RESUMEN
Canavan disease is caused by mutations in the gene encoding aspartoacylase (ASPA), a deacetylase that catabolizes N-acetylaspartate (NAA). The precise involvement of elevated NAA in the pathogenesis of Canavan disease is an ongoing debate. In the present study, we tested the effects of elevated NAA in the brain during postnatal development. Mice were administered high doses of the hydrophobic methyl ester of NAA (M-NAA) twice daily starting on day 7 after birth. This treatment increased NAA levels in the brain to those observed in the brains of Nur7 mice, an established model of Canavan disease. We evaluated various serological parameters, oxidative stress, inflammatory and neurodegeneration markers and the results showed that there were no pathological alterations in any measure with increased brain NAA levels. We examined oxidative stress markers, malondialdehyde content (indicator of lipid peroxidation), expression of NADPH oxidase and nuclear translocation of the stress-responsive transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF-2) in brain. We also examined additional pathological markers by immunohistochemistry and the expression of activated caspase-3 and interleukin-6 by Western blot. None of the markers were increased in the brains of M-NAA treated mice, and no vacuoles were observed in any brain region. These results show that ASPA expression prevents the pathologies associated with excessive NAA concentrations in the brain during postnatal myelination. We hypothesize that the pathogenesis of Canavan disease involves not only disrupted NAA metabolism, but also excessive NAA related signaling processes in oligodendrocytes that have not been fully determined and we discuss some of the potential mechanisms.
RESUMEN
BACKGROUND: The intranasal route of administration has proven to be an effective method for bypassing the blood brain barrier and avoiding first pass hepatic metabolism when targeting drugs to the brain. Most small molecules gain rapid access to CNS parenchyma when administered intranasally. However, bioavailability is affected by various factors ranging from the molecular weight of the drug to the mode of intranasal delivery. COMPARISON WITH EXISTING METHODS: We examined the effects of animal posture, intranasal application method and animal weight and age on the delivery of radiolabeled pralidoxime (3H-2-PAM) to the brain of rats. RESULTS: We found that using upright vs. supine posture did not significantly affect 3H-2-PAM concentrations in different brain regions. Older animals with higher weights required increased doses to achieve the same drug concentration throughout the brain when compared to young animals with lower body weights. The use of an intranasal aerosol propelled delivery device mainly increased bioavailability in the olfactory bulbs, but did not reliably increase delivery of the drug to various other brain regions, and in some regions of the brain delivered less of the drug than simple pipette administration. CONCLUSION: In view of the emerging interest in the use of intranasal delivery of drugs to combat cognitive decline in old age, we tested effectiveness in very old rats and found the method to be as effective in the older rats.
Asunto(s)
Envejecimiento/fisiología , Antídotos/administración & dosificación , Peso Corporal/fisiología , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Compuestos de Pralidoxima/administración & dosificación , Administración Intranasal , Vías Aferentes/efectos de los fármacos , Vías Aferentes/fisiología , Envejecimiento/efectos de los fármacos , Animales , Antídotos/farmacocinética , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Masculino , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/metabolismo , Ratas , Ratas Sprague-Dawley , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismo , Tritio/administración & dosificación , Tritio/farmacocinéticaRESUMEN
Melatonin and N-acetylserotonin (NAS) are tryptophan metabolites that have potent anti-oxidant, anti-inflammatory and neuroprotective properties in several animal models of neurological injury and disease including multiple sclerosis (MS). The therapeutic effect of NAS has not been reported previously in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. Using a MOG-peptide induced EAE mouse model we examined the effects of melatonin and NAS on clinical score, inflammatory markers, free radical generation, and sparing of axons, oligodendrocytes and myelin. We found that NAS and melatonin reduced clinical scores when administered prior to or after symptom onset. This effect was more pronounced when melatonin and NAS were administrated prior to symptom onset whereby the appearance of motor symptoms was significantly delayed. Activated microglia and CD4+ T-cells were increased in the white matter of untreated EAE mice, with a return to near control levels after melatonin or NAS treatment. The expression of the NADPH oxidase component p67phox and inducible nitric oxide synthase (iNOS) was increased in the EAE mice as compared with controls, and both drug treated groups had significant reductions in their expression. Melatonin and NAS treatment significantly reduced the loss of mature oligodendrocytes, demyelination and axonal injury. Both compounds also significantly attenuated iNOS induction and reactive oxygen species (ROS) generation in lipopolysaccharide-activated microglia in culture. Our results show for the first time the therapeutic effects of NAS and confirm previous reports on the effectiveness of melatonin in the EAE model of MS.
