated amyloid deposition occurs, and the ratios of NAA/Cr, NAA/MI and glutamate/Cr are decreased with increasing age.

One plausible hypothesis places NAA at the end of the chain of events wherein dementia results from a critical reduction in the number of functioning neuronal units. That could mean that NAA is simply a direct means of measuring neuronal number and hence quantifying the degree of neurodegeneration. An alternative hypothesis associates NAA with neuronal energetics, suggesting that reduced NAA could simultaneously reflect both reduced neuronal number, and reduced neuronal energetics in the remaining neurons. [13C] MRS has been used to demonstrate that glutamate turnover falls in Alzheimer disease in proportion to the loss of NAA concentration (Lin et al., 2003a) and that both glutamate and NAA levels are reduced in the frontal lobes of Alzheimer mice (Dedeoglu et al., 2004). Human NAA-turnover rates in the brain as measured by MRS have recently been published (Harris et al., 2006) offering a glimpse of a role for NAA metabolism, rather than steady-state concentrations of NAA in the pathobiology of Alzheimer disease. The rate of NAA-synthesis, normally about 1% of the Krebs cycle, is decreased by approximately 60% in Canavan patients (9.2 + 3.9 nmol/min/g in controls versus 3.6 + 0.1 nmol/min/g in Canavan disease) (Moreno et al., 2001). In contrast to Canavan disease where NAA synthesis rates are reduced, NAA synthesis rates appear to be modestly increased in Alzheimer disease brain (Harris et al., 2006). If confirmed, this would suggest an adaptive process which might represent a compensatory upregulation of NAA synthesis in remaining functional neurons in Alzheimer disease and perhaps other dementias.

It has been known since the 1800s that hippocampal damage is apparent in brain autopsies of epilepsy patients, a condition known as hippocampal sclerosis. In particular, neuronal loss is especially apparent in regions CA1, CA3, and the hilus of the dentate gyrus. Descriptive studies with [1H] MRS in patients with epilepsy of all types include the observation of focal or global reductions in NAA signal and NAA/Cr, regardless of etiology (Briellmann et al., 2005). However, seizures also occur in many patients with normal NAA concentrations and NAA/Cr, so that most investigations have explored focal epilepsies, including post-inflammatory (e.g., Rasmussen's encephalitis), post-tumoral and mesial temporal sclerosis induced seizures (temporal lobe epilepsy or TLE). In all of these, NAA concentrations are reduced. Perhaps the most challenging observations come from clinical studies of TLE. While NAA/Cr is systematically reduced in the affected hemisphere, the contralateral hemisphere is frequently affected as well (Vermathen et al., 2002). This certainly complicates the use of MRS–NAA data as a clinical lateralizing tool for epilepsy surgeons. But more interesting is the fact that after extirpation of the damaged tissue from the temporal lobe, NAA recovers both in the tissue behind the resection, and in the contralateral hemisphere (Serles et al., 2001). Serles and colleagues suggest that these post-operative increases in NAA levels may result from recovery of neuronal metabolism, and possibly increased dendritic sprouting, synaptogenesis, and neurogenesis. Such observations, supported by histological and anatomic MRI evidence, provide convincing evidence of NAA-recovery with time, and confirm that NAA is responsive to transient neuronal dysfunction (Briellmann et al., 2005). This aspect of NAA metabolism will be discussed further in Section 6 below.

