Oligodendrocytes Do Not Export NAA-Derived Aspartate In Vitro

Abstract

Oligodendroglial cells are known to de-acetylate the N-acetylaspartate (NAA) synthesized and released by neurons and use it for lipid synthesis. However, the role of NAA regarding their intermediary metabolism remains poorly understood. Two hypotheses were proposed regarding the fate of aspartate after being released by de-acetylation: (1) aspartate is metabolized in the mitochondria of oligodendrocyte lineage cells; (2) aspartate is released to the medium. We report here that aspartoacylase mRNA expression increases when primary rat oligodendrocyte progenitor cells (OPCs) differentiate into mature cells in culture. Moreover, characterising metabolic functions of acetyl coenzyme A and aspartate from NAA catabolism in mature oligodendrocyte cultures after 5 days using isotope-labelled glucose after 5-days of differentiation we found evidence of extensive NAA metabolism. Incubation with [1,6-13C]glucose followed by gas chromatography–mass spectrometry and high performance liquid chromatography analyses of cell extracts and media in the presence and absence of NAA established that the acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of acetyl coenzyme A. We also resolved the controversy concerning the possible release of aspartate to the medium: aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. Therefore we propose that: aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate–aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate.

Introduction

The brain is an organ with exceptionally high energy demands and relies on an uninterrupted supply of substrates for oxidative phosphorylation in mitochondria. Around 25 % of the body’s total glucose budget is spent on processes in the brain, including the generation of action potentials and synaptic transmission [1]. Glucose-derived energy is therefore of utmost importance for maintaining physiological function of the brain. In contrast, many neurodegenerative diseases including Alzheimer’s disease [2] are associated with compromised glucose metabolism and markers of low energy status. Quantification of N-acetylaspartate (NAA) has often been used to assess the metabolic integrity of neurons [3, 4]. NAA can be detected by 1H-magnetic resonance spectroscopy, and has been applied as a non-invasive quantitative method for detecting progression, recovery, and remission in an ever-increasing catalogue of disorders of the brain [5]. However, the fundamental role of NAA in the brain remains elusive and the available evidence for its function is limited to a role in providing acetyl groups for lipid synthesis [6].

NAA is amongst the most abundant amino acid derivatives in the brain and is synthesized from aspartate and acetyl coenzyme A (CoA) by aspartate N-acetyltransferase [7]. After birth, NAA content in the brain is subject to a rapid increase to reach concentrations of 5 to 10 mM [8, 9], being especially concentrated in gray matter-rich regions [912]. NAA synthesis is dependent on mitochondrial integrity [7, 13] and fluctuations in concentration can occur in parallel with changes in adenosine triphosphate (ATP) [14], suggesting an intimate relationship with metabolic energy.

NAA is produced by and released from neurons. These depend on the supply of precursor molecules provided by astrocytes to synthesize the aspartate necessary for NAA production [15, 16]. NAA is metabolized in oligodendrocytes, which contain aspartoacylase (ASPA) [1719], the only known NAA-catabolizing enzyme in the brain. The importance of ASPA to myelination is highlighted by the severely dys-myelinated phenotype of the inherited human paediatric leukodystrophy, Canavan disease (CD), which results from the loss of ASPA function. The abnormally high levels of NAA in CD are in contrast to abnormally low levels of NAA that typically are seen in practically all other neurodegenerative diseases. Considering the importance of NAA as a prognostic marker of metabolic function across a wide pathological range the view that NAA solely acts as a shuttle for acetyl groups during lipid synthesis may therefore be insufficient. Multiple roles have been suggested for NAA such as: being an osmolyte that acts as a “molecular water pump” to remove metabolically produced water from neurons [20, 21]; in addition to moving acetate groups across the mitochondrial membrane system, NAA also acts by moving nitrogen groups to the cytoplasm [22]; NAA is involved in facilitating glutamine/glutamate oxidation in neuronal mitochondria, while bypassing the glutamate dehydrogenase reaction and therefore avoiding ammonia production [23]; being a storage and transport form of acetate [24]. Other proposed roles include the involvement in histone and protein acetylation reactions [25]; altering metabolism (aerobic glycolysis and Warburg effect) in cancer cells [26, 27] and, more recently, it was linked to neuronal differentiation [28].

In order to investigate the role of NAA in intermediary metabolism, we incubated cultures enriched for mature oligodendrocytes in medium containing [1,6-13C]glucose for 8 and 24 h in the presence and absence of NAA and analysed cell extracts and media using gas chromatography–mass spectrometry (GC–MS) and high performance liquid chromatography (HPLC). We found that the acetyl CoA produced by hydrolysis of NAA does not enter mitochondrial metabolism. We propose that the aspartate joins the aspartate pool active in the malate–aspartate shuttle and we established that aspartate is not released to the medium in amounts detectable by our methods.