Asunto(s)
Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Melatonina/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Serotonina/análogos & derivados , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/patología , Serotonina/uso terapéutico , Resultado del TratamientoRESUMEN
We hypothesize that: (1) L-tryptophan (Trp) is greatly utilized and not depleted in pregnancy; (2) fetal tolerance is achieved in part through immunosuppressive kynurenine (Kyn) metabolites produced by the flux of plasma free (non-albumin-bound) Trp down the Kyn pathway; (3) the role of indoleamine 2,3-dioxygenase (IDO) in infection is not related to limitation of an essential amino acid, but is rather associated with stress responses and the production of Kyn metabolites that regulate the activities of antigen presenting cells and T-cells, as well as increased NAD(+) synthesis in IDO-expressing cells; (4) Trp depletion is not a host defence mechanism, but is a consequence of Trp utilization. We recommend that future studies in normal and abnormal pregnancies and in patients with infections or cancer should include measurements of plasma free Trp, determinants of Trp binding (albumin and non-esterified fatty acids), total Trp, determinants of activities of the Trp-degrading enzymes Trp 2,3-dioxygenase (TDO) (cortisol) and IDO (cytokines) and levels of Kyn metabolites. We also hypothesize that abnormal pregnancies and failure to combat infections or cancer may be associated with excessive Trp metabolism that can lead to pathological immunosuppression by excessive production of Kyn metabolites. Mounting evidence from many laboratories indicates that Trp metabolites are key regulators of immune cell behaviour, whereas Trp depletion is an indicator of extensive utilization of this key amino acid.
Asunto(s)
Enfermedades Transmisibles/metabolismo , Feto/metabolismo , Tolerancia Inmunológica , Complicaciones del Embarazo/metabolismo , Triptófano/metabolismo , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Enfermedades Transmisibles/sangre , Enfermedades Transmisibles/inmunología , Femenino , Feto/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/inmunología , Quinurenina/metabolismo , NAD/metabolismo , Estrés Oxidativo , Embarazo , Complicaciones del Embarazo/sangre , Complicaciones del Embarazo/inmunología , Unión Proteica , Linfocitos T/inmunología , Linfocitos T/metabolismo , Triptófano/sangre , Triptófano/deficiencia , Triptófano/inmunología , Triptófano Oxigenasa/metabolismoRESUMEN
Intranasal delivery is an emerging method for bypassing the blood brain barrier (BBB) and targeting therapeutics to the CNS. Oximes are used to counteract the effects of organophosphate poisoning, but they do not readily cross the BBB. Therefore, they cannot effectively counteract the central neuropathologies caused by cholinergic over-activation when administered peripherally. For these reasons we examined intranasal administration of oximes in an animal model of severe organophosphate poisoning to determine their effectiveness in reducing mortality and seizure-induced neuronal degeneration. Using the paraoxon model of organophosphate poisoning, we administered the standard treatment (intramuscular pralidoxime plus atropine sulphate) to all animals and then compared the effectiveness of intranasal application of obidoxime (OBD) to saline in the control groups. Intranasally administered OBD was effective in partially reducing paraoxon-induced acetylcholinesterase inhibition in the brain and substantially reduced seizure severity and duration. Further, intranasal OBD completely prevented mortality, which was 41% in the animals given standard treatment plus intranasal saline. Fluoro-Jade-B staining revealed extensive neuronal degeneration in the surviving saline-treated animals 24h after paraoxon administration, whereas no detectable degenerating neurons were observed in any of the animals given intranasal OBD 30min before or 5min after paraoxon administration. These findings demonstrate that intranasally administered oximes bypass the BBB more effectively than those administered peripherally and provide an effective method for protecting the brain from organophosphates. The addition of intranasally administered oximes to the current treatment regimen for organophosphate poisoning would improve efficacy, reducing both brain damage and mortality.