There are five basic changes in the MRS signal associated with brain tumors; NAA is decreased, lactate is increased, lipid is increased, creatine plus phosphocreatine are decreased, and choline is increased (Danielsen and Ross, 1999). NAA is reduced or even absent from most brain tumor spectra, as well as from most space-occupying brain lesions, whether benign or malignant (Arnold et al., 1990b, Barker et al., 2006, Bruhn et al., 1989a, Danielsen and Ross, 1999, Fulham et al., 1992). The explanation for the lack of NAA signal lies in the cell lines of origin for almost all primary and secondary brain tumors, which do not express the biosynthetic enzyme for NAA, Asp-NAT (see Section 2.1 below). Interestingly, central neurocytoma (a rare neuronal tumor accounting for less than 0.5% of CNS tumors) have been reported by some investigators to exhibit detectable NAA levels, but reduced NAA/Cr (Chuang et al., 2005, Jayasundar et al., 2003). Hypothalamic hamartomas are benign congenital malformations that are disorganized in cytological architecture, and can contain predominantly glial or neuronal populations. Amstutz and coworkers have recently shown that hypothalamic hamartomas exhibit reduced NAA/Cr and increased mI/Cr ratios as compared with normal gray matter (Amstutz et al., 2006). Further, using histological analyses of these tumors, they show that the NAA signal is greater in hamartomas with predominantly neuronal content, and lower in hamartomas with predominantly glial content. This finding confirms the utility of proton MRS to differentiate tumors with different cellular populations. One of the most promising uses for MRS in neuro-oncology may be to follow the course of recovery after radiation therapy for glioma, permitting the differentiation of active glioma from radiation effects in the surrounding tissue (Fan, 2006).

Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the CNS which links axonal damage to reduced NAA levels in gray and white matter (Criste and Trapp, 2006). Clinical studies (Bruhn et al., 1992, Davie et al., 1994, Larsson et al., 1991, Leary et al., 1999) as well as a recent meta-analysis of the use of MRS in multiple sclerosis (Caramanos et al., 2005) universally show decreased NAA levels associated with the progression of the disease. Reduced amounts of NAA and NAA/Cr in MR-visible lesions and in normal appearing white matter are readily documented (De Stefano et al., 2001, Fu et al., 1998, Larsson et al., 1991, Tedeschi et al., 2002). In this central inflammatory disease, additional MRS changes in cerebral choline, myoinositol, lactate, and lipid have provided insights and diagnostic value. With the investigations of Trapp and colleagues demonstrating transection of axons throughout the brain of MS patients, these MRS observations are explained as axonal injury (Criste and Trapp, 2006, Trapp et al., 1998). NAA also falls in gray matter, perhaps accounting for cognitive defects often recorded in multiple sclerosis patients (Staffen et al., 2005). Assays of whole-brain NAA using MRI have shown that cognitive impairment in multiple sclerosis correlates with reductions in NAA content (Mathiesen et al., 2006). While atrophy and loss of NAA are both features of multiple sclerosis, the degree of loss of whole-brain NAA exceeds the development of atrophy by several fold, encouraging the conclusion that neuronal dysfunction may precede tissue loss in multiple sclerosis (Mathiesen et al., 2006). Studies employing NAA measurements appear to provide a better correlation with disability in MS patients than do MRI measures of lesion load (Wolinsky and Narayana, 2002). Other neurodegenerative diseases such as amyotrophic lateral sclerosis also show reductions in NAA in affected brain regions (Kalra and Arnold, 2004, Kalra and Arnold, 2006, Rooney et al., 1998), demonstrating the usefulness of MRS for clinical observations on the progression of neurodegenerative disorders of all types.

Axonal injury begins early in multiple sclerosis (De Stefano et al., 2002), and cumulative axon loss results in progressive disability. However, the disease often goes through phases of remission and relapse, and white matter plaques visible in magnetic resonance images can wane with remission of symptoms. MRS studies show that NAA levels can be associated with neuronal dysfunction, as well as neuronal death, because levels have been shown to recover when MRI visible plaques resolve (Arnold et al., 1990a). Partial recovery of NAA levels has also been reported after treatment of patients with interferon beta-1b (Narayanan et al., 2001) glatiramer acetate (Khan et al., 2005) or fluoxetine (Mostert et al., 2006) suggesting that NAA levels reflect not only neuronal and axonal integrity, but also may reflect improvements in neuronal energetics and possibly remyelination.