Materials and Methods

Materials

Cell culture reagents were purchased from Sigma (Dorset, UK)—Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium Eagle (MEM), l-glutamine, poly-l-lysine (PLL), papain, NAA—or Life Technologies (Paisley, UK)—fetal bovine serum (FBS), penicillin–streptomycin (pen–strep), trypsin–EDTA, phosphate buffered saline (PBS). 13C-labelled compounds were obtained from Cambridge Isotope Laboratories, MA, USA. The mass spectrometry derivatization reagents MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide), MSTFA (N-methyl-N-(trimethylsilyl) trifluoroacetamide) and the t-BDMS-Cl (tert-butyldimethylchlorosilane) were purchased from Regis Technologies, Inc. (Morton Grove, IL, USA). Recombinant human PDGF-AA and Recombinant human FGF-basic were purchased from PeproTech (Rocky Hill, NJ). All other chemicals were of the purest grade available from Sigma (Dorset, UK).

Primary Cultures of Rat Oligodendrocytes

Primary mixed glia cultures were isolated from neonatal Sprague Dawley rat (postnatal day 0–2) forebrains following established protocols [29]. Briefly, pups were euthanized according to “Schedule 1” regulations from the Home Office Animal Procedures Committee UK. Cells were cultured for 10–15 days in DMEM supplemented with 10 % FBS, 1 % pen-strep and 4 mM glutamine, and kept under a humidified atmosphere at 37 °C and 7 % CO2. Oligodendrocyte precursor cells (OPCs) were subsequently isolated using a step-based shake-off protocol and cultured in Sato’s medium on PLL-coated plates [29]. To obtain immature OPCs, cells were cultured for 1 day in Sato’s medium supplemented with human PDGF-AA and human recombinant FGF at 10 ng/ml. To induce differentiation, OPCs were cultured in Sato’s medium supplemented with 0.5 % fetal calf serum (FCS) for 1 or 5 days. The cell culture medium was replaced by fresh medium at day two of differentiation. For all experiments only cultures with >93 % purity (determined based on O4 immunostaining) were used [30].

Quantitative Reverse Transcriptase PCR

To determine the mRNA levels of ASPA during OPC differentiation, cells were cultured either for 1 day in proliferation medium, or for 1 or 5 days in differentiation medium. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Manchester, UK). cDNA was synthesized from 20 ng RNA per sample using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA USA). Quantitative PCR (qPCR) was conducted as previously outlined [29] on an Applied Biosystems 7500HT Fast Real-time PCR system. The primers used were: ASPA F-AGACGTGGCTGCTGTTATCC; ASPA R-GATCTCCAGGGTGCAATGGT; beta actin F-CATGGCATTGTGATGGACT; beta actin R-ACGGATGTCAACGTCACACT. Values are represented as ASPA/beta actin ratios. Measurements were made on 8–11 samples obtained from three independently generated cultures.

Incubations with 13C Labelled Glucose and NAA

OPCs isolated from mixed glia cultures were cultured in 6 well plates at a cell density of 4 × 105 cells/well and allowed to differentiate for 5 days (see above for details). Prior to incubation, cells were washed once with PBS and incubated with 2 ml Sato’s medium prepared from a glucose, glutamine and pyruvate-free DMEM (Sigma D5030, Dorset, UK) supplemented with 0.5 % FCS and 2 mM [1,6-13C]glucose, 2 mM glutamine and 2 mM NAA (controls did not have NAA in the incubation medium) for 8 and 24 h. Samples of medium were collected before and after the incubation period and subsequently analysed by mass spectrometry. To stop the incubation, cells were washed twice with cold PBS and the intracellular metabolites extracted with 70 % ethanol [31]. Experiments were performed in four independently generated cultures with, at least, two replicate wells per condition.

Glucose and Lactate Analyses

Glucose and lactate levels in the cell culture medium were analysed at the Core Biochemical Assay Laboratory, Clinical Biochemistry, Addenbrooke’s Hospital using automated assays on a Siemens Dimension RxL analyser. The rate of glucose and lactate net change relative to cells over time (µmol/106 cells/24 h) was calculated by subtracting the value measured at the end of the experiment (T = 24 h) from the one measured in a sample of medium collected at the onset of the incubation, and dividing the resulting value by the amount of cells in each experiment, multiplied by the experimental volume (2 ml). The cell number considered in the calculations was the cell number at plating since oligodendrocytes do not proliferate. Analyses were performed on 9–12 samples, which derived from four independently generated cultures.

High Performance Liquid Chromatography (HPLC)

HPLC was used to quantify the total amounts of amino acids in samples of cell extracts. Samples were lyophilized and re-suspended in 0.01 M HCl and subsequently derivatised with o-Phthaldialdehyde [32] using an automated method prior to injection into the HPLC column. Amino acid concentrations were determined by comparison to a calibration curve of standard solutions of amino acids run after every 12 samples. Analyses were performed on 6–11 samples derived from three independently generated cultures. For details see Amaral et al. [32].

