Aspartate in the Brain: A Review

Abstract

L-Aspartate (aspartic acid; C₄H₇NO₄; 2-aminobutanedoic acid) is a non-essential α-amino acid found ubiquitously throughout the body, including in the brain. Aspartate is one of the protein-forming amino acids and the formation of tRNA-aspartate complex is catalysed by aspartyl tRNA synthetase. Free aspartate, which is the main subject of this review, plays key roles in metabolism, as an amino donor and acceptor. It contributes to the synthesis of protein, arginine and nitric oxide, asparagine, N-acetylaspartate and N-methyl-D-aspartate. Its major metabolic role in the brain is recycling reducing equivalents (protons) between the cytoplasm and mitochondrial matrix as part of the malate-aspartate shuttle. L-Aspartate's actions on synaptic receptors, as well as its possible presence in nerve terminals and synaptic vesicles, are, in principle, consistent with a role as an excitatory neurotransmitter. The evidence is far from conclusive and at times controversial. The role of D-aspartate in brain function is even less certain but, it appears that, rather than being a minor neurotransmitter, D-aspartate is more likely to be involved in fine regulation of endocrine and homeostatic processes. Much research remains to be done in this area. The diversity of its functions and chemistry make aspartate a complex molecule to investigate and measure in vivo. Perturbations of aspartate metabolism have been described in a range of neurological deficits, particularly those of white matter. Here, we examine what is known about the various roles of aspartate in brain, its metabolism, transport and compartmentation, its role as a neurotransmitter or a more general signalling molecule, and what is currently known about its role(s) in disease processes.

Keywords: d-aspartate; Energy metabolism; Malate aspartate shuttle; Neurotransmitter.

Fig. 1

Scheme showing the malate aspartate shuttle. A proton (followed in red) produced by oxidation of glucose in the glycolysis pathway is transferred to the cofactor NAD⁺ forming NADH. This proton is transferred to malate via cytosolic malate dehydrogenase (MDH1; EC1.1.1.37). The malate is then transported into the mitochondrion in exchange for 2-oxoglutarate via the oxoglutarate carrier protein (OGCP) where it is converted to oxaloacetate by mitochondrial malate dehydrogenase (MDH2; EC1.1.1.37) and the proton transferred to NAD⁺ once more. By this mechanism, the cytosolic proton produced by oxidation of glucose can be transferred into the mitochondrion for subsequent introduction into the mitochondrial electron transport chain (ETC). The malate aspartate shuttle is then completed by the transamination, by mitochondrial aspartate transaminase (GOT2; EC 2.6.1.1), of the oxaloacetate to aspartate which is then electrogenically exchanged for glutamate via the aspartate-glutamate carrier (AGC). In the cytosol, the aspartate is converted back to oxaloacetate by cytosolic aspartate aminotransferase (GOT1; EC 2.6.1.1) forming oxaloacetate. This can then be converted to malate, picking up another proton from glycolysis in the process and starting the shuttle all over again. AGC contains calcium-binding domains and is stimulated by mitochondrial Ca2⁺, with activation enhancing the transport of aspartate and glutamate. Ca2⁺ activation of the 2-oxoglutarate dehydrogenase complex (OGDC; EC 1.2.4.2) drives the reaction towards glutamate, lowering the local concentration of 2-oxoglutarate which supports the transamination of oxaloacetate to l-Aspartate. Serine biosynthesis is indirectly influenced by the MAS as phosphoglycerate dehydrogenase (PHGDH; EC 1.1.1.95) activity is dependent on the NAD +/NADH ratio. When MAS activity is low, alternative NAD⁺ regeneration pathways, including lactate dehydrogenase (LDH; EC 1.1.1.27) and cytosolic glycerol-3-phosphate dehydrogenase (cGPD; EC 1.1.1.8), may partially compensate to support PHGDH function and sustain serine biosynthesis. Aspartate (red circle), glutamate (blue circle), 2-oxoglutarate (purple circle), and malate (orange circle)

Fig. 2

Scheme showing conversion of [2-¹³C,¹⁵N]alanine to [2-¹³C,¹⁵N]aspartate. The ¹³C is transferred by alanine aminotransferase from [2-¹³C,¹⁵N]alanine to [2-¹³C]pyruvate, which is converted to [2-¹³C]oxaloacetate by the glial enzyme pyruvate carboxylase. This carbon backbone is re-aminated by aspartate aminotransferase using [¹⁵N]glutamate to form [2-¹³C,¹⁵N]aspartate. Meanwhile, the ¹⁵N from [2-¹³C,¹⁵N]alanine is transferred to [¹⁵N]glutamate by alanine aminotransferase, thence to [2-¹³C]oxaloacetate by aspartate aminotransferase. The high level of ¹⁵N labelling in aspartate C2 indicated that these reactions were tightly coupled, and the need for pyruvate carboxylase showed that this tight coupling took place in glial cells. Scheme adapted from [65]