Human immunodeficiency virus (HIV) infection in and of itself does not result in reduced NAA levels in the brain, but many HIV-associated encephalopathies do result in altered brain metabolite levels, including reduced NAA (Paley et al., 1996). In addition to AIDS-dementia complex, a condition which significantly reduces regional concentrations of NAA (Meyerhoff et al., 1993, Paley et al., 1996, Vion-Dury et al., 1994), a number of associated conditions stemming from immune-incompetence also result in local or global reductions in NAA and NAA/Cr (Schuff et al., 2006). These include lymphoma, in which the brain spectrum closely resembles that from other brain neoplasms, and toxoplasmosis, an opportunistic intracellular parasitic infection which reduces all cerebral metabolite concentrations including NAA (Chinn et al., 1995). JC-virus induced progressive multifocal leukoencephalopathy is associated with moderate reductions in NAA, and increased choline and myo-inositol (Chang et al., 1997). Progressive multifocal leukoencephalopathy is a fatal demyelinating disease of the central nervous system that predominantly affects immunocompromised individuals (Hou and Major, 2000). This CNS viral disease may now be arrested or even reversed by introduction of effective treatments termed HAART (highly active anti-retroviral therapy), in which cases, the decline in cerebral NAA may also be arrested.

Viral encephalopathies including herpes encephalitis (Danielsen and Ross, 1999, Menon et al., 1990), and prion diseases such as Creutzfeldt-Jakob disease (Oppenheim et al., 2004), result in reductions in NAA and NAA/Cr over quite large volumes of the affected brain, and may provide clues as to the nature of the disease process. In hamsters inoculated with Creutzfeldt-Jakob disease, large reductions in NAA were observed in cortex only in later stages of the disease process (135 days post-inoculation) (Behar et al., 1998) suggesting that the reductions were associated with tissue loss. Bacterial and fungal abscesses all result in local reductions in NAA concentration and NAA/Cr ratio by the means of tissue destruction (Gujar et al., 2005, Harada et al., 1994, Kadota et al., 2001, Nakaiso et al., 2002).

NAA levels as measured by MRS have proven to be a sensitive measure of neuronal compromise after traumatic brain injury (TBI) (Danielsen and Ross, 1999). Appearances on CT and MRI scans after TBI, while of great assistance in management of individual clinical problems, do not always correlate well with neurological deficit during the acute phase after brain injury, and give even less guidance on long-term outcome for patients. In contrast, the use of proton MRS to determine NAA content locally, and distant to the site of injury, has proved of considerable value in the clinical setting (Brooks et al., 2001) based upon the simplifying hypothesis that in white matter, loss of NAA defines diffuse axonal injury, and in gray matter, loss of neurons. However, the heterogeneous nature of the neuropathological response to trauma has stimulated interest in other interpretations. In infants subject to “shaken baby syndrome” loss of NAA appears to follow a cascade of neurochemical events in which activated phospholipases are hypothesized to release lipids and macromolecules visible in the proton brain spectrum (Haseler et al., 1997). Childhood traumatic brain injury may result in a reversible loss of NAA which recovers approximately with the time-scale of recovery from the syndrome of inappropriate anti-diuretic hormone (SIADH) (Ross et al., 1998). Endocrine response to trauma, in the form of SIADH, is seen in 40–50% of children with head injury, and often results in reduced blood and brain osmolytes, or hyponatremia. Effects of brain hyponatremia are seen in MRS in the form of decreased myo-inositol, choline, creatine, and NAA (Danielsen and Ross, 1999). Despite potential complicating factors such as hyponatremia, MRS measurements of NAA levels in the brain after TBI provide significant prognostic value relative to long-term outcome, wherein higher post-injury NAA levels correlate with significantly better neurological recoveries.

MRS observations in humans after TBI or hypoxia suggest a time course of hours or days for substantial loss of NAA from affected brain tissue. However, animal studies of moderate to severe TBI have shown more rapid declines in NAA levels which were paralleled by decreases in ATP levels. Using HPLC analyses of brain extracts, Signoretti and colleagues reported significant and concomitant drops in NAA and ATP within 10 min of injury, and partial recovery of both compounds by 5 days in less severe injuries (Signoretti et al., 2001). In more severe TBI injuries, and those exacerbated by hypoxia-hypotension, recovery of NAA and ATP levels was not observed. In accord with human MRS studies, the lowest NAA levels attained after severe TBI were detected at the longest time period examined (5 days). These data provide strong support for the idea that NAA levels are linked to ATP levels, and that both can recover after injuries that do not involve substantial, permanent brain tissue destruction. In summary, MRS measurements of NAA provide an invaluable tool for assessing the degree and potential recoverability of brain damage following head injury.