Gas Chromatography–Mass Spectrometry (GC–MS)

For analysis of percent enrichment with 13C in lactate, amino acids (aspartate, glutamate and glutamine) and TCA cycle intermediate (citrate) after incubation with [1,6-13C]glucose ± NAA, cell extracts and samples of medium were lyophilized and re-suspended in 0.01 M HCl. Derivatisation with MTBSTFA in the presence of 1 % t-BDMS-Cl [33] was performed as described in Amaral et al. [30]. The samples were analysed on an Agilent 6890 gas chromatograph connected to an Agilent 5975B mass spectrometer (Agilent Technologies, Palo Alto, CA). The parent ion (M) and atom percent excess for one 13C atom (M + 1) values for lactate, citrate, glutamate, glutamine and aspartate were calculated from the GC–MS data using the MassHunter software supplied by Agilent (Agilent Technologies, Palo Alto, CA) and correcting for the naturally abundant 13C by using non-enriched standards [34]. Analyses were performed on 6–12 samples of medium and 8–11 samples of extracts derived from four independently generated cultures.

Statistical Analysis

Statistical analysis was conducted using unpaired two-tailed Student t tests (confidence interval = 95 %) to compare the effect of NAA on the intracellular amounts of amino acids, on enrichment in 13C in extracellular and intracellular metabolites and on the glucose consumption and lactate release rates. To evaluate differences in the expression of ASPA mRNA levels at different stages of OPC maturation in culture, a one-way ANOVA (alpha = 0.05) followed by Tukey’s multiple comparison test was performed.

Results

To investigate how NAA is metabolised by mature oligodendrocytes, we incubated primary OPC cultures maintained in differentiation medium for 5 days with [1,6-13C]glucose in the presence or absence of NAA. By assessing differences in metabolite enrichment in 13C, this strategy enabled us to study aspartate and acetate, both metabolic products of NAA, in metabolic pathways of glucose. For details on 13C labelling Fig. 1a. [1,6-13C]Glucose is taken up by oligodendrocytes and converted to 2 molecules of [3-13C]pyruvate via glycolysis. Lactate dehydrogenase converts [3-13C]pyruvate to [3-13C]lactate or [3-13C]pyruvate, which can enter the mitochondria to be further metabolized to [2-13C]acetyl CoA and enter the TCA cycle. The subsequent TCA cycle metabolites and amino acids derived from oxaloacetate and α-ketoglutarate, aspartate and glutamate, respectively, will be labelled with one 13C atom, resulting in mass M + 1. When a labelled acetyl CoA molecule condenses with a labelled oxaloacetate molecule the subsequent metabolites will contain two 13C atoms which will result in mass M + 2. Apart from its role in the TCA cycle, citrate is used to funnel acetyl CoA units into lipid synthesis. Figure 1b provides an overview of the metabolic pathways assessed in the present study. We postulated that oligodendrocytes possess distinct mitochondria or TCA cycles that are specialised for lipid synthesis and others that prioritise energy and amino acid production (Fig. 1b). When NAA enters the cell it is hydrolyzed into acetyl CoA and aspartate by ASPA. Whereas acetate is used for lipid synthesis (Fig. 1b), aspartate can then participate in the malate–aspartate shuttle (for details see [32]) or/and enter the TCA cycle after conversion to oxaloacetate.

Fig. 1
figure1

a 13C glucose labelling patterns in oligodendrocytes. [1,6-13C]Glucose is taken up by oligodendrocytes and converted to 2 molecules of [3-13C]pyruvate via glycolysis. Lactate dehydrogenase converts [3-13C]pyruvate to [3-13C]lactate (LAC) and alanine aminotransferase to alanine (ALA). Alternatively, [3-13C]pyruvate can enter the mitochondria to be metabolized to [2-13C]acetyl CoA which can enter the TCA cycle. The subsequent TCA cycle metabolites such as: citrate, malate (MAL) and oxaloacetate (OAA) and amino acids such as aspartate (ASP), glutamate (GLU) and glutamine (GLN), will be labelled with one 13C atom, resulting in an increase in the parent ion M mass to M + 1, which can be detected by GC–MS. b Schematic presentation of glucose metabolism in oligodendrocytes in the presence of NAA. We propose that at least two distinct TCA cycles exist: (1) one TCA cycle (depicted on the left) in which most of the energy is produced and in which only a fraction of citrate leaves the cycle and contributes to lipid synthesis. This “energy producing” TCA cycle is halted by the presence of NAA, which results in increased labelling of e.g. glutamate and glutamine and, to a lesser degree, citrate. The aspartate derived from NAA does not enter into this (1) TCA cycle during the initial phase (up to 8 h), since this would reduce labelling of glutamate and glutamine. In contrast, we found that labelling of glutamate and glutamine increased but labelling of aspartate did not increase. These findings indicate that the aspartate derived from NAA enters the malate–aspartate shuttle and dilutes aspartate labelling. (2) A second TCA cycle is implied by the increased 13C labelling of citrate in the medium, which occurred much more extensively than labelling of other metabolites after 24 h incubation with [1,6-13C]glucose. The citrate producing TCA cycle for lipid synthesis was not affected by the presence of NAA as the unchanged labelling of the major pool of citrate suggests

In order to verify that the oligodendrocyte cultures used in the present study expressed the enzyme responsible for NAA hydrolysis, we quantified the ASPA mRNA levels. This demonstrated an increase in ASPA mRNA expression from day 1 to day 5 of differentiation (Fig. 2). All subsequent experiments were performed on 5-day-old cultures.

