Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid CompositionClick to copy article linkArticle link copied!
- František SedlákFrantišek SedlákInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaInstitute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 2 110 01, CzechiaFirst Department of Internal Medicine - Hematology, Charles University General Hospital in Prague, Prague 110 01, CzechiaMore by František Sedlák
- Aleš KvasničkaAleš KvasničkaLaboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, and Faculty of Medicine and Dentistry, Palacký University Olomouc, Zdravotníku° 248/7, Olomouc 779 00, CzechiaMore by Aleš Kvasnička
- Barbora MarešováBarbora MarešováInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaInstitute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 2 110 01, CzechiaMore by Barbora Marešová
- Radana BrumarováRadana BrumarováLaboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, and Faculty of Medicine and Dentistry, Palacký University Olomouc, Zdravotníku° 248/7, Olomouc 779 00, CzechiaMore by Radana Brumarová
- Dana DobešováDana DobešováLaboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, and Faculty of Medicine and Dentistry, Palacký University Olomouc, Zdravotníku° 248/7, Olomouc 779 00, CzechiaMore by Dana Dobešová
- Kateřina DostálováKateřina DostálováLaboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, and Faculty of Medicine and Dentistry, Palacký University Olomouc, Zdravotníku° 248/7, Olomouc 779 00, CzechiaMore by Kateřina Dostálová
- Karolína ŠrámkováKarolína ŠrámkováInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaMore by Karolína Šrámková
- Martin PehrMartin PehrInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaThird Department of Medicine − Department of Endocrinology and Metabolism of the first Faculty of Medicine and General University Hospital in Prague, Charles University, Prague 110 01, CzechiaMore by Martin Pehr
- Pavel ŠáchaPavel ŠáchaInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaMore by Pavel Šácha
- David Friedecký*David Friedecký*Email: [email protected]. Tel.: +420588442619.Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, and Faculty of Medicine and Dentistry, Palacký University Olomouc, Zdravotníku° 248/7, Olomouc 779 00, CzechiaMore by David Friedecký
- Jan Konvalinka*Jan Konvalinka*Email: [email protected]. Tel.: +420220183218.Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6 166 10, CzechiaDepartment of Biochemistry, Faculty of Science, Charles University, Hlavova 8, Prague 128 00, CzechiaMore by Jan Konvalinka
Abstract
Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, −/–; one allele deficient in Folh1, +/–; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.
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You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.
License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Attribution (BY): Credit must be given to the creator.
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1. Introduction
2. Results
2.1. Targeted Metabolomic Analysis
metabolite group | number of metabolites | |||
---|---|---|---|---|
urine | plasma | CSF | brain | |
amines | 10 | 10 | 9 | 11 |
proteinogenic amino acids | 19 | 20 | 19 | 18 |
conjugates of amino acids | 30 | 35 | 11 | 16 |
dipeptides | 6 | 4 | 3 | 3 |
coenzymes & vitamins | 9 | 9 | 6 | 7 |
hydroxyl chain acylcarnitines | 5 | 11 | 1 | 7 |
long/very long chain acylcarnitines | 7 | 16 | 1 | 12 |
medium chain acylcarnitines | 8 | 7 | 0 | 5 |
short chain acylcarnitines | 6 | 7 | 5 | 6 |
organic acids | 33 | 23 | 8 | 13 |
phosphosaccharides | 4 | 0 | 4 | 9 |
purine/pyrimidine bases and ribosides | 28 | 21 | 19 | 15 |
purine/pyrimidine conjugates | 1 | 0 | 0 | 4 |
purine/pyrimidine nucleotides | 2 | 0 | 3 | 11 |
saccharides | 16 | 14 | 11 | 9 |
total | 184 | 177 | 100 | 146 |
2.2. Targeted Lipidomic Analysis
lipid class | abbreviation | number of lipids | ||
---|---|---|---|---|
plasma | CSF | brain | ||
cholesteryl esters | CE | 0 | 4 | 0 |
ceramides | Cer | 4 | 4 | 6 |
free fatty acids | FA | 21 | 21 | 22 |
dihexosylceramides | Hex2Cer | 0 | 0 | 1 |
monohexosylceramides | HexCer | 2 | 2 | 6 |
lysophosphatidylcholine | LPC | 32 | 9 | 21 |
lysophosphatidylcholine plasmanyls | LPCO | 6 | 0 | 5 |
lysophosphatidylethanolamines | LPE | 14 | 7 | 15 |
lysophosphatidylethanolamine plasmanyls | LPEO | 0 | 0 | 1 |
phosphatidylcholines | PC | 123 | 30 | 64 |
phosphatidylcholine plasmanyls/plasmalogens | PCO/PCP | 71 | 15 | 22 |
phosphatidylethanolamines | PE | 28 | 12 | 27 |
phosphatidylethanolamine plasmanyls/plasmalogens | PEO/PEP | 20 | 5 | 40 |
phosphatidylglycerols | PG | 0 | 0 | 2 |
phosphatidylinositols | PI | 14 | 0 | 4 |
phosphatidylserines | PS | 4 | 0 | 10 |
sphingomyelins | SM | 42 | 17 | 22 |
total | 381 | 126 | 268 |
2.3. N-Acetylaspartylglutamic Acid in Biofluids and Brain Tissue
3. Discussion
3.1. Metabolomic Analysis Revealed Mainly Age- and Sex-Specific Differences
3.2. Alterations Occur in the Brain Lipidome of GCPII-Deficient Mice
3.3. NAAG and BCG Concentrations in the Biofluids and in the Brain Tissue of GCPII/III-Deficient Mice
3.4. Limitations of the Study
3.5. Conclusions
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Animals
4.3. Sample Collection
4.4. Sample Preparation
4.5. Targeted Metabolomic Analysis
4.6. Targeted Lipidomic Analysis
4.7. Design of Experiment, Data Treatment, and Statistical Analysis
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00494.