Asunto(s)
Encéfalo/enzimología , Enfermedades del Sistema Nervioso Central/prevención & control , Reactivadores de la Colinesterasa/uso terapéutico , Cloruro de Obidoxima/uso terapéutico , Intoxicación por Organofosfatos , Acetilcolinesterasa/metabolismo , Administración Intranasal , Animales , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Enfermedades del Sistema Nervioso Central/etiología , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Masculino , Intoxicación por Organofosfatos/complicaciones , Intoxicación por Organofosfatos/tratamiento farmacológico , Intoxicación por Organofosfatos/mortalidad , Compuestos de Pralidoxima/metabolismo , Compuestos de Pralidoxima/farmacocinética , Ratas , Ratas Sprague-Dawley , Estadísticas no Paramétricas , Tritio/farmacocinéticaRESUMEN
Metabolic networks are significantly altered in neoplastic cells. This altered metabolic program leads to increased glycolysis and lipogenesis and decreased dependence on oxidative phosphorylation and oxygen consumption. Despite their limited mitochondrial respiration, cancer cells, nonetheless, derive sufficient energy from alternative carbon sources and metabolic pathways to maintain cell proliferation. They do so, in part, by utilizing fatty acids, amino acids, ketone bodies, and acetate, in addition to glucose. The alternative pathways used in the metabolism of these carbon sources provide opportunities for therapeutic manipulation. Acetate, in particular, has garnered increased attention in the context of cancer as both an epigenetic regulator of posttranslational protein modification, and as a carbon source for cancer cell biomass accumulation. However, to date, the data have not provided a clear understanding of the precise roles that protein acetylation and acetate oxidation play in carcinogenesis, cancer progression or treatment. This review highlights some of the major issues, discrepancies, and opportunities associated with the manipulation of acetate metabolism and acetylation-based signaling in cancer development and treatment.
Asunto(s)
Epigénesis Genética , Neoplasias/tratamiento farmacológico , Procesamiento Proteico-Postraduccional , Acetato CoA Ligasa/fisiología , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Animales , Carcinogénesis/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/dietoterapia , Neoplasias/enzimología , Transducción de SeñalRESUMEN
BACKGROUND: The blood brain barrier (BBB) is critical for maintaining central nervous system (CNS) homeostasis by restricting entry of potentially toxic substances. However, the BBB is a major obstacle in the treatment of neurotoxicity and neurological disorders due to the restrictive nature of the barrier to many medications. Intranasal delivery of active enzymes to the brain has therapeutic potential for the treatment of numerous CNS enzyme deficiency disorders and CNS toxicity caused by chemical threat agents. NEW METHOD: The aim of this work is to provide a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain with therapeutic bioavailability. RESULTS: We tested intranasal delivery of chloramphenicol acetyltransferase (CAT), a relatively large (75kD) enzyme, in its active form into different regions of the brain. CAT was delivered intranasally to anaesthetized rats and enzyme activity was measured in different regions using a highly specific High Performance Thin Layer Chromatography (HP-TLC)-radiometry coupled assay. Active enzyme reached all examined areas of the brain within 15min (the earliest time point tested). In addition, the yield of enzyme activity in the brain was almost doubled in the brains of rats pre-treated with matrix metalloproteinase-9 (MMP-9). COMPARISON WITH EXISTING METHOD (S): Intranasal administration of active enzymes in conjunction with MMP-9 to the CNS is both rapid and effective. CONCLUSION: The present results suggest that intranasal enzyme therapy is a promising method for counteracting CNS chemical threat poisoning, as well as for treating CNS enzyme deficiency disorders.
Asunto(s)
Encéfalo/metabolismo , Cloranfenicol O-Acetiltransferasa/administración & dosificación , Cloranfenicol O-Acetiltransferasa/farmacocinética , Terapia Enzimática/métodos , Metaloproteinasa 9 de la Matriz/farmacología , Administración Intranasal , Animales , Disponibilidad Biológica , Masculino , Metaloproteinasa 9 de la Matriz/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes. RESULTS: Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 µm vs. 1.77 ± 0.01 µm in control), with a near linear dose response in the 1-10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca(2+) uptake. Moreover, pretreatment of cells with the mitochondrial Ca(2+) uptake blocker, Ru-360 (10 µM), markedly suppressed the effect of acetate on contraction. CONCLUSIONS: Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca(2+) uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.
Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Contracción Miocárdica , Acetato de Sodio/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Acetato de Sodio/farmacologíaRESUMEN
Cancer is associated with globally hypoacetylated chromatin and considerable attention has recently been focused on epigenetic therapies. N-acetyl-L-aspartate (NAA), the primary storage form of acetate in the brain, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis to generate acetate and ultimately acetyl-Coenzyme A for histone acetylation, are reduced in oligodendroglioma. The short chain triglyceride glyceryl triacetate (GTA), which increases histone acetylation and inhibits histone deacetylase expression, has been safely used for acetate supplementation in Canavan disease, a leukodystrophy due to ASPA mutation. We demonstrate that GTA induces cytostatic G0 growth arrest of oligodendroglioma-derived cells in vitro, without affecting normal cells. Sodium acetate, at doses comparable to that generated by complete GTA catalysis, but not glycerol also promoted growth arrest, whereas long chain triglycerides promoted cell growth. To begin to elucidate its mechanism of action, the effects of GTA on ASPA and acetyl-CoA synthetase protein levels and differentiation of established human oligodendroglioma cells (HOG and Hs683) and primary tumor-derived oligodendroglioma cells that exhibit some features of cancer stem cells (grade II OG33 and grade III OG35) relative to an oligodendrocyte progenitor line (Oli-Neu) were examined. The nuclear localization of ASPA and acetyl-CoA synthetase-1 in untreated cells was regulated during the cell cycle. GTA-mediated growth arrest was not associated with apoptosis or differentiation, but increased expression of acetylated proteins. Thus, GTA-mediated acetate supplementation may provide a safe, novel epigenetic therapy to reduce the growth of oligodendroglioma cells without affecting normal neural stem or oligodendrocyte progenitor cell proliferation or differentiation.
Asunto(s)
Acetatos/farmacología , Antígenos/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Madre Neoplásicas/patología , Oligodendroglioma/patología , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Acetilación/efectos de los fármacos , Amidohidrolasas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Mesodermo/efectos de los fármacos , Mesodermo/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Oligodendroglioma/enzimología , Fenotipo , Transporte de Proteínas/efectos de los fármacosRESUMEN
Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.
Asunto(s)
Ácido Aspártico/análogos & derivados , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Dipéptidos/farmacología , Células Madre Neoplásicas/metabolismo , Fármacos Neuroprotectores/farmacología , Oligodendroglioma/metabolismo , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Animales , Ácido Aspártico/farmacología , Línea Celular Transformada , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Oligodendroglioma/tratamiento farmacológico , Oligodendroglioma/genética , Oligodendroglioma/patologíaRESUMEN
N-acetylaspartate (NAA) is recognized as a noninvasive diagnostic neuronal marker for a host of neuropsychiatric disorders using magnetic resonance spectroscopy (MRS). Numerous correlative clinical studies have found significant decreases in NAA levels in specific neuronal systems in an array of neuropsychiatric and substance-abuse disorders. We have recently identified the methamphetamine-induced neuronal protein known as "shati" as the NAA biosynthetic enzyme (aspartate N-acetyltransferase [Asp-NAT]; gene Nat8l). We have generated an Nat8l transgenic knockout mouse line to study the functions of NAA in the nervous system. We were unable to breed homozygous Nat8l knockout mice successfully for study and so used the heterozygous mice (Nat8l(+/-) ) for initial characterization. MRS analysis of the Nat8l(+/-) mice indicated significant reductions in NAA in cortex (-38%) and hypothalamus (-29%) compared with wild-type controls, which was confirmed using HPLC (-29% in forebrain). The level of the neuromodulator N-acetylaspartylglutamate (NAAG), which is synthesized from NAA, was decreased by 12% in forebrain as shown by HPLC. Behavioral analyses of the heterozygous animals indicated normal behavior in most respects but reduced vertical activity in open-field tests compared with age- and sex-matched wild-type mice of the same strain. Nat8l(+/-) mice also showed atypical locomotor responses to methamphetamine administration, suggesting that NAA is involved in modulating the hyperactivity effect of methamphetamine. These observations add to accumulating evidence suggesting that NAA has specific regulatory functional roles in mesolimbic and prefrontal neuronal pathways either directly or indirectly through impact on NAAG synthesis
Asunto(s)
Ácido Aspártico/análogos & derivados , Acetiltransferasas/metabolismo , Análisis de Varianza , Animales , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Cromatografía Líquida de Alta Presión , Dipéptidos/deficiencia , Dipéptidos/genética , Dopaminérgicos/farmacología , Conducta Exploratoria/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Locomoción/efectos de los fármacos , Locomoción/genética , Espectroscopía de Resonancia Magnética , Metanfetamina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury.