Fig. 2
figure2

Aspartoacylase (ASPA) mRNA levels during OPC differentiation in vitro. Cells were cultured either for 1 day in proliferation medium, or for 1 or 5 days in differentiation medium. Values are represented as ASPA/beta actin ratios. Measurements were made on 8–12 samples obtained from three independently generated cultures (for details see “Methods” section)

Cells were incubated in medium containing [1,6-13C]glucose for 8 and 24 h in the presence and absence of NAA. The presence of NAA increased glucose consumption and lactate production (Fig. 3a, b). The theoretical maximum enrichment of lactate from [1,6-13C]glucose is 100 %. However, after 8 h only approximately 50 % of lactate was 13C labelled, reaching approximately 65 % after 24 h (Fig. 3c, d). The addition of NAA did not affect 13C enrichment in lactate (Fig. 3c, d). Similarly, 13C-enriched citrate was released to the medium, but its enrichment was not affected by the addition of NAA (Fig. 3e, f).

Fig. 3
figure3

Effect of NAA on glucose consumption, lactate release, and enrichment with 13C of extracellular metabolites in mature oligodendrocyte cultures. Oligodendrocytes were incubated in medium containing [1,6-13C]glucose, 2 mM glutamine and 2 mM N-acetyl aspartate (NAA). Controls did not have NAA in the incubation medium. Samples of medium were collected and subsequently analysed by gas chromatography–mass spectrometry. % 13C enrichment above natural abundance is given for lactate (LAC) and citrate (CIT) after 8 or 24 h of incubation. Experiments were performed on 6–12 samples, which derived from four independently generated cultures. Glucose consumption and lactate release were measured in 9–12 samples obtained from four independently generated cultures after 24 h of incubation (for details see “Methods” section)

The intracellular amounts of aspartate, glutamine and glutamate after 8 and 24 h incubation are provided in Fig. 4. Aspartate was increased in the presence of NAA whereas glutamine and glutamate were not (Fig. 4).

Fig. 4
figure4

Effect of NAA on the intracellular levels of amino acids in mature oligodendrocyte cultures. HPLC was used to quantify the amounts of aspartate (ASP), glutamate (GLU) and glutamine (GLN) in samples of cell extracts of oligodendrocytes cultured for 5 days in differentiation medium and incubated for 24 h in the presence or absence of 2 mM N-acetyl aspartate (NAA). Analyses were performed on 6–11 samples, which derived from three independently generated cultures. Amounts are given in nmol/106 cells (for details see “Methods” section)

Analysis of 13C incorporation from [1,6-13C]glucose into intracellular metabolites was conducted after 8 and 24 h in the absence or presence of NAA (Fig. 5). No differences were detected in 13C enrichment in any metabolite analysed after 24 h (Fig. 5) and aspartate and citrate were also not affected at 8 h (Fig. 5). All other metabolites displayed an increase in 13C enrichment after 8 h incubation with [1,6-13C]glucose and NAA as compared to cultures that did not receive NAA (Fig. 5).

Fig. 5
figure5

Effect of NAA on [1,6-13C]glucose-derived enrichment with 13C in intracellular metabolites of mature oligodendrocytes. Oligodendrocytes differentiated for 5 days were incubated in medium containing [1,6-13C]glucose, 2 mM glutamine and 2 mM N-acetyl aspartate (NAA). Controls did not have NAA in the incubation medium. Cell extracts were collected and subsequently analysed by gas chromatography–mass spectrometry. % 13C enrichment above natural abundance is given for aspartate (ASP), glutamate (GLU), glutamine (GLN), citrate (CIT) and malate (MAL) after 8 or 24 h of incubation. Experiments were performed on 8–11 samples, which derived from four independently generated cultures

Discussion

Glucose metabolism in neurons and astrocytes has been studied extensively and much is known about the interaction between these two cell types in the brain [35]. However, until very recently oligodendrocytes have not been studied in this context [30, 3538].