Lipid retention pattern plots, overview of changes in the brain and CSF lipidome across all lipid classes as comparison between female (+/+♀Y) and male (+/+♂Y) data sets and comparison of the (−/–♂Y/+/+♂Y) and (−/–♀Y/+/+♀Y), quantitative analysis of NAAG and BCG in the CSF and brain from mice of different NAALAD2 genotypes (PDF)
MS parameters, retention time, and data from metabolomic and lipidomic analysis of plasma, urine, CSF, and brain tissue after processing, normalization, and transformation (XLSX)
Normality testing (Shapiro–Wilk) of the data from metabolomic and lipidomic analysis of plasma, urine, CSF, and brain tissue after processing, normalization, and transformation (XLSX)
Results of univariate statistical analysis (t test and fold change) for metabolomic and lipidomic data, calculation of the cumulative p value (using Fisher’s method) to evaluate systematic changes in lipid classes in the brain tissue and CSF (XLSX)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
2-PMPA | 2-(phosphonomethyl)pentanedioic acid |
ACN | acetonitrile |
AmAc | ammonium acetate |
BCG | β-citryl-glutamate |
BF | Bonferroni correction |
CE | cholesterol ester |
Cer | ceramide |
CSF | cerebrospinal fluid |
CV | coefficient of variation |
FA | free fatty acid |
FC | fold-change |
FOLH1 | folyl-poly-γ-glutamyl hydrolase I |
GCPII | glutamate carboxypeptidase II |
GCPIII | glutamate carboxypeptidase III |
Hex2Cer | dihexosylceramide |
HexCer | hexosylceramide |
IPA | 2-propanol |
LC–MS | liquid chromatography–tandem mass spectrometry |
LPC | lysophosphatidylcholine |
LPCO | lysophosphatidylcholine plasmanyl |
LPE | lysophosphatidylethanolamine |
LPEO | lysophosphatidylethanolamine plasmanyl |
MeOH | methanol |
mGluR3 | metabotropic glutamate receptors |
MRM | multiple reaction monitoring |
MTBE | tert-butyl methyl ether |
NAA | N-acetyl-aspartyl-aspartate |
NAAG | N-acetyl-aspartyl-glutamate |
NAAGS | N-acetylaspartylglutamate synthetase |
NAALadase I | N-acetylated-alpha-linked acidic dipeptidase I |
NAT8L | N-acetyltransferase-8-like enzyme |
OPLS-DA | orthogonal partial least-squares discriminant analysis |
PC | phosphatidylcholine |
PCA | principal component analysis |
PCO | phosphatidylcholine plasmanyl |
PCP | phosphatidylcholine plasmalogen |
PE | phosphatidylethanolamine |
PEO | phosphatidylethanolamine plasmanyl |
PEP | phosphatidylethanolamine plasmalogen |
PG | phosphatidylglycerol |
PI | phosphatidylinositol |
PS | phosphatidylserine |
PSMA | prostate specific membrane antigen |
QC | quality control |
SM | sphingomyelin |
References
This article references 67 other publications.
- 1Robinson, M. B.; Blakely, R. D.; Couto, R.; Coyle, J. T. Hydrolysis of the Brain Dipeptide N-Acetyl-L-Aspartyl-L-Glutamate. Identification and Characterization of a Novel N-Acetylated Alpha-Linked Acidic Dipeptidase Activity from Rat Brain. J. Biol. Chem. 1987, 262 (30), 14498– 14506, DOI: 10.1016/S0021-9258(18)47823-4Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXlvFersrc%253D&md5=27492dd297c2a204212efa2c51d4c8bbHydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated α-linked acidic dipeptidase activity from rat brainRobinson, Michael B.; Blakely, Randy D.; Couto, Renee; Coyle, Joseph T.Journal of Biological Chemistry (1987), 262 (30), 14498-506CODEN: JBCHA3; ISSN:0021-9258.HPLC studies documented the presence of an enzyme activity, N-acetylated α-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatog., which quant. sepd. [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, it was found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain was characterized. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37°. Eadie-Hofstee anal. of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 μM and 1 mM, resp. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by Mn. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated α-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degrdn. of N-acetyl-L-aspartyl-L-glutamate.
- 2Halsted, C. H.; Ling, E. H.; Luthi-Carter, R.; Villanueva, J. A.; Gardner, J. M.; Coyle, J. T. Folylpoly-Gamma-Glutamate Carboxypeptidase from Pig Jejunum. Molecular Characterization and Relation to Glutamate Carboxypeptidase II. J. Biol. Chem. 1998, 273 (32), 20417– 20424, DOI: 10.1074/jbc.273.32.20417Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlsVWgtbs%253D&md5=ec8c60eab7b3348a72060331af48ce1bFolylpoly-γ-glutamate carboxypeptidase from pig jejunum. Molecular characterization and relation to glutamate carboxypeptidase IIHalsted, Charles H.; Ling, Erh-Hsin; Luthi-Carter, Ruth; Villanueva, Jesus A.; Gardner, John M.; Coyle, Joseph T.Journal of Biological Chemistry (1998), 273 (32), 20417-20424CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Jejunal folylpoly-γ-glutamate carboxypeptidase hydrolyzes dietary folates prior to their intestinal absorption. The complete folylpoly-γ-glutamate carboxypeptidase cDNA was isolated from a pig jejunal cDNA library using an amplified homologous probe incorporating primer sequences from prostate-specific membrane antigen, a protein capable of folate hydrolysis. The cDNA encodes a 751-amino acid polypeptide homologous to prostate-specific membrane antigen and rat brain N-acetylated α-linked acidic dipeptidase. PC3 transfectant membranes exhibited activities of folylpoly-γ-carboxypeptidase and N-acetylated α-linked acidic dipeptidase, while immunoblots using monoclonal antibody to native folypoly-γ-glutamate carboxypeptidase identified a glycoprotein at 120 kDa and a polypeptide at 84 kDa. The kinetics of native folylpoly-γ-carboxypeptidase were expressed in membranes of PC3 cells transfected with either pig folylpoly-γ-carboxypeptidase or human prostate-specific membrane antigen. Folylpoly-γ-carboxypeptidase transcripts were identified at 2.8 kilobase pairs in human and pig jejunum, human and rat brain, and human prostate cancer LNCaP cells. Thus, pig folylpoly-γ-carboxypeptidase, rat N-acetylated α-linked acidic dipeptidase, and human prostate-specific membrane antigen appear to represent varied expressions of the same gene in different species and tissues. The discovery of the jejunal folylpoly-γ-carboxypeptidase gene provides a framework for future studies on relationships among these proteins and on the mol. regulation of intestinal folate absorption.