RESUMEN
Aspartoacylase (ASPA) catalyzes deacetylation of N-acetylaspartate (NAA) to generate acetate and aspartate. Mutations in the gene for ASPA lead to reduced acetate availability in the CNS during development resulting in the fatal leukodystrophy Canavan disease. Highly specific polyclonal antibodies to ASPA were used to examine CNS expression in adult rats. In white matter, ASPA expression was associated with oligodendrocyte cell bodies, nuclei, and some processes, but showed a dissimilar distribution pattern to myelin basic protein and oligodendrocyte specific protein. Microglia expressed ASPA in all CNS regions examined, as did epiplexus cells of the choroid plexus. Pial and ependymal cells and some endothelial cells were ASPA positive, as were unidentified cellular nuclei throughout the CNS. Astrocytes did not express ASPA in their cytoplasm. In some fiber pathways and nerves, particularly in the brainstem and spinal cord, the axoplasm of many neuronal fibers expressed ASPA, as did some neurons. Acetyl coenzyme A synthase immunoreactivity was also observed in the axoplasm of many of the same fiber pathways and nerves. All ASPA-immunoreactive elements were unstained in brain sections from tremor rats, an ASPA-null mutant. The strong expression of ASPA in oligodendrocyte cell bodies is consistent with a lipogenic role in myelination. Strong ASPA expression in cell nuclei is consistent with a role for NAA-derived acetate in nuclear acetylation reactions, including histone acetylation. Expression of ASPA in microglia may indicate a role in lipid synthesis in these cells, whereas expression in axons suggests that some neurons can both synthesize and catabolize NAA.
Asunto(s)
Amidohidrolasas/metabolismo , Sistema Nervioso Central/enzimología , Animales , Astrocitos/enzimología , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Ratas , Temblor/enzimología , Temblor/patologíaRESUMEN
Acetyl coenzyme A synthetase-1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. By using an affinity-purified antibody generated against an 18-mer peptide sequence of AceCS1 and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons, and oligodendrocytes. Some nonneuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18-day-old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially up-regulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons.
Asunto(s)
Encéfalo/enzimología , Núcleo Celular/enzimología , Coenzima A Ligasas/biosíntesis , Citoplasma/enzimología , Animales , Western Blotting , Encéfalo/anatomía & histología , Lesiones Encefálicas/enzimología , Corteza Cerebral/lesiones , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Técnicas para Inmunoenzimas , Inmunohistoquímica , Inmunoprecipitación , Masculino , Datos de Secuencia Molecular , Fibras Nerviosas/enzimología , Vías Nerviosas/citología , Vías Nerviosas/enzimología , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Genetic mutations that severely diminish the activity of aspartoacylase (ASPA) result in the fatal brain dysmyelinating disorder, Canavan disease. There is no effective treatment. ASPA produces free acetate from the concentrated brain metabolite, N-acetylaspartate (NAA). Because acetyl coenzyme A is a key building block for lipid synthesis, we postulated that the inability to catabolize NAA leads to a brain acetate deficiency during a critical period of CNS development, impairing myelination and possibly other aspects of brain development. We tested the hypothesis that acetate supplementation during postnatal myelination would ameliorate the severe phenotype associated with ASPA deficiency using the tremor rat model of Canavan disease. Glyceryltriacetate (GTA) was administered orally to tremor rats starting 7 days after birth, and was continued in food and water after weaning. Motor function, myelin lipids, and brain vacuolation were analyzed in GTA-treated and untreated tremor rats. Significant improvements were observed in motor performance and myelin galactocerebroside content in tremor rats treated with GTA. Further, brain vacuolation was modestly reduced, and these reductions were positively correlated with improved motor performance. We also examined the expression of the acetyl coenzyme A synthesizing enzyme acetyl coenzyme A synthase 1 and found upregulation of expression in tremor rats, with a return to near normal expression levels in GTA-treated tremor rats. These results confirm the critical role played by NAA-derived acetate in brain myelination and development, and demonstrate the potential usefulness of acetate therapy for the treatment of Canavan disease.