It was evident that the metabolism of N-acetylaspartylglutamate (NAAG) has a tri-cellular compartmentation, however, this was not clear in regard to NAA [3840]. Both NAA and NAAG are synthesized by neurons in the brain. Neurons release both NAA and NAAG to the extracellular space upon stimulation, where astrocytes, the target cells for NAAG, hydrolyse it, releasing NAA, which is subsequently catabolised in oligodendrocytes. It has been proposed that oligodendrocytes release the aspartate obtained from NAA for recycling to neurons [39]. This hypothesis assumes that aspartate produced from NAA is passed from oligodendrocytes back to neurons to be reutilized for re-synthesis of NAA and NAAG. This exchange of metabolites would be in analogy with the well-established glutamine–glutamate cycle between glia and neurons [35]. Existing evidence suggests that the aspartate derived by catabolism of NAA in the brain may not be used for the re-synthesis of NAA [22]. Studies demonstrated rapid transamination of doubly labelled (3H and 15N) aspartate from [3H2 15N]NAA, in which the amino group was transferred to glutamate, in the absence of 15N-NAA production. However, Miller et al. [22] could not follow the carbon skeleton of NAA since the nitrogen was labelled, not the carbon atoms. Our results, using a 13carbon labelled precursor ([1,6-13C]glucose), demonstrate that aspartate is not released to the medium by oligodendrocytes in culture, disproving the hypothesis of aspartate recycling between neurons and oligodendrocytes [39]. What we observed was an increase in aspartate content in the oligodendrocytes but the % 13C labelling remained unchanged. It is likely that aspartate is converted to oxaloacetate and further to malate in the cytosol, steps in the energy producing malate-aspartate shuttle for transporting reducing equivalents from NADH from glycolysis into the mitochondria [35]. The malate–aspartate shuttle is tightly coupled to glycolysis in that reducing equivalents are transported into the mitochondria for oxidative phosphorylation. Therefore, ATP is produced and NAD, which is necessary for glycolysis, is re-generated in the cytosol. The increased glucose consumption and lactate production observed in the presence of NAA in the present study is indicative of an increased aerobic glycolysis (Warburg effect) also seen in cancer cells. In this context it is noteworthy that exogenously applied NAA has been found to promote growth in several cancer cell lines [41]. A potential explanation consists in a more efficient NAD regeneration due to an increase in the capacity of the malate–aspartate shuttle due to aspartate generation from NAA. Entry of aspartate from NAA into the TCA cycle appears to be slow due to the expected dilution of 13C labelling of compounds derived from this cycle, and consequently % enrichment, was not decreased after 8 h of incubation. Aspartate labelling would be expected to increase together with glutamate and glutamine at 8 h, but this was not observed. Indeed, % 13C labelling of aspartate was unchanged, indicating that aspartate synthesis from NAA took place in a restricted compartment, which we hypothesise to be the malate-aspartate shuttle. However, after 24 h incubation with NAA the labelling of glutamate and glutamine was not found to be different from the one observed in cells cultured in the absence of NAA, indicating that aspartate was participating in the TCA cycle. This observation may also be related to the slow turnover of NAA detected in vivo. The reported turnover rates of NAA C6 and C3 in rat brain range between 0.7 ± 0.1 and 0.6 ± 0.1 µmol/(g h) with the time constants 14 ± 2 and 13 ± 2 h, respectively, with an estimated pool size of 8 µmol/g [42]. These results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. This is in agreement with a slow incorporation of aspartate into the oligodendrocyte TCA cycle in order to maintain homeostasis between synthesis and degradation. A potential explanation for the increase in % 13C labelling in glutamate and glutamine is a decrease in the amount of 13C labelled citrate leaving the TCA cycle as the acetyl CoA content in the cytosol is increased due to the catabolism of NAA. The reduced leakage of labelled substance is expected to cause an overall increase in labelling.

Percent 13C labelling of glutamate and glutamine were increased in the presence of NAA. This may be caused by a decrease in 13C labelled citrate efflux from the TCA cycle to the cytosol. Citrate is the most common precursor for acetyl CoA production in the cytosol, and also acts as a substrate for lipid synthesis. High levels of NAA in the cytosol of oligodendrocytes incubated with NAA could support acetyl CoA production from acetate generated by hydrolysis of NAA. This is a possibility since ACSS2, the gene for the nuclear-cytosolic form of acyl-CoA synthetase short-chain family member, is expressed in oligodendrocytes to a high extent as reported in transcriptomics studies of the mouse brain [43, 44]. This would decrease the need for citrate catabolism for fatty acid synthesis. In contrast to the increased enrichment of glutamate and glutamine, no differences in the labelling of citrate inside the cells and in the medium were detected. This indicates that the majority of citrate was produced in a separate compartment. The notion of compartmentation is also supported by the much larger % 13C enrichment in citrate in the medium compared to that of intracellular metabolites.

It is unlikely that the acetate moiety of NAA released by ASPA entered the mitochondria and was converted to acetyl CoA since this would have resulted in decreased 13C labelling of the metabolites, and this was not observed. This is in agreement with a report that oligodendrocytes do not have the mitochondrial enzyme AceCS2 [4]. However, in the mitochondria of oligodendrocytes in culture, we have shown that acetate from the medium is indeed converted to acetyl CoA [30]. As previously mentioned, the most likely fate of the resulting acetyl CoA is its entry into lipid synthesis [6].

Tri-Cellular Compartmentation of NAA Metabolism?