- 3Pangalos, M. N.; Neefs, J. M.; Somers, M.; Verhasselt, P.; Bekkers, M.; van der Helm, L.; Fraiponts, E.; Ashton, D.; Gordon, R. D. Isolation and Expression of Novel Human Glutamate Carboxypeptidases with N-Acetylated Alpha-Linked Acidic Dipeptidase and Dipeptidyl Peptidase IV Activity. J. Biol. Chem. 1999, 274 (13), 8470– 8483, DOI: 10.1074/jbc.274.13.8470Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitFyrs7g%253D&md5=8fbd95de336d72d0d4d8d1440cd18b76Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated α-linked acidic dipeptidase and dipeptidyl peptidase IV activityPangalos, Menelas N.; Neefs, Jean-Marc; Somers, Marijke; Verhasselt, Peter; Bekkers, Mariette; Van der Helm, Liesbet; Fraiponts, Erwin; Ashton, David; Gordon, Robert D.Journal of Biological Chemistry (1999), 274 (13), 8470-8483CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Hydrolysis of the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) by N-acetylated α-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a no. of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homolog glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, the authors describe the cloning of human ileal 100-kDa protein, which the authors have called a NAALADase-"like" (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection expts. Furthermore, the authors describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which the authors have localized to chromosome 11 by fluorescent in situ hybridization anal. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas.
- 4Collard, F.; Vertommen, D.; Constantinescu, S.; Buts, L.; Van Schaftingen, E. Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3. J. Biol. Chem. 2011, 286 (44), 38220– 38230, DOI: 10.1074/jbc.M111.287318Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlyktbrM&md5=fa7065a9e255e4a1639e1fcaa4ccb119Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3Collard, Francois; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, EmileJournal of Biological Chemistry (2011), 286 (44), 38220-38230CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)β-Citrylglutamate (BCG), a compd. present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca2+, the purified enzyme specifically hydrolyzed BCG and did not act on N-acetylaspartylglutamate (NAAG). However, both compds. were hydrolyzed in the presence of Mn2+. This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of NAAG. The mouse tissue distribution of β-citrylglutamate hydrolase was found to be strikingly similar to that of glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed BCG in the presence of Ca2+, and acted on both NAAG and BCG in the presence of Mn2+, whereas recombinant GCP2 only hydrolyzed NAAG and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis expts. revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) was largely responsible for GCP3 being able to hydrolyze BCG. Based on the crystal structure of GCP3 and kinetic anal., the authors propose that GCP3 forms a labile catalytic Zn-Ca cluster that is crit. for its β-citrylglutamate hydrolase activity.
- 5Navrátil, M.; Tykvart, J.; Schimer, J.; Pachl, P.; Navrátil, V.; Rokob, T. A.; Hlouchová, K.; Rulíšek, L.; Konvalinka, J. Comparison of Human Glutamate Carboxypeptidases II and III Reveals Their Divergent Substrate Specificities. FEBS J. 2016, 283 (13), 2528– 2545, DOI: 10.1111/febs.13761Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptFWgu70%253D&md5=5e6dba4900f70da471495c1319e7c883Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificitiesNavratil, Michal; Tykvart, Jan; Schimer, Jiri; Pachl, Petr; Navratil, Vaclav; Rokob, Tibor Andras; Hlouchova, Klara; Rulisek, Lubomir; Konvalinka, JanFEBS Journal (2016), 283 (13), 2528-2545CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)Glutamate carboxypeptidase III (GCPIII) is best known as a homolog of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurol. disorders and overexpressed in a no. of solid cancers. However, mouse GCPIII was recently shown to cleave β-citryl-L-glutamate (BCG), suggesting that these 2 closely related enzymes have distinct functions. Here, to develop a tool to dissect, evaluate, and quantify the activities of human GCPII and GCPIII, the authors analyzed the catalytic efficiencies of these enzymes toward 3 physiol. substrates (BCG, N-acetyl-L-aspartyl-L-glutamate, and polyglutamated folic acid). The authors obsd. a high efficiency of BCG cleavage by GCPIII but not GCPII. The authors also identified a strong modulation of GCPIII enzymic activity by divalent cations, while they did not observe this effect for GCPII. Addnl., the authors used x-ray crystallog. and computational modeling (QM/MM calcns.) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, resp. Finally, the authors took advantage of the substantial differences in the enzymic efficiencies of GCPII and GCPIII toward their substrates, using enzymic assays for specific detection of these proteins in human tissues. The findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiol. function related to BCG metab. in the human body.
