Drosophila melanogaster Uncoupling Protein-4A (UCP4A) Catalyzes a Unidirectional Transport of Aspartate.

Uncoupling proteins (UCPs) form a distinct subfamily of the mitochondrial carrier family (MCF) SLC25. Four UCPs, DmUCP4A-C and DmUCP5, have been identified in Drosophila melanogaster on the basis of their sequence homology with mammalian UCP4 and UCP5. In a Parkinson's disease model, DmUCP4A showed a protective role against mitochondrial dysfunction, by increasing mitochondrial membrane potential and ATP synthesis. To date, DmUCP4A is still an orphan of a biochemical function, although its possible involvement in mitochondrial uncoupling has been ruled out. Here, we show that DmUCP4A expressed in bacteria and reconstituted in phospholipid vesicles catalyzes a unidirectional transport of aspartate, which is saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. Swelling experiments carried out in yeast mitochondria have demonstrated that the unidirectional transport of aspartate catalyzed by DmUCP4 is not proton-coupled. The biochemical function of DmUCP4A has been further confirmed in a yeast cell model, in which growth has required an efflux of aspartate from mitochondria. Notably, DmUCP4A is the first UCP4 homolog from any species to be biochemically characterized. In Drosophila melanogaster, DmUCP4A could be involved in the transport of aspartate from mitochondria to the cytosol, in which it could be used for protein and nucleotide synthesis, as well as in the biosynthesis of ß-alanine and N-acetylaspartate, which play key roles in signal transmission in the central nervous system.

Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius.

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate ß-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD+ over NADP+, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD+-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection.

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice.

Marked elevation in the brain concentration of N-acetyl-L-aspartate (NAA) is a characteristic feature of Canavan disease, a vacuolar leukodystrophy resulting from deficiency of the oligodendroglial NAA-cleaving enzyme aspartoacylase. We now demonstrate that inhibiting NAA synthesis by intracisternal administration of a locked nucleic acid antisense oligonucleotide to young-adult aspartoacylase-deficient mice reverses their pre-existing ataxia and diminishes cerebellar and thalamic vacuolation and Purkinje cell dendritic atrophy. Ann Neurol 2020;87:480-485.

Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis.

RATIONALE: Hypertrophied hearts switch from mainly using fatty acids (FAs) to an increased reliance on glucose for energy production. It has been shown that preserving FA oxidation (FAO) prevents the pathological shift of substrate preference, preserves cardiac function and energetics, and reduces cardiomyocyte hypertrophy during cardiac stresses. However, it remains elusive whether substrate metabolism regulates cardiomyocyte hypertrophy directly or via a secondary effect of improving cardiac energetics. OBJECTIVE: The goal of this study was to determine the mechanisms of how preservation of FAO prevents the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: We cultured adult rat cardiomyocytes in a medium containing glucose and mixed-chain FAs and induced pathological hypertrophy by phenylephrine. Phenylephrine-induced hypertrophy was associated with increased glucose consumption and higher intracellular aspartate levels, resulting in increased synthesis of nucleotides, RNA, and proteins. These changes could be prevented by increasing FAO via deletion of ACC2 (acetyl-CoA-carboxylase 2) in phenylephrine-stimulated cardiomyocytes and in pressure overload-induced cardiac hypertrophy in vivo. Furthermore, aspartate supplementation was sufficient to reverse the antihypertrophic effect of ACC2 deletion demonstrating a causal role of elevated aspartate level in cardiomyocyte hypertrophy. 15N and 13C stable isotope tracing revealed that glucose but not glutamine contributed to increased biosynthesis of aspartate, which supplied nitrogen for nucleotide synthesis during cardiomyocyte hypertrophy. CONCLUSIONS: Our data show that increased glucose consumption is required to support aspartate synthesis that drives the increase of biomass during cardiac hypertrophy. Preservation of FAO prevents the shift of metabolic flux into the anabolic pathway and maintains catabolic metabolism for energy production, thus preventing cardiac hypertrophy and improving myocardial energetics.

Effects of the antidepressant medication duloxetine on brain metabolites in persistent depressive disorder: A randomized, controlled trial.