MRS is capable of detecting both endogenous compounds in the brain as well as consumed or administered drugs ranging from anti-cancer agents (Port and Wolf, 2003), to alcohol ingested in excess (Danielsen and Ross, 1999) to anti-epileptic medications (e.g., valproic acid) administered in therapeutic doses (Bluml et al., 2002). Chronic alcoholism is accompanied by loss of NAA (Jagannathan et al., 1996, Meyerhoff et al., 2004) which in some studies is reported to recover after prolonged abstinence (Parks et al., 2002).

In the great majority of neuropsychiatric disorders listed in DSM-IV 1R, the accepted compendium of clinical psychiatric diseases, there has been little incontrovertible evidence of abnormalities in the concentration of NAA in the brain. The neuropsychiatric condition most studied by MRS is schizophrenia, wherein the majority of published papers report small, regional reductions in NAA or NAA/Cr (Bertolino et al., 1996, Callicott et al., 2000a, Deicken et al., 2001). Often the targeted regions differ from one study to the next so that we obtain no clear picture of the full extent of the loss or its regional distribution (Deicken et al., 2000b), and some studies have found no significant alterations in NAA or NAA/Cr in schizophrenic patients. A number of groups have reviewed the use of MRS to analyze alterations in NAA and other metabolites in schizophrenia (Abbott and Bustillo, 2006, Bertolino and Weinberger, 1999, Keshavan et al., 2000, Marenco et al., 2004, Rowland et al., 2001, Stanley et al., 2000). A recent meta-analysis of MRS studies on NAA levels in schizophrenia indicated that most studies did not include enough subjects for substantial statistical power, but that the majority of studies indicated gray matter reductions of approximately 5–10% in the frontal lobes (Steen et al., 2005). A number of studies have found that NAA levels and NAA/Cr ratios in the medial temporal lobe and the prefrontal cortex are reduced in schizophrenia (Abbott and Bustillo, 2006), and that these reductions parallel alterations in cerebral blood flow measured with PET and functional MRI (Callicott et al., 2000b, Marenco et al., 2006a). In a recent MRS study comparing NAA concentrations in 14 schizophrenics with 13 control subjects it was found that NAA was decreased significantly in the frontal lobe of affected patients (average, 7.94 mmol/L, compared with healthy subjects average of 8.45 mmol/L, P < 0.05) (Tanaka et al., 2006). The reduced NAA levels correlated with the severity of negative symptoms and poor performance in the Wisconsin Card Sorting Test. Other brain regions where NAA reductions have been reported in schizophrenia include the thalamus (Deicken et al., 2000a, Ende et al., 2003, Jakary et al., 2005), anterior cingulate cortex (Deicken et al., 1997) and the cerebellum (Ende et al., 2005).

Because schizophrenia may be associated with volume reductions in certain brain regions (Selemon et al., 2002, Selemon and Goldman-Rakic, 1999, van Haren et al., 2003), such as the superior temporal gyrus, the medial temporal lobe, and prefrontal cortex (Shenton et al., 2001), MRS data are usually normalized for tissue volume (Ohrmann et al., 2006). Nonetheless, it is difficult to completely separate the issues of tissue volume and metabolite concentrations as measured by MRI and MRS, and some differences observed between schizophrenic patients and controls may have a volume component. Early signs of schizophrenia have been associated with reductions in NAA or NAA/Cr. For example, NAA/Cr was reduced in the left frontal lobe of patients who were considered at risk for developing schizophrenia, as well as in schizophrenics (Jessen et al., 2006). Wood et al. found that the only MRS measure which correlated with poor outcome 18 months after a first psychotic incident in schizophrenia patients was a low NAA/Cr ratio detected in the prefrontal cortex (Wood et al., 2006). Further, reductions in NAA levels in the dorsolateral prefrontal cortex of schizophrenic patients have been correlated with poorer performance in the Auditory Verbal Learning Task (AVLT) indicating connections between cognitive performance and NAA levels (Ohrmann et al., 2006).