- 6Wroblewska, B.; Santi, M. R.; Neale, J. H. N-Acetylaspartylglutamate Activates Cyclic AMP-Coupled Metabotropic Glutamate Receptors in Cerebellar Astrocytes. Glia 1998, 24 (2), 172– 179, DOI: 10.1002/(SICI)1098-1136(199810)24:2<172::AID-GLIA2>3.0.CO;2-6Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1czps12ltg%253D%253D&md5=2e2c0f07b9e087565a76a407d6b1866eN-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytesWroblewska B; Santi M R; Neale J HGlia (1998), 24 (2), 172-9 ISSN:0894-1491.N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide in the mammalian nervous system. NAAG meets the traditional criteria of a neurotransmitter, including localization in synaptic vesicles, depolarization-induced release, low potency activation of some N-methyl-D-aspartate receptors, and highly selective activation of a cAMP-coupled metabotropic glutamate receptor (mGluR) with potency approaching that of glutamate. The peptide is present in cultured cortical glia in high concentration and is hydrolyzed by cell surface peptidase activity. In the present work, we tested the hypothesis that NAAG selectively activates a subclass of metabotropic receptors on cultured rat cerebellar glia, primarily astrocytes. These glial cells express mRNA for mGluR subtypes 1, 3, 4, 5, 6, 7, and 8. We were not able to detect message for mGluR2 in these cells using the reverse transcriptase-polymerase chain reaction. Cerebellar glia responded to NAAG, glutamate, and trans-ACPD with a decrease in forskolin-stimulated cAMP formation. AP4, an agonist of the group III receptors mGluR4, mGluR6, mGluR7, and mGluR8, had little or no effect on stimulated cAMP levels. Treatment with low micromolar NAAG significantly increased uptake of radioactive thymidine by cultured astrocytes through activation of metabotropic glutamate receptors. Antagonists of group II mGluRs prevented the decrease in cAMP and the increase in uptake of thymidine by NAAG. Cultured cerebellar astrocytes expressed 20 pmol NAAG per mg protein, a value that is at least 30-fold lower than that expressed by mixed glial cultures prepared from mouse cortex. We conclude that cerebellar astrocytes respond to NAAG via the mGluR3 receptor and that the peptide may selectively activate this receptor subtype in astrocytes following release from neurons or glia.
- 7Wroblewska, B. NAAG as a Neurotransmitter. Adv. Exp. Med. Biol. 2006, 576, 317– 325, DOI: 10.1007/0-387-30172-0_23Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFygtrs%253D&md5=19e8133bd82cc9e117e85bcded7f9b2dNAAG as a neurotransmitterWroblewska, BarbaraAdvances in Experimental Medicine and Biology (2006), 576 (N-Acetylaspartate), 317-325CODEN: AEMBAP; ISSN:0065-2598. (Springer)A review. A review on the role of N-acetylaspartylglutamate (NAAG) as a neurotransmitter. It discusses NAAG in the context of pain models and schizophrenia.
- 8Romei, C.; Raiteri, M.; Raiteri, L. Glycine Release Is Regulated by Metabotropic Glutamate Receptors Sensitive to mGluR2/3 Ligands and Activated by N-Acetylaspartylglutamate (NAAG). Neuropharmacology 2013, 66, 311– 316, DOI: 10.1016/j.neuropharm.2012.05.030Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XoslGls7o%253D&md5=ab561f27a0d7ffa91fbacb727970ccffGlycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG)Romei, Cristina; Raiteri, Maurizio; Raiteri, LucaNeuropharmacology (2013), 66 (), 311-316CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [3H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY 379268 inhibited the K+-evoked overflow of [3H]glycine in a concn.-dependent manner (EC50 about 0.2 nM). The effect of LY 379268 was prevented by the selective mGluR2/3 antagonist LY 341495 (IC50 about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [3H]glycine overflow with extraordinary potency (EC50 about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of com. NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY 341495 antagonized the NAAG inhibition of [3H]glycine release. The effect of a combination of maximally effective concns. of LY 379268 and NAAG exhibited no additivity. The non-hydrolyzable NAAG analog N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY 379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacol.
- 9Ghose, S.; Wroblewska, B.; Corsi, L.; Grayson, D. R.; De Blas, A. L.; Vicini, S.; Neale, J. H. N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABA(A) alpha6 Subunit in Cerebellar Granule Cells. J. Neurochem. 1997, 69 (6), 2326– 2335, DOI: 10.1046/j.1471-4159.1997.69062326.xGoogle Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXntlKlsL8%253D&md5=271106d6c26559d0c3d6166eb0c66590N-Acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABAA α6 subunit in cerebellar granule cellGhose, Subroto; Wroblewska, Barbara; Corsi, Lorenzo; Grayson, Dennis R.; De Blas, Angel L.; Vicini, Stefano; Neale, Joseph H.Journal of Neurochemistry (1997), 69 (6), 2326-2335CODEN: JONRA9; ISSN:0022-3042. (Lippincott-Raven Publishers)We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 μM) results in the transient increase in content of GABAA α6 subunit mRNA. Using quant. PCR, this increase was detd. to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)- 2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors neg. linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABAB receptors. Forskolin stimulation of cAMP levels abolished the effect of NAAG. The change in α6 levels induced by 30 μM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concn.-dependent manner, indicating an increase in functional α6-contg. GABAA receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABAA α6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABAA receptor subunit compn.