Tri-cellular compartmentation is necessary for catabolism of NAAG but not for NAA. For the latter, interactions between neurons and oligodendrocytes appear to be sufficient. However, it is important to note that aspartate production in neurons is only possible with the help of glutamine from other cells. Neurons do not express pyruvate carboxylase, the anaplerotic enzyme in the brain [15] and, thus, cannot produce “de novo” aspartate. In order to export NAA, neurons have to import glutamine from external sources [45]. Aspartate is synthesized in the TCA cycle as a result of multiple conversion steps involving glutamate and oxaloacetate. So far only astrocytes are known to synthesise glutamine “de novo” and to release it to the medium [35]. We have recently demonstrated that oligodendrocytes are capable of anaplerosis [32] and therefore meet one of the requirements for supporting neurons. However, it remains unknown whether they are capable of exporting glutamine and, so, support transfer of NAA. Our present findings indicate a tri-cellular compartmentation of NAA metabolism, which starts with astrocytes releasing glutamine, which is taken by neurons, and converted into NAA. Subsequently, NAA released into the extracellular space is taken up and metabolized by oligodendrocytes. Figure 6 summarizes the findings described in the present work. A similar multicellular metabolism of NAA has been proposed depicting some of the metabolite trafficking between various cell types in the brain associated with NAA synthesis and breakdown [4, 46]. The authors stressed that the slow rate of NAA synthesis in neurons and breakdown and utilization in oligodendrocytes is suggestive of non-energy derivation roles in lipid synthesis and protein acetylation reactions under normal conditions, with a shift to much more rapid metabolism in response to injury or disease. The rate of synthesis and breakdown would also be much greater during postnatal myelination than in the normal adult brain and the oligodendrocyte cultures used in the present study might reflect this.

Fig. 6
figure6

Schematic overview of the metabolic interactions involving N-acetyl aspartate (NAA) and N-acetyl aspartate glutamate (NAAG) between neurons, oligodendrocytes and astrocytes. Glucose from the blood is taken up by neurons, astrocytes and oligodendrocytes and can be metabolized via glycolysis giving rise to pyruvate formation. Pyruvate can be carboxylated in astrocytes via pyruvate carboxylase and glutamine (GLN) can be formed eventually (for details see [26]). In brain, the predominant cell type for NAA synthesis is neurons that synthesize it from aspartate (ASP) and Acetyl-CoA. NAAG is also synthesized in neurons and converted to NAA in astrocytes. NAA is taken up and metabolized by oligodendrocytes. The acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of Acetyl-CoA it is used for lipid synthesis. Aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. We propose that aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate–aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate

Conclusion

In addition to the results from a previous study [47], this study provides new evidence that cultured oligodendrocytes hydrolyse NAA. The resulting aspartate and the acetate moieties remain within the cells. As the acetate is not incorporated into TCA cycle intermediates, it is likely to be used in lipid synthesis. The aspartate entity is likely to affect the malate–aspartate shuttle, glycolysis and energy production.

References

  1. 1.

    Attwell D, Laughlin SB (2001) An energy budget for signaling in the grey matter of the brain. J Cerebr Blood F Met 21(10):1133–1145

    CAS  Article  Google Scholar 

  2. 2.

    Zaroff S, Leone P, Markov V, Francis JS (2015) Transcriptional regulation of N-acetylaspartate metabolism in the 5xFAD model of Alzheimer’s disease: evidence for neuron-glia communication during energetic crisis. Mol Cell Neurosci 65:143–152

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Liu W, Yu H, Jiang B, Pan B, Yu S, Li H, Zheng L (2015) The predictive value of baseline NAA/Cr for treatment response of first-episode schizophrenia: a (1)H MRS study. Neurosci Lett 600:199–205

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Moffett JR, Arun P, Ariyannur PS, Namboodiri AM (2013) N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation. Front Neuroenergetics 5:11

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Sherry EB, Lee P, Choi IY (2015) In vivo NMR studies of the brain with hereditary or acquired metabolic disorders. Neurochem Res 40(12):2647–2685

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    D’Adamo AF Jr, Gidez LI, Yatsu FM (1968) Acetyl transport mechanisms. Involvement of N-acetyl aspartic acid in de novo fatty acid biosynthesis in the developing rat brain. Exp Brain Res 5(4):267–273

    PubMed  Google Scholar 

  7. 7.

    Patel TB, Clark JB (1979) Synthesis of N-acetyl-L-aspartate by rat brain mitochondria and its involvement in mitochondrial/cytosolic carbon transport. Biochem J 184(3):539–546

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Tkáč I, Rao R, Georgieff MK, Gruetter R (2003) Developmental and regional changes in the neurochemical profile of the rat brain determined by in vivo 1 H NMR spectroscopy. Magn Reson Med 50(1):24–32

    Article  PubMed  Google Scholar 

  9. 9.

    Tallan HH (1957) Studies on the distribution of N-acetyl-L-aspartic acid in brain. J Biol Chem 224(1):41–45

    CAS  PubMed  Google Scholar 

  10. 10.