Intriguing functional correlates of the observed NAA reductions in schizophrenia have emerged over the last several years. Ohrmann and coworkers used MRS to show that glutamate and glutamine levels in the frontal lobes of schizophrenia patients were reduced in conjunction with the loss of NAA, suggesting a connection between NAA levels and glutamatergic neurotransmission (Ohrmann et al., 2005). A number of studies have found that the expression or activity of proteins and genes associated with glutamatergic neurotransmission are altered in schizophrenia patients (Moghaddam, 2003, Tsai et al., 1995, Tsai et al., 1998). Metabotropic glutamate receptors are involved in regulating neurotransmitter release, including the release of glutamate (Cartmell and Schoepp, 2000), and disorders of the NMDA type of glutamate receptor have been strongly implicated in the etiology of schizophrenia (Moghaddam and Jackson, 2003). In individuals at risk for schizophrenia Egan and colleagues found variations in a particular allele of GRM3, the gene encoding the type 2/3 metabotropic glutamate receptor (mGluR2/3) (Egan et al., 2004). These GRM3 alleles were associated with reduced levels of NAA in prefrontal cortex, and reduced expression of a glial glutamate transporter. MRS studies which examined individuals with the schizophrenia associated GRM3 allele confirmed reductions in NAA levels in the right prefrontal cortex (Marenco et al., 2006b). NAA reductions in prefrontal cortex have been associated with a dysregulation of dopamine release in the striatum of schizophrenia patients (Bertolino et al., 2000). It is interesting to note that NAAG, which is synthesized from NAA, acts to regulate glutamate and dopamine release, most likely via activation of presynaptic mGluR2/3 receptors.

Additional work with larger groups will be required to confirm and expand these results and determine the full degree and localization of perturbations in NAA metabolism in schizophrenia patients. Further work will also be required to ascertain if NAA metabolic disturbances are etiologically involved in the development of schizophrenia, or are a secondary consequence of other factors. Nonetheless, as techniques improve, MRS will certainly become an invaluable tool in the diagnosis and treatment of schizophrenia (Abbott and Bustillo, 2006).

It is important to mention before concluding this section that NAA levels in the brain as measured by MRS, particularly levels in white matter, have been positively correlated with general measures of intellectual functioning (Yeo et al., 2006). In fact, the regions of white matter that were found to correlate with general measures of intelligence were reported to be different in the brains of men and women (Jung et al., 2005). These findings highlight the sensitivity and utility of MRS techniques for studying normal and pathological brain function non-invasively, but also raise additional questions concerning the role of NAA in brain activity and cognition.

NAA has presented neuroscientists with a particularly perplexing subject of study in part because application of NAA to various cell types often does not elicit a detectable response. Based on a relatively small number of published papers, four primary hypotheses have been proposed for the role of NAA in the nervous system. These include: (1) NAA acts as an organic osmolyte that counters the “anion deficit” in neurons, or a co-transport substrate for a proposed “molecular water pump” that removes metabolic water from neurons, (2) NAA is an immediate precursor for the enzyme-mediated biosynthesis of the important neuronal dipeptide N-acetylaspartylglutamate (NAAG), (3) NAA provides a critical source of acetate for myelin lipid synthesis in oligodendrocytes, and (4) NAA is involved in facilitating energy metabolism in neuronal mitochondria.

In addition, a limited number of reports suggest other possible roles for NAA. In a single study, NAA was suggested to form a complex with transfer RNA, which then might be involved in the initiation of protein synthesis (Clarke et al., 1975). NAA has also been reported to increase cAMP and cGMP levels in minced cortical preparations (Burgal et al., 1982). In another more recent study, NAA was proposed to be an endogenous ligand for G protein-coupled metabotropic glutamate receptors (Yan et al., 2003). In this study, NAA acted in a dose-dependent manner to induce neuronal depolarization in dissociated hippocampal neurons, but this finding has not yet been confirmed in other laboratories. Finally, NAA has been reported to be present in and released by peritoneal mast cells, implicating it in possible immune functions (Burlina et al., 1997). It is interesting to note that NAAG has potent anti-allergic actions (Bonnet et al., 1985, Chevance and Etievant, 1986, Jambou and Lapalus, 1990, Lapalus et al., 1986, Miadonna et al., 1998).