- 10Thomas, A. G.; Liu, W.; Olkowski, J. L.; Tang, Z.; Lin, Q.; Lu, X. C.; Slusher, B. S. Neuroprotection Mediated by Glutamate Carboxypeptidase II (NAALADase) Inhibition Requires TGF-Beta. Eur. J. Pharmacol. 2001, 430 (1), 33– 40, DOI: 10.1016/S0014-2999(01)01239-0Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotFWiur8%253D&md5=7cc06bc1105eaa01d53d18302f6de90fNeuroprotection mediated by glutamate carboxypeptidase II (NAALADase) inhibition requires TGF-βThomas, Ajit G.; Liu, Weilin; Olkowski, Jennifer L.; Tang, Zhaocheng; Lin, Qian; Lu, Xi-Chun M.; Slusher, Barbara S.European Journal of Pharmacology (2001), 430 (1), 33-40CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier Science B.V.)Inhibition of glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), also termed N-acetylated alpha-linked acidic dipeptidase (NAALADase), has been shown to protect against ischemic injury presumably via decreasing glutamate and increasing N-acetyl-aspartyl-glutamate (NAAG). NAAG is a potent and selective mGlu3 receptor agonist. Activation of glial mGlu3 receptors has been shown to protect against NMDA toxicity by releasing transforming growth factors, TGF-βs. We hypothesized that GCP II inhibition could be neuroprotective also via TGF-βs, due to increased NAAG. To verify this, Enzyme-Linked Immunosorbent Assays (ELISAs) were performed on media from both control and ischemic cultures treated with the GCP II inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). We found that 2-PMPA attenuated ischemia-induced declines in TGF-β. To further assess the role of TGF-βs in 2-PMPA-mediated neuroprotection, a neutralizing antibody to TGF-β (TGF-β Ab) was used. In both in vitro and in vivo models of cerebral ischemia, TGF-β Ab reversed the neuroprotection by 2-PMPA. Antibodies to other growth factors had no effect. Data suggests that neuroprotection by GCP II inhibition may be partially mediated by promoting TGF-β release.
- 11Vornov, J. J.; Wozniak, K.; Lu, M.; Jackson, P.; Tsukamoto, T.; Wang, E.; Slusher, B. Blockade of NAALADase: A Novel Neuroprotective Strategy Based on Limiting Glutamate and Elevating NAAG. Ann. N.Y. Acad. Sci. 1999, 890, 400– 405, DOI: 10.1111/j.1749-6632.1999.tb08019.xGoogle Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnsVGhtQ%253D%253D&md5=93aa2b9a1609c5675a0ba5aea5a0d7eeBlockade of NAALADase: a novel neuroprotective strategy based on limiting glutamate and elevating NAAGVornov, James J.; Wozniak, Krystyna; Lu, May; Jackson, Paul; Tsukamoto, Takashi; Wang, Eric; Slusher, BarbaraAnnals of the New York Academy of Sciences (1999), 890 (Neuroprotective Agents), 400-405CODEN: ANYAA9; ISSN:0077-8923. (New York Academy of Sciences)Excessive glutamate receptor activation is thought to be involved in the neuronal injury caused by stroke. Based on the hypothesis that N-acetyl-aspartyl-glutamate (NAAG) is a modulatory neurotransmitter or storage form of glutamate, the authors have pursued a novel strategy of therapeutic intervention, blockade of N-acetylated alpha-linked acidic dipeptidase (NAALADase), the enzyme that hydrolyzes NAAG to liberate glutamate. Using the suture model of transient middle cerebral artery occlusion (MCAO) in rats, the prototype NAALADase inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) dramatically reduced extracellular glutamate accumulation measured by microdialysis both during a 2-h occlusion and during reperfusion, consistent with an effect on glutamate supply. During reperfusion, the decrease in glutamate was accompanied by an equimolar, reciprocal rise in extracellular NAAG. NAALADase inhibition may prove to be a well tolerated therapy for cerebral ischemia. In addn., NAALADase inhibitors should prove to be important tools in understanding the physiol. role of NAAG in the brain.
- 12Williams, A. J.; Lu, X. M.; Slusher, B.; Tortella, F. C. Electroencephalogram Analysis and Neuroprotective Profile of the N-Acetylated-Alpha-Linked Acidic Dipeptidase Inhibitor, GPI5232, in Normal and Brain-Injured Rats. J. Pharmacol. Exp. Ther. 2001, 299 (1), 48– 57Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXntFWltro%253D&md5=016872fdcd14c17504959e5b03601b31Electroencephalogram analysis and neuroprotective profile of the N-acetylated-α-linked acidic dipeptidase inhibitor, GPI5232, in normal and brain-injured ratsWilliams, A. J.; Lu, X. M.; Slusher, B.; Tortella, F. C.Journal of Pharmacology and Experimental Therapeutics (2001), 299 (1), 48-57CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)We have evaluated the effects of the N-acetylated-α-linked acidic dipeptidase (NAALADase) inhibitor, GPI5232 [2-([(pentafluorophenylmethyl)hydroxyphosphinyl]methyl)-pentanedioic acid], to not only decrease brain injury but also to alter the inherent electroencephalog. (EEG) changes obsd. in a rat model of transient middle cerebral artery occlusion (MCAo). Continuous i.v. infusion of GPI5232 starting 1 h after injury resulted in more than a 50% redn. in brain infarct vol. caused by 2 h of MCAo. This effect was dose-dependent and significant even when first treatment was delayed for 2 h post-MCAo. At 24 h post-MCAo, EEG spectral anal. of the injured hemisphere revealed functional improvement in GPI5232-treated rats. Significant recovery in high-frequency EEG power (8-30 Hz) was measured in GPI5232-treated animals in both parietal and temporal brain regions but not in vehicle-treated animals. MCAo-injured rats were also predisposed to developing cortical brain seizures, and GPI5232-treated rats had significantly fewer brain seizures than vehicle-treated animals. In sep. expts., acute high doses of GPI5232 in normal rats did not significantly alter EEG brain activity as evaluated by spectral anal. and did not produce any signs of seizure activity or behavioral abnormalities. These results show GPI5232 to be an effective neuroprotective treatment when given postinjury by reducing brain infarction and ameliorating the pathol. EEG assocd. with focal brain ischemia.