Asunto(s)
Acetatos/uso terapéutico , Ácido Aspártico/análogos & derivados , Enfermedad de Canavan/terapia , Mutación , Animales , Ácido Aspártico/metabolismo , Ácido Aspártico/uso terapéutico , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Femenino , Heterocigoto , Lípidos/química , Masculino , Vaina de Mielina/química , Fenotipo , Ratas , Resultado del TratamientoRESUMEN
N-acetylaspartate (NAA) is a concentrated, neuron-specific brain metabolite routinely used as a magnetic resonance spectroscopy marker for brain injury and disease. Despite decades of research, the functional roles of NAA remain unclear. Biochemical investigations over several decades have associated NAA with myelin lipid synthesis and energy metabolism. However, studies have been hampered by an inability to identify the gene for the NAA biosynthetic enzyme aspartate N-acetyltransferase (Asp-NAT). A very recent report has identified Nat8l as the gene encoding Asp-NAT and confirmed that the only child diagnosed with a lack of NAA on brain magnetic resonance spectrograms has a 19-bp deletion in this gene. Based on in vitro Nat8l expression studies the researchers concluded that many previous biochemical investigations have been technically flawed and that NAA may not be associated with brain energy or lipid metabolism. In studies done concurrently in our laboratory we have demonstrated via cloning, expression, specificity for acetylation of aspartate, responsiveness to methamphetamine treatment, molecular modeling and comparative immunolocalization that NAT8L is the NAA biosynthetic enzyme Asp-NAT. We conclude that NAA is a major storage and transport form of acetyl coenzyme A specific to the nervous system, thus linking it to both lipid synthesis and energy metabolism.
Asunto(s)
Acetilcoenzima A/metabolismo , Acetiltransferasas/biosíntesis , Ácido Aspártico/análogos & derivados , Metanfetamina/farmacología , Neuronas/enzimología , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Ácido Aspártico/biosíntesis , Línea Celular , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/enzimología , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato/genéticaRESUMEN
N-acetylaspartate (NAA) is present at very high concentrations in the brain and is used as a non-invasive marker of neuronal viability in magnetic resonance spectroscopy. N-acetylaspartylglutamate (NAAG) is an acetylated dipeptide formed from NAA, and may be an agonist of the mGluR3 receptor. Both NAA and NAAG are synthesized primarily in neurons. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in motor neuron death, and progressive paralysis. Levels of both NAA and NAAG are reported to be decreased in ALS. Riluzole is a glutamatergic modulating agent used to treat ALS, but there are conflicting results in the literature concerning the recovery of NAA after riluzole treatment. We studied the effects of riluzole on the biosynthesis of both NAA and NAAG in SH-SY5Y human neuroblastoma cells. We used two methodologies to examine the effect; one involving radiolabel incorporation from corresponding substrates into NAA and NAAG, and the other involving the measurement of endogenous NAA and NAAG levels using HPLC. We show that riluzole treatment, which decreases glutamatergic neuronal excitation, decreases the synthesis and levels of both NAA and NAAG in SH-SY5Y cells in a dose and time dependant manner. These results suggest that the synthesis of NAA and NAAG may be coupled to glutamatergic neurotransmission, and further investigations along these lines are warranted.