It is likely that the list of biological functions served by NAA will grow with additional research. For further background information, two previous reviews on the state of knowledge about NAA are available (Baslow, 2003b, Tsai and Coyle, 1995), and a recent international symposium on NAA has been published (Moffett et al., 2006).

Section snippets

NAA synthesis

Studies on the biosynthesis of NAA had a controversial beginning. In 1959, Goldstein reported that in the brain NAA is synthesized through the acetylation of aspartate by a soluble enzyme, l-aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) (Goldstein, 1959). This result was subsequently disputed on the grounds of incomplete product identification and the fact that a membrane bound enzyme was found which could use acetyl CoA and aspartate to form NAA (Goldstein, 1969, Knizley, 1967). It was

NAA and Canavan disease

A rare leukodystrophy, or spongy degenerative disease involving brain white matter was described in an infant by Myrtelle May Moore Canavan in 1931, which she tentatively identified as Schilder's disease, or diffuse cerebral sclerosis of Schilder (Canavan, 1931). This fatal infantile condition was later determined to be a unique autosomal inheritable white matter degenerative disease by van Bogaert and Bertrand in 1949 (van Bogaert and Bertrand, 1949). Despite the fact that these clinicians

NAA and NAAG biosynthesis

NAAG is the most concentrated neuropeptide in the human brain. 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, Moffett and Namboodiri, 2006, Tieman et al., 1991, Tieman and Tieman, 1996). NAAG is released from synapses in a calcium dependent manner (Williamson et

NAA breakdown and myelin lipid synthesis

Canavan disease affects children throughout the world, but is most common in Ashkenazi Jewish and Saudi Arabian families (Adachi et al., 1973, Matalon et al., 1995). It is not known how many children are born each year with Canavan disease, and many cases certainly go undiagnosed. Currently there is no effective treatment available for restoring proper myelination and motor function. The pathogenic mechanisms operative in Canavan disease currently remain a matter of debate, but it has been

NAA in neuronal energy metabolism

The earliest indication that NAA could be involved in energy metabolism was the second report by Harris Tallan in which he showed that NAA was present at high concentrations in the brains of birds and mammals, and that the distribution pattern closely paralleled the distribution of “respiratory activity” (Tallan, 1957). A subsequent report by Buniatian and coworkers in 1965 also indicated that NAA might be involved in brain energy metabolism (Buniatian et al., 1965). Using rat brain cortical

Integrating various NAA functions in the CNS

Recently, Madhavarao and coworkers proposed a model whereby NAA has two primary roles in the nervous system; facilitation of energy metabolism in neuronal mitochondria, and a source of acetate for fatty acid and steroid synthesis in oligodendrocytes. In this model, Asp-NAT facilitates removal of excess aspartate from the matrix of neuronal mitochondria via acetylation, thus favoring alpha ketoglutarate formation from glutamate, and energy production via the citric acid cycle. Anaplerosis and

Summary and future directions

NAA remains an enigmatic molecule, but researchers are beginning to decipher the complex web of CNS biochemistry in which it is actively involved. NAA is one of the most prominent metabolites in magnetic resonance spectrograms of brain, and the measured levels are highly sensitive to brain injury or disease, providing an invaluable tool for diagnosis and evaluation in clinical settings. Increases in MRS sensitivity and reduced voxel size, in conjunction with emerging scanner technologies and

Acknowledgements

This work is dedicated to the memory of Dr. Suzannah Bliss Tieman. We would like to thank Dr. C. Demougeot for commenting on the manuscript. We apologize to any researchers whose relevant work we did not directly cite. This work was supported by a grant to MAAN from the NIH (Grant # RO1: NS39387), and by grants from the Samueli Institute for Information Biology and Jacob's Cure. CNM was supported partially by a fellowship from the American Academy of Neurology Foundation, co-sponsored by the

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