- 13Cai, Z.; Lin, S.; Rhodes, P. G. Neuroprotective Effects of N-Acetylaspartylglutamate in a Neonatal Rat Model of Hypoxia-Ischemia. Eur. J. Pharmacol. 2002, 437 (3), 139– 145, DOI: 10.1016/S0014-2999(02)01289-XGoogle Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhvVWrt7o%253D&md5=571c0efc81a95837323203319f9f9065Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemiaCai, Zhengwei; Lin, Shuying; Rhodes, Philip G.European Journal of Pharmacology (2002), 437 (3), 139-145CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier Science B.V.)Neuroprotective effects of N-acetylaspartylglutamate (NAAG), the precursor of glutamate and a selective agonist at the Group II metabotropic glutamate (mGlu) receptor, against hypoxic-ischemic brain injury were examd. in a neonatal rat model of cerebral hypoxia-ischemia. The neonatal hypoxia-ischemia procedure (unilateral carotid artery ligation followed by exposure to an 8% oxygen hypoxic condition for 1.5 h) was performed in 7-day-old rat pups. Following unilateral carotid artery ligation, NAAG (0.5 to 20 mg/kg, i.p.) was administered before or after the hypoxic exposure. Brain injury was examd. 1-wk later by wt. redn. in the ipsilateral brain and by neuron d. in the hippocampal CA1 area. In the saline-treated rat, neonatal hypoxia-ischemia resulted in severe brain injury as indicated by a 24% redn. in the ipsilateral brain wt. Low doses of NAAG (2-10 mg/kg, but not 0.5 mg/kg), administered before or even if 1 h after the hypoxic exposure, greatly reduced hypoxia-ischemia-induced brain injury (3.8-14.2% redn. in the ipsilateral brain wt.). A high dose of NAAG (20 mg/kg) was ineffective. While l(+)-2-Amino-4-phosphonobutyric acid (L-AP4) and trans-[1S,3R]-1-Amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) were unable to provide protection against hypoxic-ischemic brain injury, 2-(phosphonomethyl) pentanedioic acid (2-PMPA), an inhibitor of N-acetylated alpha-linked acidic dipeptidase (NAALADase), which hydrolyzes endogenous NAAG into N-acetyl-aspartate and glutamate, significantly reduced neonatal hypoxia-ischemia-induced brain injury. (αS)-α-Amino-α-[(1S, 2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), a selective antagonist at the mGlu2/3 receptor, prevented the neuroprotective effect of NAAG. Neuron d. data measured in the hippocampal CA1 area confirmed that ipsilateral brain wt. redn. was a valid measure for hypoxic-ischemic brain injury. Neonatal hypoxia-ischemia stimulated an elevation of cAMP concn. in the saline-treated rat brain. NAAG, L-AP4 and t-ACPD all significantly decreased hypoxia-ischemia-induced elevation of cAMP. LY341495 blocked the effect of NAAG, but not of L-AP4 or t-ACPD, on hypoxia-ischemia-stimulated cAMP elevation. The overall results suggest that the neuroprotective effect of NAAG is largely assocd. with activation of mGlu2/3 receptor.
- 14Zhong, C.; Zhao, X.; Sarva, J.; Kozikowski, A.; Neale, J. H.; Lyeth, B. G. NAAG Peptidase Inhibitor Reduces Acute Neuronal Degeneration and Astrocyte Damage Following Lateral Fluid Percussion TBI in Rats. J. Neurotrauma 2005, 22 (2), 266– 276, DOI: 10.1089/neu.2005.22.266Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2M%252Fos12gtw%253D%253D&md5=9213155428f974705aa703a2fe2871cfNAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in ratsZhong Chunlong; Zhao Xueren; Sarva Jayaprakash; Kozikowski Alan; Neale Joseph H; Lyeth Bruce GJournal of neurotrauma (2005), 22 (2), 266-76 ISSN:0897-7151.Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate associated with excitotoxicity and secondary brain pathology. The peptide neurotransmitter Nacetylaspartylglutamate (NAAG) suppresses glutamate transmission through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Thus, inhibition of NAAG peptidase activity and the prolong presence of synaptic NAAG were hypothesized to have significant potential for cellular protection following TBI. In the present study, a novel NAAG peptidase inhibitor, ZJ-43, was used in four different doses (0, 50, 100, or 150 mg/kg). Each dose was repeatedly administered i.p. (n=5/group) by multiple injections at three times (0 time, 8 h, 16 h) after moderate lateral fluid percussion TBI in the rat. An additional group was co-administered ZJ-43 (150 mg/kg) and the Group II mGluR antagonist, LY341495 (1 mg/kg), which was predicted to abolish any protective effects of ZJ-43. Rats were euthanized at 24 h after TBI, and brains were processed with a selective marker for degenerating neurons (Fluoro-Jade B) and a marker for astrocytes (GFAP). Ipsilateral neuronal degeneration and bilateral astrocyte loss in the CA2/3 regions of the hippocampus were quantified using stereological techniques. Compared with vehicle, ZJ-43 significantly reduced the number of the ipsilateral degenerating neurons (p<0.01) with the greatest neuroprotection at the 50 mg/kg dose. Moreover, LY341495 successfully abolished the protective effects of ZJ-43. 50 mg/kg of ZJ-43 also significantly reduced the ipsilateral astrocyte loss (p<0.05). We conclude that the NAAG peptidase inhibitor ZJ-43 is a potential novel strategy to reduce both neuronal and astrocyte damage associated with the glutamate excitotoxicity after TBI.
- 15Ghadge, G. D.; Slusher, B. S.; Bodner, A.; Canto, M. D.; Wozniak, K.; Thomas, A. G.; Rojas, C.; Tsukamoto, T.; Majer, P.; Miller, R. J.; Monti, A. L.; Roos, R. P. Glutamate Carboxypeptidase II Inhibition Protects Motor Neurons from Death in Familial Amyotrophic Lateral Sclerosis Models. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (16), 9554– 9559, DOI: 10.1073/pnas.1530168100Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmtlyku7k%253D&md5=9142ca962162bd94ee0d25e0bf3f1516Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis modelsGhadge, Ghanashyam D.; Slusher, Barbara S.; Bodner, Amos; Dal Canto, Mauro; Wozniak, Krystyna; Thomas, Ajit G.; Rojas, Camilo; Tsukamoto, Takashi; Majer, Pavel; Miller, Richard J.; Monti, Anna Liza; Roos, Raymond P.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (16), 9554-9559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Approx. 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and ≈ 20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.