BACKGROUND: To assess whether patients with Persistent Depressive Disorder (PDD) have abnormal levels of N-acetyl-aspartate (NAA) and whether those levels normalize following treatment with the antidepressant medication duloxetine. Furthermore, we conducted post hoc analyses of other important brain metabolites to understand better the cellular and physiological determinants for changes in NAA levels. METHODS: We acquired proton (1H) magnetic resonance spectroscopic imaging (MRSI) data on a 3 Tesla (3T), GE Magnetic Resonance Imaging (MRI) scanner in 41 patients (39.9±10.4 years, 22 males) with PDD at two time points: before the start and at the end of a 10-week, placebo-controlled, double-blind, randomized controlled trial (RCT) of the antidepressant medication duloxetine. Patients were randomized such that 21 patients received the active medication and 20 patients received placebo during the 10 week period of the trial. In addition, we acquire 1H MRSI data once in 29 healthy controls (37.7±11.2 years, 17 males). FINDINGS: Patients had significantly higher baseline concentrations of NAA across white matter (WM) pathways and subcortical gray matter, and in direct proportion to the severity of depressive symptoms. NAA concentrations declined in duloxetine-treated patients over the duration of the trial in the direction toward healthy values, whereas concentrations increased in placebo-treated patients, deviating even further away from healthy values. Changes in NAA concentration did not mediate medication effects on reducing symptom severity, however; instead, changes in symptom severity partially mediated the effects of medication on NAA concentration, especially in the caudate and putamen. INTERPRETATION: These findings, taken together, suggest that PDD is not a direct consequence of elevated NAA concentrations, but that a more fundamental pathophysiological process likely causes PDD and determines the severity of its symptoms. The findings also suggest that although duloxetine normalized NAA concentrations in patients, it did so by modulating the severity of depressive symptoms. Medication presumably reduced depressive symptoms through other, as yet unidentified, brain processes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00360724.

One-Pot Biosynthesis of l-Aspartate from Maleate via an Engineered Strain Containing a Dual-Enzyme System.

l-Aspartate has been widely used in medicine and the food and chemical industries. In this study, Serratia marcescens maleate cis-trans isomerase (MaiA) and Escherichia coli aspartase (AspA) were coupled and coexpressed in an engineered E. coli strain in which the byproduct metabolic pathway was inactivated. The engineered E. coli strain containing the dual-enzyme system (pMA) was employed to bioproduce l-aspartate from maleate with a conversion of 98%. We optimized the activity ratio of double enzymes through ribosome binding site (RBS) regulation and molecular modification of MaiA, resulting in an engineered strain: pMA-RBS4-G27A/G171A. The conversion of l-aspartate biotransformed from maleate using the pMA-RBS4-G27A/G171A strain was almost 100%. It required 40 min to complete the whole-cell catalysis, without the intermediate product and byproduct, compared to 120 min before optimization. The induction timing and the amount of inducer in a 5-liter fermentor were optimized for scale-up of the production of l-aspartate. The amount of produced l-aspartate using the cells obtained by fermentation reached 419.8 g/liter (3.15 M), and the conversion was 98.4%. Our study demonstrated an environmentally responsible and efficient method to bioproduce l-aspartate from maleate and provided an available pathway for the industrial production of l-aspartate. This work should greatly improve the economic benefits of l-aspartate, which can now be simply produced from maleate by the engineered strain constructed based on dual-enzyme coupling.IMPORTANCE l-Aspartate is currently produced from fumarate by biological methods, and fumarate is synthesized from maleate by chemical methods in industry. We established a biosynthesis method to produce l-aspartate from maleate that is environmentally responsible, convenient, and efficient. Compared to conventional l-aspartate production, no separation and purification of intermediate products is required, which could greatly improve production efficiency and reduce costs. As environmental issues are attracting increasing attention, conventional chemical methods gradually will be replaced by biological methods. Our results lay an important foundation for the industrialization of l-aspartate biosynthesis from maleate.

Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative ß-alanine with high stoichiometric yield.

L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2 mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01 mol L-aspartate/mol glucose and 1.52 mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249 mM (33.1 g/L) L-aspartate or 424 mM (37.7 g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

Preventing BRCA1/ZBRK1 repressor complex binding to the GOT2 promoter results in accelerated aspartate biosynthesis and promotion of cell proliferation.

Breast cancer susceptibility gene 1 (BRCA1) has been implicated in modulating metabolism via transcriptional regulation. However, direct metabolic targets of BRCA1 and the underlying regulatory mechanisms are still unknown. Here, we identified several metabolic genes, including the gene which encodes glutamate-oxaloacetate transaminase 2 (GOT2), a key enzyme for aspartate biosynthesis, which are repressed by BRCA1. We report that BRCA1 forms a co-repressor complex with ZBRK1 that coordinately represses GOT2 expression via a ZBRK1 recognition element in the promoter of GOT2. Impairment of this complex results in upregulation of GOT2, which in turn increases aspartate and alpha ketoglutarate production, leading to rapid cell proliferation of breast cancer cells. Importantly, we found that GOT2 can serve as an independent prognostic factor for overall survival and disease-free survival of patients with breast cancer, especially triple-negative breast cancer. Interestingly, we also demonstrated that GOT2 overexpression sensitized breast cancer cells to methotrexate, suggesting a promising precision therapeutic strategy for breast cancer treatment. In summary, our findings reveal that BRCA1 modulates aspartate biosynthesis through transcriptional repression of GOT2, and provides a biological basis for treatment choices in breast cancer.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

ß-Hydroxyaspartic acid in siderophores: biosynthesis and reactivity.