Asunto(s)
Ácido Aspártico/análogos & derivados , Dipéptidos/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Riluzol/farmacología , Ácido Aspártico/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Humanos , Neuroblastoma , Factores de TiempoRESUMEN
Patients suffering from traumatic brain injury (TBI) have decreased markers of energy metabolism, including N-acetylaspartate (NAA) and ATP. In the nervous system, NAA-derived acetate provides acetyl-CoA required for myelin lipid synthesis. Acetate can also be oxidized in mitochondria for the derivation of metabolic energy. In the current study, using the controlled cortical impact model of TBI in rats, we investigated the effects of the hydrophobic acetate precursor, glyceryltriacetate (GTA), as a method of delivering metabolizable acetate to the injured brain. We found that GTA administration significantly increased the levels of both NAA and ATP in the injured hemisphere 4 and 6 days after injury, and also resulted in significantly improved motor performance in rats 3 days after injury.
Asunto(s)
Acetatos/farmacología , Ácido Acético/metabolismo , Acetilcoenzima A/biosíntesis , Lesiones Encefálicas/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Triacetina/farmacología , Acetatos/uso terapéutico , Animales , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/fisiología , Masculino , Lípidos de la Membrana/biosíntesis , Vaina de Mielina/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas Sprague-Dawley , Resultado del Tratamiento , Triacetina/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
N-acetylaspartate (NAA) is synthesized predominantly in neurons, and brain homogenate subfractionation studies suggest that biosynthesis occurs at both microsomal (cytoplasmic) and mitochondrial sites. We investigated NAA synthesis in SH-SY5Y human neuroblastoma cells using distinct metabolic precursors that are preferentially metabolized in mitochondria and cytoplasm. Incorporation of (14)C-aspartate and (14)C-malate into NAA was examined in the presence and absence of an inhibitor (aminooxyacetic acid, AOAA) of aspartate aminotransferase (AAT), a central enzyme involved in the biosynthesis of aspartate in mitochondria, and degradation of aspartate in the cytoplasm. AOAA increased the incorporation of (14)C-aspartate into NAA, reflecting direct aspartate-->NAA synthesis in an extramitochondrial location. As expected, AOAA decreased incorporation of (14)C-malate into NAA, reflecting NAA synthesis in mitochondria via the malate-->oxaloacetate-->aspartate-->NAA pathway. When (14)C-malate was used as substrate, the (14)C-NAA/(14)C-aspartate ratio was over 20-fold higher than the ratio obtained with (14)C-aspartate. Because NAA can only be synthesized from aspartate, the higher (14)C-NAA/(14)C-aspartate (product/substrate) ratio obtained with (14)C-malate suggests greater NAA biosynthetic activity. We conclude that NAA biosynthesis occurs in both the cytoplasm and mitochondria of SH-SY5Y cells, and that the contribution from the mitochondrial compartment is quantitatively larger than that in the extramitochondrial compartment.
Asunto(s)
Ácido Aspártico/análogos & derivados , Encéfalo/metabolismo , Citoplasma/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Ácido Aminooxiacético/farmacología , Aspartato Aminotransferasas/antagonistas & inhibidores , Aspartato Aminotransferasas/metabolismo , Ácido Aspártico/biosíntesis , Ácido Aspártico/metabolismo , Encéfalo/ultraestructura , Radioisótopos de Carbono/metabolismo , Compartimento Celular/fisiología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Citoplasma/ultraestructura , Inhibidores Enzimáticos/farmacología , Humanos , Malatos/metabolismo , Mitocondrias/ultraestructura , Neuronas/ultraestructura , Ácido Oxaloacético/metabolismoRESUMEN
Several reports during the last three decades have indicated that biosynthesis of N-acetylaspartate (NAA) occurs primarily in the mitochondria. But a recent report by Lu et al. in this journal [2004; 122: 71-78] and subsequent two reports that cited those data suggested a predominant microsomal localization of the NAA biosynthetic enzyme, which is surprising in view of what is known about the biological functions of NAA. Therefore we reinvestigated this issue in rat brain homogenates using a similar fractionation procedure used by Lu et al. but without the loss of enzyme activity that they have encountered. We found that about 70% of the total Asp-NAT activity in the crude supernatant was present in the mitochondrial fraction which is about 5 times more than that in the microsomes. We found similar results in the case of the enzyme from bovine brain. In subsequent studies, we also have found that Asp-NAT activity in the bovine brain is very similar to that in the rat brain in substrate specificity and chromatographic characteristics including the high molecular weight pattern (approx. 670 kD) on size-exclusion HPLC.