- 16Yamamoto, T.; Kozikowski, A.; Zhou, J.; Neale, J. H. Intracerebroventricular Administration of N-Acetylaspartylglutamate (NAAG) Peptidase Inhibitors Is Analgesic in Inflammatory Pain. Mol. Pain 2008, 4, 31, DOI: 10.1186/1744-8069-4-31Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1crit1Cqug%253D%253D&md5=d4b7128c2e8ad234ac923de798c3da4cIntracerebroventricular administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory painYamamoto Tatsuo; Kozikowski Alan; Zhou Jia; Neale Joseph HMolecular pain (2008), 4 (), 31 ISSN:.BACKGROUND: The peptide neurotransmitter N-Acetylaspartylglutamate (NAAG) is the third most prevalent transmitter in the mammalian central nervous system. Local, intrathecal and systemic administration of inhibitors of enzymes that inactivate NAAG decrease responses to inflammatory pain in rat models. Consistent with NAAG's activation of group II metabotropic glutamate receptors, this analgesia is blocked by a group II antagonist. RESULTS: This research aimed at determining if analgesia obtained following systemic administration of NAAG peptidase inhibitors is due to NAAG activation of group II mGluRs in brain circuits that mediate perception of inflammatory pain. NAAG and NAAG peptidase inhibitors, ZJ43 and 2-PMPA, were microinjected into a lateral ventricle prior to injection of formalin in the rat footpad. Each treatment reduced the early and late phases of the formalin-induced inflammatory pain response in a dose-dependent manner. The group II mGluR antagonist reversed these analgesic effects consistent with the conclusion that analgesia was mediated by increasing NAAG levels and the peptide's activation of group II receptors. CONCLUSION: These data contribute to proof of the concept that NAAG peptidase inhibition is a novel therapeutic approach to inflammatory pain and that these inhibitors achieve analgesia by elevating synaptic levels of NAAG within pain processing circuits in brain.
- 17Yamamoto, T.; Saito, O.; Aoe, T.; Bartolozzi, A.; Sarva, J.; Zhou, J.; Kozikowski, A.; Wroblewska, B.; Bzdega, T.; Neale, J. H. Local Administration of N-Acetylaspartylglutamate (NAAG) Peptidase Inhibitors Is Analgesic in Peripheral Pain in Rats. Eur. J. Neurosci. 2007, 25 (1), 147– 158, DOI: 10.1111/j.1460-9568.2006.05272.xGoogle Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s%252Fkt1Skug%253D%253D&md5=0dc9a94990e727813453ae3aba93dae7Local administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in ratsYamamoto Tatsuo; Saito Osamu; Aoe Tomohiko; Bartolozzi Alessandra; Sarva Jayaprakash; Zhou Jia; Kozikowski Alan; Wroblewska Barbara; Bzdega Tomasz; Neale Joseph HThe European journal of neuroscience (2007), 25 (1), 147-58 ISSN:0953-816X.The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) selectively activates group II metabotropic glutamate receptors (mGluRs). Systemic administration of inhibitors of the enzymes that inactivate NAAG results in decreased pain responses in rat models of inflammatory and neuropathic pain. These effects are blocked by a group II mGluR antagonist. This research tested the hypothesis that some analgesic effects of NAAG peptidase inhibition are mediated by NAAG acting on sensory neurite mGluRs at the site of inflammation. Group II mGluR agonists, SLx-3095-1, NAAG and APDC, or NAAG peptidase inhibitors, ZJ-43 and 2-PMPA, injected into the rat footpad reduced pain responses in carrageenan or formalin models. The analgesic effects of SLx-3095-1, APDC, ZJ-43, 2-PMPA and NAAG were blocked by co-injection of LY341495, a selective group II mGluR antagonist. Injection of group II mGluR agonists, NAAG or the peptidase inhibitors into the contralateral rat footpad had no effect on pain perception in the injected paw. At 10-100 microm ZJ-43 and 2-PMPA demonstrated no consistent agonist activity at mGluR2 or mGluR3. Consistent with the conclusion that peripherally administered NAAG peptidase inhibitors increase the activation of mGluR3 by NAAG that is released from peripheral sensory neurites, we found that the tissue average concentration of NAAG in the unstimulated rat hind paw was about 6 microm. These data extend our understanding of the role of this peptide in sensory neurons and reveal the potential for treatment of inflammatory pain via local application of NAAG peptidase inhibitors at doses that may have little or no central nervous system effects.
- 18Zhang, W.; Murakawa, Y.; Wozniak, K. M.; Slusher, B.; Sima, A. A. F. The Preventive and Therapeutic Effects of GCPII (NAALADase) Inhibition on Painful and Sensory Diabetic Neuropathy. J. Neurol. Sci. 2006, 247 (2), 217– 223, DOI: 10.1016/j.jns.2006.05.052Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpsVKksrY%253D&md5=31c8dadecc4fb2c34346f4fbc1c38e7fThe preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathyZhang, W.; Murakawa, Y.; Wozniak, K. M.; Slusher, B.; Sima, A. A. F.Journal of the Neurological Sciences (2006), 247 (2), 217-223CODEN: JNSCAG; ISSN:0022-510X. (Elsevier B.V.)Excitotoxic glutamate release occurs in several neurol. disorders. One source is derived from the hydrolysis of the neuropeptide N-acetyl aspartyl glutamate (NAAG) by glutamate carboxypeptidase II (GCPII, also known as NAALADase). Drugs that attenuate glutamate transmission have been shown to relieve neuropathic pain, however side effects have limited their clin. use. It appears that GCPII is exclusively recruited to provide a glutamate source in hyperglutamatergic, excitotoxic conditions and therefore would be devoid of such side effects. Here we report on the therapeutic effects of an orally bio-available GCP II inhibitor on established painful and sensory neuropathy in the spontaneously diabetic BB/Wor rat. It significantly improved hyperalgesia, nerve conduction velocity and underlying myelinated fiber atrophy. The data suggest that GCP II inhibition may provide a meaningful and effective approach to the treatment of painful diabetic neuropathy.