    Simmons ML, Frondoza CG, Coyle JT (1991) Immunocytochemical localization of N-acetyl-aspartate with monoclonal antibodies. Neuroscience 45(1):37–45

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Moffett JR, Namboodiri MA, Cangro CB, Neale JH (1991) Immunohistochemical localization of N-acetylaspartate in rat brain. Neuroreport 2(3):131–134

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Nadler JV, Cooper JR (1972) N-acetyl-L-aspartic acid content of human neural tumours and bovine peripheral nervous tissues. J Neurochem 19(2):313–319

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Bates TE, Strangward M, Keelan J, Davey GP, Munro PM, Clark JB (1996) Inhibition of N-acetylaspartate production: implications for 1 H MRS studies in vivo. Neuroreport 7(8):1397–1400

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Signoretti S, Marmarou A, Tavazzi B, Lazzarino G, Beaumont A, Vagnozzi R (2001) N-Acetylaspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury. J Neurotrauma 18(10):977–991

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41(5):1484–1487

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Norenberg MD (1979) Distribution of glutamine synthetase in the rat central nervous system. J Histochem Cytochem 27(3):756–762

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Chakraborty G, Mekala P, Yahya D, Wu G, Ledeen RW (2001) Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J Neurochem 78(4):736–745

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Madhavarao CN, Arun P, Moffett JR, Szucs S, Surendran S, Matalon R, Garbern J, Hristova D, Johnson A, Jiang W, Namboodiri MA (2005) Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan’s disease. Proc Natl Acad Sci USA 102(14):5221–5226

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Madhavarao CN, Moffett JR, Moore RA, Viola RE, Namboodiri MA, Jacobowitz DM (2004) Immunohistochemical localization of aspartoacylase in the rat central nervous system. J Comp Neurol 472(3):318–329

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Baslow MH (1999) Molecular water pumps and the aetiology of Canavan disease: a case of the Sorcerer’s apprentice. J Inherit Metab Dis 22(2):99–101

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Baslow MH (2003) Brain N-acetylaspartate as a molecular water pump and its role in the etiology of Canavan disease: a mechanistic explanation. J Mol Neurosci 21(3):185–190

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Miller SL, Daikhin Y, Yudkoff M (1996) Metabolism of N-acetyl-L-aspartate in rat brain. Neurochem Res 21(5):615–618

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Madhavarao CN, Chinopoulos C, Chandrasekaran K, Namboodiri MA (2003) Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain. J Neurochem 86(4):824–835

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Ariyannur PS, Moffett JR, Manickam P, Pattabiraman N, Arun P, Nitta A, Nabeshima T, Madhavarao CN, Namboodiri AM (2010) Methamphetamine-induced neuronal protein NAT8L is the NAA biosynthetic enzyme: implications for specialized acetyl coenzyme A metabolism in the CNS. Brain Res 1335:1–13

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Moffett JR, Ariyannur PS, Arun P, Namboodiri MA (2014) N-Acetylaspartate and N-acetylaspartylglutamate in central nervous system health and disease. In: Stagg C, Rothman D (eds) Magnetic resonance spectroscopy: tools for neuroscience research and emerging clinical applications. Academic Press, San Diego, CA, pp 71–99

    Chapter  Google Scholar 

  26. 26.

    Wynn ML, Yates JA, Evans CR, Van Wassenhove L, Wu ZF, Bridges S, Bao L, Fournier C, Ashrafzadeh S, Merrins MJ, Satin LS, Schnell S, Burant CF, Merajver SD (2016) RhoC is a potent regulator of glutamine metabolism and N-acetylaspartate production in inflammatory breast cancer cells. J Biol Chem. 291:13715–13729

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Zand B, Previs RA, Zacharias NM, Rupaimoole R, Mitamura T, Nagaraja AS, Guindani M, Dalton HJ, Yang L, Baddour J, Achreja A, Hu W, Pecot CV, Ivan C, Wu SY, McCullough CR, Gharpure KM, Shoshan E, Pradeep S, Mangala LS, Rodriguez-Aguayo C, Wang Y, Nick AM, Davies MA, Armaiz-Pena G, Liu J, Lutgendorf SK, Baggerly KA, Eli MB, Lopez-Berestein G, Nagrath D, Bhattacharya PK, Sood AK (2016) Role of increased N-acetylaspartate levels in cancer. J Natl Cancer Inst 108(6):djv426

  28. 28.

    Mazzoccoli C, Ruggieri V, Tataranni T, Agriesti F, Laurenzana I, Fratello A, Capitanio N, Piccoli C (2016) N-acetylaspartate (NAA) induces neuronal differentiation of SH-SY5Y neuroblastoma cell line and sensitizes it to chemotherapeutic agents. Oncotarget 7(18):26235–26246

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Baer AS, Syed YA, Kang SU, Mitteregger D, Vig R, Ffrench-Constant C, Franklin RJ, Altmann F, Lubec G, Kotter MR (2009) Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling. Brain 132 (Pt 2):465–481

    PubMed  PubMed Central  Google Scholar 

  30. 30.