Fig. 3

Results from MRI investigations in a patient with AGC1 deficiency before and 6 months after treatment [110]. Before treatment, T₂ axial imaging A, B and T₂ coronal imaging D, E showed lack of myelination and progressively reduced supratentorial cerebral volume. At 6 years and 7 months of age, after 6 months of treatment with the ketogenic diet, T₂ axial imaging (C) and T₂ coronal imaging F show that the previously high signal corresponding to white matter is lower, and that the ventricles and subarachnoid spaces are less prominent, indicating reversal of the volume loss. Tx, duration of treatment with the ketogenic diet. Reproduced from [110] with permission

Fig. 4

Time courses of glutamate (Glu) and aspartate (Asp) concentrations during a visual stimulation paradigm averaged across subjects (N = 12). Error bars indicate s.e.m., while shaded areas indicate the stimulation (STIM) periods. P-values correspond to statistical evaluation of differences between STIM and subsequent REST (resting) periods (paired t-tests, mean values from the second half of each period). Figure adapted from [13], with thanks to Silvia Mangia and Petr Bednařík (CMRR, U Minnesota)

Fig. 5

Metabolism of aspartate. Aspartate plays a central role in brain metabolism. This figure highlights several key metabolic pathways of aspartate metabolism that correspond to Sects. “Degradation of Aspartate” and “Metabolism of Aspartate” of the review. 5.3. Malate-Aspartate Shuttle: Components of the shuttle are covered in Fig. 1. Key enzymes involved include alanine aminotransferase (ALT; EC 2.6.1.2), pyruvate carboxylase (PC; EC 6.41.1), aspartate aminotransferase or glutamate-oxalate transaminase 1 and 2 (GOT1/2; EC 2.6.1.1). malate dehydrogenase 1 and 2 (MDH1/2; EC 1.1.1.37), d-aspartate oxidase (DDO EC 1.4.3.1), aspartate-glutamate carrier (AGC), 2-oxoglutarate-malate carrier (OGC), fumarase (EC 4.2.1.2). 4.0 Degradation of aspartate: Malic enzyme (ME1; EC 1.1.1.40) is both anaplerotic and cataplerotic, contributing to neurotransmitter formation via pyruvate carboxylase (PC; EC 6.4.1.1). ME2 (EC 1.1.1.38) and ME3 (EC 1.1.1.39) are primarily involved in mitochondrial pyruvate metabolism and Krebs cycle intermediate regulation. 5.5 Aspartate and N-acetylaspartate: NAA acts as an aspartate reservoir and is synthesised via acetylation of aspartate by N-Acetyltransferase-8 Like Protein (NAT8L; EC 2.3.1.17). In oligodendrocytes, NAA is hydrolysed by aspartoacylase (ASPA; EC 3.5.1.15) into aspartate and acetate. Aspartate may reenter the malate-aspartate shuttle (MAS), whereas acetyl-CoA synthesised from acetate by (ACSS2; EC 6.2.1.1) contributes to fatty acid (FA) synthesis via acetyl-CoA carboxylase 1 (ACC1; EC:6.4.1.2). NAA and glutamate may be metabolised into N-acetylaspartylglutamate (NAAG) by NAAG synthetase (NAAGS; EC 6.3.2.41). 5.6 Interaction with L-arginine metabolism: Aspartate combines with citrulline via argininosuccinate synthetase (ASS; EC 6.3.4.5) to form argininosuccinate, which is cleaved by argininosuccinate lyase (ASL; EC 4.3.2.1) into fumarate that can be recycled in the malate aspartate shuttle, and arginine. Arginine is a metabolised by arginine:glycine aminotransferase (AGAT; EC 2.1.4.1) which converts glycine and arginine into ornithine and guanidinoacetate, with the latter being a precursor for creatine. Arginine can also be hydrolysed to ornithine by Arginase 1 and 2 (ARG1/2; EC 3.5.3.10) yielding urea. Arginine can be metabolised by nitric oxide synthase (NOS; EC 1.14.13.39) into citrulline and nitric oxide (NO). 5.7 Polyamine metabolism: Ornithine is converted into putrescene via Ornithine Decarboxylase (ODC; EC 4.1.1.17). Putrescine is oxidized by diamine oxidase (DAO; EC 1.4.3.22) to form γ-aminobutyraldehyde (γ-ABAL), which is converted into γ-aminobutyric acid (GABA) by aminobutyraldehyde dehydrogenase (ABALDH; EC 1.2.1.19). Alternatively, ornithine and 2-oxoglutarate can be metabolised into glutamate-5-semialdehyde (GSSA) and glutamate by ornithine δ-aminotransferase (OAT, EC 2.6. 1.13). GSSA can be further metabolised by glutamate-5-semialdehyde dehydrogenase (P5CDH; EC 1.2.1.88) to glutamate. Glutamate can be converted into GABA by Glutamic acid decarboxylase (GAD; EC 4.1.1.15). 5.8 Synthesis of l-Asparagine: Asparagine synthetase (ASNS; EC 6.3.5.4) hydrolyses glutamine, producing glutamate and releasing ammonia, ATP is then used to activate aspartate forming a β-aspartyl-AMP intermediate, which subsequently reacts with ammonia to generate asparagine and AMP. In brain, the source of this ammonia remains unclear. As phosphate activated glutaminase (PAG; EC 3.5.1.2) is localised within the mitochondria, any ammonia produced by this enzyme would need to be transported across the mitochondrial membrane. 5.9 Synthesis of purine nucleotides: Adenylosuccinate synthetase (ADSS; EC 6.3.4.4) binds inosine monophosphate (IMP) and GTP is hydrolysed to GDP and Pi to condense aspartate with IMP to form adenylosuccinate (S-AMP). Adenylosuccinate lyase (ADSL; EC 4.3.2.2) then catalyses’ the cleavage of adenylosuccinate releasing AMP and fumarate. The fumarate can be recycled through the MAS and Krebs cycle, linking purine biosynthesis to energy metabolism. 5.10 Aspartyl-tRNA synthesis and defects: Aspartyl-tRNA synthetases (DARS; EC 4.2.1.2) ensures proper aminoacylation of tRNA during protein synthesis (Asp moiety, red circle, other amino acids, grey circles). Aspartate racemase (AspR; EC 5.1.1.12) catalyses the conversion of l-Aspartate and d-aspartate. d-aspartate may act as an agonist at NMDA receptors, though its precise role in neurotransmission remains under investigation. d-Aspartate can be converted into N-methyl-d-aspartate (NMDA) by the enzyme d-aspartate Methyltransferase (DDNMT), which uses S-adenosyl-L-methionine (SAM) as a methyl donor. This conversion allows NMDA (orange circle) to act directly on NMDA receptors (NMDAR)