A growing number of siderophores are found to contain ß-hydroxyaspartic acid (ß-OH-Asp) as a functional group for Fe(III) coordination, along with the more common catechol and hydroxamic acid groups. This review covers the structures, biosynthesis, and reactions of peptidic ß-OH-Asp siderophores. Hydroxylation of Asp in siderophore biosynthesis is predicted to be carried out either through discrete aspartyl ß-hydroxylating enzymes or through hydroxylating domains within non-ribosomal peptide synthetases, both of which display sequence homology to known non-heme iron(II), α-ketoglutarate-dependent dioxygenases. Ferric complexes of ß-OH-Asp siderophores are photoreactive, resulting in reduction of Fe(III) and oxidative cleavage of the siderophore to yield distinct types of photoproducts. Probing the photoreactivity of synthetic Fe(III)-α-hydroxycarboxylate clusters yields mechanistic insights into the different photoproducts observed for ß-OH-Asp and other α-hydroxycarboxylate siderophore Fe(III) complexes.

Effects of nitrogen combined with zinc application on glutamate, glutamine, aspartate and asparagine accumulation in two winter wheat cultivars.

Zinc (Zn) deficiency remarkably depresses the protein concentration in the grain of winter wheat. Cultivar 'Pingan 8' showed lower Zn concentrations in the grain than did cultivar 'Yangao 006' after nitrogen (N) combined with Zn application. However, little is known about how amino acids are influenced by Zn combined with N application or about the differences in amino acid accumulation between the two winter wheat cultivars. A pot experiment was conducted to characterize amino acid accumulation in the low Zn-accumulating cultivar 'Pingan 8' and the high Zn-accumulating cultivar 'Yangao 006' at various growth stages (seedling, jointing, grain filling and maturity) as influenced by N and Zn supply. The N (N0.2) combined with Zn (Zn10) application significantly increased grain yields and the concentrations of N, Zn and crude protein in the grain of both wheat cultivars. N combined with Zn application significantly increased the concentrations of glutamate (Glu) and asparagine (Asn) but decreased the concentrations of glutamine (Gln) and aspartate (Asp) in cultivar 'Yangao 006'; the N combined with Zn application decreased the concentrations of Glu and Gln but increased the concentrations of Asp and Asn in cultivar 'Pingan 8' at the jointing, grain filling and mature stages. Correlation analysis results showed that there were significant relationships between grain yields, spike number, grain number and Zn, N, crude protein, Glu, Gln, Asp and Asn concentrations in the shoots and grain of winter wheat at different growth stages. These results demonstrate that N combined with Zn application enhanced protein synthesis by altering amino acid accumulation in both winter wheat cultivars. Cultivar 'Pingan 8' had lower Gln, Asp and Asn concentrations and higher Glu concentrations than did cultivar 'Yangao 006' after the N0.05 treatment but had higher Glu, Gln, Asp, and Asn concentrations and lower Glu concentrations than did cultivar 'Yangao 006' after the N0.2 treatment. These results revealed that the difference in amino acid concentrations between the two cultivars was related to the N application level.

Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria.

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

Biocontrol of Monilinia laxa by Aureobasidium pullulans strains: Insights on competition for nutrients and space.

Two Aureobasidium pullulans strains (L1 and L8), able to prevent postharvest fruit decay, were evaluated in order to elucidate how the competition for nutrients and space was involved in their activity against Monilinia laxa, the causal agent of peach brown rot. The competition for nutrients was studied by co-culturing pathogen conidia and antagonists in different conditions of nutrient availability and avoiding contact between them. Both antagonists prevented the germination of conidia of M. laxa in water, reducing germination rate by >35%. However, L1 and L8 showed the lowest inhibition of conidial germination in peach juice at 5%, with a reduction of 12.6% and 13.9% respectively. HPLC amino acid analysis of peach juice revealed that the addition of the yeast suspension greatly modified their composition: asparagine was completely depleted soon after 12h of incubation and was probably hydrolyzed to aspartic acid by the yeasts, as aspartic acid content markedly increased. Pure asparagine and aspartic acid were tested by in vitro trials at the concentrations found in peach juice: both influenced M. laxa growth, but in opposite ways. Asparagine stimulated pathogen growth; conversely, amended medium with aspartic acid significantly inhibited the conidia germination and mycelial development of M. laxa. Scanning Electron Microscopy revealed that both strains showed a great capability to compete with M. laxa for space (starting 8h after treatment), colonizing the wound surface and inhibiting pathogen growth. This study clearly showed that A. pullulans L1 and L8 strains could compete with M. laxa for nutrients and space; this mode of action may play an important role in the antagonistic activity, especially in the first hours of tritrophic host-pathogen-antagonist interaction, although several other mechanisms can interact each other.

Aspartate Glycosylation Triggers Isomerization to Isoaspartate.

O-Linked ß-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Suppressing N-Acetyl-l-Aspartate Synthesis Prevents Loss of Neurons in a Murine Model of Canavan Leukodystrophy.

Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in AspaNur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like. SIGNIFICANCE STATEMENT: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.

Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin.

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism.