Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

Lysosome inhibition sensitizes pancreatic cancer to replication stress by aspartate depletion.

Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.

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.

Ablating N-acetylaspartate prevents leukodystrophy in a Canavan disease model.

Canavan disease is caused by inactivating ASPA (aspartoacylase) mutations that prevent cleavage of N-acetyl-L-aspartate (NAA), resulting in marked elevations in central nervous system (CNS) NAA and progressively worsening leukodystrophy. We now report that ablating NAA synthesis by constitutive genetic disruption of Nat8l (N-acetyltransferase-8 like) permits normal CNS myelination and prevents leukodystrophy in a murine Canavan disease model.

[Physiological functions of L-ornithine and L-aspartate in the body and the efficacy of administration of L-ornithine-L-aspartate in conditions of relative deficiency]. / Fizjologiczne funkcje L-ornityny i L-asparaginianu oraz celowosc podawania asparaginianu ornityny w stanach wzglednego niedoboru.

L-ornithine-L-aspartate (LOLA) is a stable salt of two natural nonessential L-amino acids: ornithine and aspartic acid. It is formulated and marketed in low and high doses. Low doses are used as a food supplement and high doses (above 5 g) as a medicinal product to lower blood ammonia concentration and to eliminate symptoms of hepatic encephalopathy associated with liver cirrhosis. The aim of this review is to present physiological roles of L-ornithine and L-aspartate in the human body, to assess conditions under which these amino acids could be deficient, to analyze consequences of these deficiencies, and to review the current state of knowledge on the effects of LOLA administration. The data used in this publication result from searches of different electronic databases such as Cochrane Trials Register, MEDLINE, PubMed, Medscape, or Google Scholar, with a cut-off date of November 29, 2009, using terms: L-ornithine-L-aspartate, ornithine aspartate, ornithine, Hepa-Merz, ornithine deficiency, hyperammonemia, hepatic encephalopathy, and liver cirrhosis. Both amino acids play key roles in ammonia detoxification and in proline and polyamine biosyntheses. Polyamines are considered critical for DNA synthesis and cell replication and have been shown to stimulate hepatic regeneration. Supplementation with ornithine in animal models demonstrated enhanced wound breaking strength and collagen deposition. It has been shown in vitro, in vivo and in perfused organs that urea synthesis from ammonia is limited by endogenous ornithine and that ornithine can pharmacologically promote urea formation to a greater degree than any ammonia supply. Administration of LOLA in high doses reduced high blood ammonia induced either by ammonium chloride or protein ingestion or existing as a clinical complication of cirrhosis. In health and with proper diet, L-ornithine and L-aspartate are synthesized de novo in sufficient quantities, but in the states of disease, tissue damage, organ insufficiency, excessive metabolic demand, growth, pregnancy, or urea cycle enzyme deficiencies, these amino acids need to be supplemented with the food. The review of available data indicate that there is direct and indirect (resulting from physiology) scientific rationale for dietary use of LOLA, depending on an individual's physiological, metabolic or pathological conditions. In conditional ornithine deficiency, daily supplementation with LOLA at doses about 1 g/day is safe and, as demonstrated in vitro, should be sufficient to saturate tissue ornithine concentration to prevent postprandial hyperammonemia and to stimulate tissue regeneration.

Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia.

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

Adaptation to a long term (4 weeks) arginine- and precursor (glutamate, proline and aspartate)-free diet.

BACKGROUND & AIMS: It is not known whether arginine homeostasis is negatively affected by a "long term" dietary restriction of arginine and its major precursors in healthy adults. To assess the effects of a 4-week arginine- and precursor-free dietary intake on the regulatory mechanisms of arginine homeostasis in healthy subjects. METHODS: Ten healthy adults received a complete amino acid diet for 1 week (control diet) and following a break period, six subjects received a 4-week arginine, proline, glutamate and aspartate-free diet (APF diet). The other four subjects continued for 4 weeks with the complete diet. On days 4 and 7 of the first week and days 25 and 28 of the 4-week period, the subjects received 24-h infusions of arginine, citrulline, leucine and urea tracers. RESULTS: During the 4-week APF, plasma arginine fluxes for the fed state, were significantly reduced. There were no significant differences for citrulline, leucine or urea fluxes. Arginine de novo synthesis was not affected by the APF intake. However, arginine oxidation was significantly decreased. CONCLUSIONS: In healthy adults, homeostasis of arginine under a long term arginine- and precursor-free intake is achieved by decreasing catabolic rates, while de novo arginine synthesis is maintained.

A decrease in N-acetylaspartate and an increase in myoinositol in the anterior cingulate gyrus are associated with behavioral and psychological symptoms in Alzheimer's disease.

BACKGROUND AND PURPOSE: The cognitive decline in Alzheimer's disease (AD) patients has been reported to involve alterations in the medial temporal lobe and the posterior cingulate gyrus. On the other hand, the neurochemical pathologies of the behavioral and psychological symptoms of dementia (BPSD) have not been sufficiently discussed. The aim of this study was to clarify the pathologies of BPSD in AD patients. METHODS: Thirty patients with probable AD were included and underwent the following assessments: Mini Mental State Examination (MMSE), Clock Drawing Test (CDT), Story Recall Test (SRT), Behavioral pathology in Alzheimer's disease (BEHAVE-AD) and proton MRS ((1)H-MRS). None of them had been medicated for BPSD. RESULTS: The MRS study revealed that MMSE, CDT, and SRT scores were positively related to N-acetyl-aspartate (NAA)/creatine(Cr) and negatively related to myoinositol (mI)/Cr in the posterior cingulate gyrus, but not in the anterior cingulate gyrus. On the other hand, the scores obtained in two categories of BEHAVE-AD (delusional thought/ activity disturbances) were negatively related with NAA/Cr and positively related with mI/Cr in the anterior cingulate gyrus, but not in the posterior cingulate gyrus. CONCLUSION: We conclude that BPSD and the decline in cognitive function in AD might have separate pathologies.

The neuronal pathology of schizophrenia: molecules and mechanisms.

There is an accumulation of evidence for abnormalities in schizophrenia of both the major neurotransmitter systems of the brain - those of GABA (gamma-aminobutyric acid) and glutamate. Initial studies have found deficits in the putative neuronal marker, N-acetylaspartate, in a number of brain regions in schizophrenia. The animal models have provided some interesting correlates and discrepancies with these findings. The deficit in inhibitory interneurons within structures implicated in schizophrenic symptomatology may well have direct functional relevance, and can be induced by animal models of the disease such as subchronic phencyclidine administration or social isolation. Their association with these animal models suggests an environmental involvement. A loss of glutamatergic function in schizophrenia is supported by decreases in markers for the neuronal glutamate transporter in striatal structures that receive cortical glutamatergic projections. Deficits in the VGluT1 (vesicular glutamate transporter-1) in both striatal and hippocampal regions support this observation, and the association of VGluT1 density with a genetic risk factor for schizophrenia points to genetic influences on these glutamatergic deficits. Further studies differentiating neuronal loss from diminished activity and improved models allowing us to determine the temporal and causal relationships between GABAergic and glutamatergic deficits will lead to a better understanding of the processes underlying the neuronal pathology of schizophrenia.

Canavan disease and the role of N-acetylaspartate in myelin synthesis.

Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.15) due to mutations. ASPA releases acetate by deacetylation of N-acetylaspartate (NAA), a highly abundant amino acid derivative in the central nervous system. CD results in spongiform degeneration of the brain and severe psychomotor retardation, and the affected children usually die by the age of 10. The pathogenesis of CD remains a matter of inquiry. Our hypothesis is that ASPA actively participates in myelin synthesis by providing NAA-derived acetate for acetyl CoA synthesis, which in turn is used for synthesis of the lipid portion of myelin. Consequently, CD results from defective myelin synthesis due to a deficiency in the supply of the NAA-derived acetate. The demonstration of the selective localization of ASPA in oligodendrocytes in the central nervous system (CNS) is consistent with the acetate deficiency hypothesis of CD. We have tested this hypothesis by determining acetate levels and studying myelin lipid synthesis in the ASPA gene knockout model of CD, and the results provided the first direct evidence in support of this hypothesis. Acetate supplementation therapy is proposed as a simple and inexpensive therapeutic approach to this fatal disease, and progress in our preclinical efforts toward this goal is presented.

What have we learned from proton magnetic resonance spectroscopy about schizophrenia? A critical update.

PURPOSE OF REVIEW: This review discusses recent studies investigating schizophrenia with proton magnetic resonance spectroscopy including the first meta-analysis [Steen RG, Hamer RM, Lieberman JA. Measurement of brain metabolites by 1H magnetic resonance spectroscopy in patients with schizophrenia: a systematic review and meta-analysis. Neuropsychology 2005; 30:1949-1962]. We also highlight methodological issues and suggest a modality for future research to further explore glutamatergic dysfunction in schizophrenia. RECENT FINDINGS: Despite methodological differences, spectroscopy studies with schizophrenia show reductions in N-acetylaspartate in the medial temporal and prefrontal regions. Other areas such as the anterior cingulate, parietal cortex thalamus, and cerebellum may also have N-acetylaspartate reductions. The proton magnetic resonance spectroscopy studies at higher fields and with shorter echo time have revealed abnormalities in glutamate and glutamine. Animal studies have shown that the discrepancies in metabolites between patients and controls are not due to antipsychotic medication exposure, and that chronic exposure to N-methyl-D-aspartate antagonists has produced decreased N-acetylaspartate in the temporal cortex. The human and animal studies both support an excitoxic glutamatergically mediated process that may explain decreased N-acetylaspartate, volume loss, and the poor outcomes of schizophrenia. SUMMARY: Use of higher field strengths and longitudinal studies may reveal a progressive excitoxic glutamatergic process that leads to N-acetylaspartate and volume reductions. This may lead to the development of neuroprotective agents that change the course of schizophrenia.

Cortical NAA deficits in HIV infection without dementia: influence of alcoholism comorbidity.

Alcoholism comorbidity is highly prevalent in individuals infected with human immunodeficiency virus (HIV). Each condition is known to affect brain structure, function, and metabolism, but the combined effects on the brain have only recently been considered. Single-voxel, proton MR spectroscopy (MRS) has yielded sensitive measures of early brain deterioration in the progression of HIV, but has limited coverage of neocortex, whereas MRS imaging (MRSI) can simultaneously interrogate large regions of cortex. Included were 15 men with HIV+alcoholism, nine men with HIV alone, eight men with alcoholism alone (abstinent for 3-17 months), and 23 controls. The two HIV groups were matched in T-cell count and were not demented; the two alcoholism groups were relatively matched in lifetime alcohol consumption. We used MRSI with a variable-density spiral sequence to quantify major proton metabolites--N-acetylaspartate (NAA), creatine (Cr), and choline (Cho)-in the superior parietal-occipital cortex. Metabolites were expressed in absolute units and as the NAA/Cr ratio. Significant group effects were present for NAA and Cr. Only the HIV+alcoholism group was significantly affected, exhibiting a 0.8 SD deficit in NAA and a 1.0 SD deficit in Cr. The deficits were not related to highly active antiretroviral therapy (HAART) status. Neither HIV infection nor alcoholism independently resulted in parietal-occipital cortical metabolite abnormalities, yet each disease carried a liability that put affected individuals at a heightened risk of neuronal compromise when the diseases were compounded. Further, the use of absolute measures revealed deficits in NAA and Cr that would have gone undetected if these metabolites were expressed as a ratio.

In vivo evidence of neuronal loss in the hypothalamus of narcoleptic patients.

A dysfunction of the orexin (hypocretin) system in the hypothalamus has recently been linked to the pathogenesis of narcolepsy. The authors used in vivo proton MR spectroscopy to assess the N-acetylaspartate (NAA) content in the hypothalamus of narcoleptic patients. Hypothalamic NAA/creatine-phosphocreatine was reduced in narcoleptic patients compared with control subjects (p < 0.01). Hypothalamic neuronal loss/damage is a central pathogenetic feature in narcolepsy.

Gray matter N-acetyl aspartate deficits in secondary progressive but not relapsing-remitting multiple sclerosis.

BACKGROUND AND PURPOSE: Spectroscopic examination of multiple sclerosis (MS) patients has revealed abnormally low N-acetyl-aspartate (NAA) signal intensity, even in brain tissue that appears normal on high-resolution structural MR images but has yielded inconclusive evidence to distinguish the well-documented clinical differences between MS subtypes. This study used proton MR spectroscopic imaging (MRSI) and high-resolution MR imaging to characterize metabolite profiles in normal-appearing brain tissue of relapsing-remitting multiple sclerosis (RRMS) and secondary progressive (SP) MS. METHODS: Volumetric spiral MRSI was used together with high-resolution MR imaging to derive absolute measures of metabolite concentrations separately in normal-appearing supratentorial cerebral gray matter and white matter in five RRMS patients, five SPMS patients, and nine age-matched controls. Structural MR images were segmented into compartments of gray matter, white matter, CSF, and lesions, and metabolite signals per unit of tissue volume were calculated for gray matter and white matter separately. RESULTS: Only the SPMS group had significantly lower NAA concentrations in normal-appearing gray matter compared with concentrations in controls. NAA in normal-appearing white matter was equally reduced in RRMS and SPMS patients. The functional relevance of this brain metabolite measure was suggested by the observed but statistically nonsignificant correlation between higher disability scores on the Expanded Disability Status Scale and lower gray matter NAA concentrations. CONCLUSION: The otherwise occult abnormality in supratentorial gray matter in SPMS but not RRMS may explain the more severe physical and cognitive impairments afflicting patients with SPMS that do not correlate well with visible lesion burden.

Selective deficit of hippocampal N-acetylaspartate in antipsychotic-naive patients with schizophrenia.

BACKGROUND: Studies using proton magnetic resonance spectroscopy in schizophrenia have demonstrated abnormality of N-acetylaspartate but are confounded by the effects of phase of illness and medication. There is mounting evidence that antipsychotic medication influences N-acetylaspartate. METHODS: A group of first-episode patients who had received no, or minimal, antipsychotic medication was examined at baseline and after 3 months treatment. Normal comparison subjects were examined at the same interval. Ratios of N-acetylaspartate, creatine plus phosphocreatine, and choline-containing compounds in the left prefrontal cortex, hippocampus, and basal ganglia were measured. RESULTS: The mean duration of symptoms for all patients was 31.6 (SD 26.1) weeks. A significant reduction of hippocampal N-acetylaspartate/creatine plus phosphocreatine was found in the antipsychotic-naive group relative to those previously treated and to controls at baseline (F = 7.3, p <.002). No group differences were found at follow-up. CONCLUSIONS: Hippocampal N-acetylaspartate/creatine plus phosphocreatine appears to be selectively affected early in the course of illness. The finding of neurochemical differences between treatment naive and previously treated patients confirms the relevance of medication status in proton magnetic resonance spectroscopy studies. Further investigation of the influence of medication at this stage of illness is warranted.

N-acetylaspartate and N-Acetylaspartylglutamate deficits in superior temporal cortex in schizophrenia and bipolar disorder: a postmortem study.

BACKGROUND: N-acetylaspartylglutamate is found in neurons and its metabolite N-acetylaspartate, which can be measured by magnetic resonance spectroscopy, is considered a marker of neuronal integrity. Several magnetic resonance spectroscopy studies have found evidence of N-acetylaspartate deficits in schizophrenia. METHODS: We employed a high-pressure liquid chromatography method to determine N-acetylaspartate and N-acetylaspartylglutamate in postmortem brain tissues taken from a well-defined series of psychiatric cases. N-acetylaspartate and N-acetylaspartylglutamate concentrations were measured in superior temporal and frontal cortices of patients with schizophrenia, bipolar disorder, and depression and control subjects. RESULTS: N-acetylaspartate was significantly decreased below controls in superior temporal cortex in schizophrenia (p <.01) and bipolar disorder (p <.01), but no deficits were found in frontal cortex. N-acetylaspartylglutamate was significantly decreased only in superior temporal cortex in schizophrenia. CONCLUSIONS: The results are consistent with evidence of superior temporal cortex abnormalities in schizophrenia. The finding in bipolar disorder suggests that temporal cortex N-acetylaspartate deficits may be a common feature of psychotic disorders.

Evidence supporting a role for N-acetyl-L-aspartate as a molecular water pump in myelinated neurons in the central nervous system. An analytical review.

Molecular water pumps (MWPs) are characterized as biochemical systems existing at a compartmental boundary of living cells that can actively pump water against its gradient. A role for the observed intercompartmental transport of N-acetyl-L-aspartate (NAA), between neurons and oligodendrocytes in the CNS, as an efflux MWP for the removal of neuronal metabolic water has been proposed. In this review, accumulating evidence in support of such a role for NAA is presented, and the dynamics of the NAA cycle in myelinated neurons are considered. Based on the results of recent investigations, it is calculated that 1 mol of NAA is synthesized for every 40 mol of glucose (Glc) equivalent oxidized in the brain, and each mol of NAA may transport 121 mol of metabolic water out of neurons. In addition, turnover of total brain NAA is very rapid and appears to be only 16.7 h. Thus, the most important characteristic of NAA in the brain may not be its static level, but a dynamic aspect related to its rapid turnover. The relationship of NAA as a potential MWP to Canavan disease (CD), a genetic spongiform leukodystrophy in which the catabolic portion of the NAA cycle is deficient, and in a newly recognized brain disorder, hypoacetylaspartia, where the anabolic portion of the NAA cycle appears to be deficient, are discussed.