    Amaral AI, Hadera MG, Tavares JM, Kotter MR, Sonnewald U (2016) Characterization of glucose-related metabolic pathways in differentiated rat oligodendrocyte lineage cells. Glia 64(1):21–34

    Article  PubMed  Google Scholar 

  31. 31.

    Amaral AI, Alves PM, Teixeira AP (2014) Metabolic fux analysis tools to investigate brain metabolism in vitro. In: Hirrlinger HW, Waagepetersen HS (eds) Brain energy metabolism, vol 90. neuromethods. Humana Press, New York, pp 107–144

    Google Scholar 

  32. 32.

    Geddes JW, Wood JD (1984) Changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate and gamma-aminobutyric acid. J Neurochem 42(1):16–24

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Mawhinney TP, Robinett RS, Atalay A, Madson MA (1986) Analysis of amino acids as their tert-butyldimethylsilyl derivatives by gas–liquid chromatography and mass spectrometry. J Chromatogr 358(1):231–242

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Biemann K (1962) The mass spectra of isotopically labeled molecules. In: Mass spectrometry; organic chemical applications. McGraw-Hill series in advanced chemistry. McGraw-Hill, New York, pp 223–227

    Google Scholar 

  35. 35.

    McKenna MC, Dienel GA, Sonnewald U, Waagepetersen HS, Schousboe A (2012) Energy metabolism of the brain. In: Siegel GJ, Albers RW, Brady ST, Price DL (eds) Basic neurochemistry, vol 8. Elsevier, Amsterdam, pp 200–299

    Chapter  Google Scholar 

  36. 36.

    Amaral AI, Meisingset TW, Kotter MR, Sonnewald U (2013) Metabolic aspects of neuron-oligodendrocyte-astrocyte interactions. Front endocrinol 4:54

    CAS  Article  Google Scholar 

  37. 37.

    Funfschilling U, Supplie LM, Mahad D, Boretius S, Saab AS, Edgar J, Brinkmann BG, Kassmann CM, Tzvetanova ID, Mobius W, Diaz F, Meijer D, Suter U, Hamprecht B, Sereda MW, Moraes CT, Frahm J, Goebbels S, Nave KA (2012) Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485(7399):517–521

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Rinholm JE, Hamilton NB, Kessaris N, Richardson WD, Bergersen LH, Attwell D (2011) Regulation of oligodendrocyte development and myelination by glucose and lactate. J Neurosci 31(2):538–548

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Baslow MH (2000) Functions of N-acetyl-L-aspartate and N-acetyl-L-aspartylglutamate in the vertebrate brain: role in glial cell-specific signaling. J Neurochem 75(2):453–459

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Moffett JR, Ross B, Arun P, Madhavarao CN, Namboodiri AM (2007) N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology. Prog Neurobiol 81(2):89–131

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Long PM, Moffett JR, Namboodiri AM, Viapiano MS, Lawler SE, Jaworski DM (2013) N-Acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) promote growth and inhibit differentiation of glioma stem-like cells. J Biol Chem 288(36):26188–26200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Choi IY, Gruetter R (2004) Dynamic or inert metabolism? Turnover of N-acetyl aspartate and glutathione from D-[1-13C]glucose in the rat brain in vivo. J Neurochem 91(4):778–787

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28(1):264–278

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O’Keeffe S, Phatnani HP, Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, Daneman R, Maniatis T, Barres BA, Wu JQ (2014) An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci 34(36):11929–11947

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Norenberg MD, Martinez-Hernandez A (1979) Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161(2):303–310

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Moffett JR, Ross B, Arun P, Madhavarao CN, Namboodiri AM (2007) N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology. Prog Neurobiol 81(2):89–131

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Kumar S, Sowmyalakshmi R, Daniels SL, Chang R, Surendran S, Matalon R, de Vellis J (2006) Does ASPA gene mutation in Canavan disease alter oligodendrocyte development? A tissue culture study of ASPA KO mice brain. Adv Exp Med Biol 576:175–182

    CAS  Article  PubMed  Google Scholar 

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Acknowledgments

This work was supported by grants from the UK Multiple Sclerosis Society and from Qatar Foundation. The work was further supported by core funding from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. The authors acknowledge the excellent technical support in GC-MS and HPLC analysis from Lars Evje (NTNU, Norway). The authors declare no conflict of interest related to this work.

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Correspondence to Mark Kotter or Ursula Sonnewald.

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I. Amaral, A., Hadera, M.G., Kotter, M. et al. Oligodendrocytes Do Not Export NAA-Derived Aspartate In Vitro. Neurochem Res 42, 827–837 (2017). https://doi.org/10.1007/s11064-016-1985-y

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Keywords

  • Brain cells
  • Glia
  • Energy metabolism
  • Glucose
  • Aspartate
  • Acetate