Fig. 6

MRI of 4 year old boy with compound heterozygous pathogenic variants in ASNS (NM_183356.3:c. [866G > C]; [1010C > T]). A sagittal and B, C axial T2-weighted images showing small cranial vault with craniofacial dysmorphism in keeping with microcephaly, enlargement of the ventricles and the supratentorial and infratentorial extra-axial spaces in keeping with global atrophy in both the supratentorial and infratentorial compartments. Also noted are a diffusely thin corpus callosum, slender brainstem and a generalized, marked reduction of white matter volume. Head circumference was between − 1 and − 2 SD from the mean at birth but rapidly decelerated to be below − 5 SD from the mean by 20 months of age [200]

Fig. 7

¹H Spectra of brain showing aspartate resonances. Panels A and B show simulated (A) and actual (B) in vivo MEGA-PRESS (TE = 150 ms, ON frequency 3.89 ppm, OFF 10.0 ppm, editing pulse 45 ms, 192 averages, VOI 27 cm³ in right centrum semiovale, 32 channel head coil) spectra of human brain at 3 Tesla. The spectrum of edited aspartate is shown in red (A). C shows a section of a spectrum of human brain in vivo acquired at 9.4 T with a home-built multi-transmit-receive coil using a metabolite-cycled semi-LASER sequence (TE = 24 ms, VOI 8 cm³ in occipital lobe, 96 averages) [22]. mI, myoinositol; Cr, creatine; PCr, phosphocreatine; GSH, glutathione; PE phosphoethanolamine; Glx, glutamate and glutamine; Tau, taurine; tCh, total choline; tNAA, N-acetylaspartate and N-acetylaspartylglutamate; MM, macromolecules. D shows a section of a high field NMR spectrum of Guinea pig cortex extracted with methanol/chloroform [396] acquired at 800 MHz (18.8 T). The high frequency resonances from the aspartate CH₂ protons are shown inside red ellipses