- 19Wozniak, K. M.; Wu, Y.; Vornov, J. J.; Lapidus, R.; Rais, R.; Rojas, C.; Tsukamoto, T.; Slusher, B. S. The Orally Active Glutamate Carboxypeptidase II Inhibitor E2072 Exhibits Sustained Nerve Exposure and Attenuates Peripheral Neuropathy. J. Pharmacol. Exp. Ther. 2012, 343 (3), 746– 754, DOI: 10.1124/jpet.112.197665Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslyjs7zN&md5=eb9a44606e863472a46b5b6ce9eca88dThe orally active glutamate carboxypeptidase II inhibitor E2072 exhibits sustained nerve exposure and attenuates peripheral neuropathyWozniak, Krystyna M.; Wu, Ying; Vornov, James J.; Lapidus, Rena; Rais, Rana; Rojas, Camilo; Tsukamoto, Takashi; Slusher, Barbara S.Journal of Pharmacology and Experimental Therapeutics (2012), 343 (3), 746-754CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)Peripheral neuropathy from nerve trauma is a significant problem in the human population and often constitutes a dose-limiting toxicity in patients receiving chemotherapy. (3-2-Mercaptoethyl)biphenyl-2,3-dicarboxylic acid (E2072) is a potent (Ki = 10 nM), selective, and orally available inhibitor of glutamate carboxypeptidase II (GCPII). Here, we report that E2072 attenuates hyperalgesia and nerve conduction velocity deficits in preclin. rodent models of neuropathic pain and oxaliplatin-induced neuropathy. In the chronic constrictive injury model, orally administered E2072 reversed pre-existing thermal hyperalgesia in rats in a dose-dependent fashion with a minimally ED of 0.1 mg/kg/day. It is noteworthy that multiple days of dosing of E2072 were required before analgesia was realized even though GCPII inhibitory exposures were achieved on the 1st day of dosing. In addn., analgesia was found to persist for ≤ 7 days after cessation of dosing, consistent with E2072's pharmacokinetic profile and sustained exposure. Furthermore, in a chronic oxaliplatin-induced neuropathy model (6 mg/kg i.p. oxaliplatin twice weekly for 4 wk), female BALB/c mice receiving daily oral E2072 at 1.0 and 0.1 mg/kg displayed no deficits in either caudal or digital velocity compared with significant deficits obsd. in mice treated with oxaliplatin alone (12 and 9%, resp.). Similar findings were seen with oxaliplatin-induced digital and caudal amplitude deficits. It is noteworthy that E2072 showed no interference with the antineoplastic efficacy of oxaliplatin in mice bearing leukemia (L1210), even at doses 100 times its neuroprotective/analgesic dose, indicating a selective effect on neuropathy. These data support the therapeutic utility of GCPII inhibitors in neuropathy and neuropathic pain.
- 20Olszewski, R. T.; Bukhari, N.; Zhou, J.; Kozikowski, A. P.; Wroblewski, J. T.; Shamimi-Noori, S.; Wroblewska, B.; Bzdega, T.; Vicini, S.; Barton, F. B.; Neale, J. H. NAAG Peptidase Inhibition Reduces Locomotor Activity and Some Stereotypes in the PCP Model of Schizophrenia via Group II mGluR. J. Neurochem. 2004, 89 (4), 876– 885, DOI: 10.1111/j.1471-4159.2004.02358.xGoogle Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXksVyksLg%253D&md5=4ca607a68135b77ae0cd997eed5d2566NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluROlszewski, Rafal T.; Bukhari, Noreen; Zhou, Jia; Kozikowski, Alan P.; Wroblewski, Jarda T.; Shamimi-Noori, Susan; Wroblewska, Barbara; Bzdega, Tomasz; Vicini, Stefano; Barton, Franca B.; Neale, Joseph H.Journal of Neurochemistry (2004), 89 (4), 876-885CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)Phencyclidine (PCP) administration elicits pos. and neg. symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochem. effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. The authors synthesized and characterized a urea-based NAAG analog, ZJ43. This novel compd. is a potent inhibitor of enzymes, glutamate carboxypeptidase II (Ki = 0.8 nM) and III (Ki = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 μM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Addnl., LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.
- 21Profaci, C. P.; Krolikowski, K. A.; Olszewski, R. T.; Neale, J. H. Group II mGluR Agonist LY354740 and NAAG Peptidase Inhibitor Effects on Prepulse Inhibition in PCP and D-Amphetamine Models of Schizophrenia. Psychopharmacology 2011, 216 (2), 235– 243, DOI: 10.1007/s00213-011-2200-0Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFKltLo%253D&md5=486c472ac2b6bd6a95da455150ef7576Group II mGluR agonist LY354740 and NAAG peptidase inhibitor effects on prepulse inhibition in PCP and D-amphetamine models of schizophreniaProfaci, Caterina P.; Krolikowski, Kristyn A.; Olszewski, Rafal T.; Neale, Joseph H.Psychopharmacology (Heidelberg, Germany) (2011), 216 (2), 235-243CODEN: PSCHDL; ISSN:0033-3158. (Springer)Rationale: Group II metabotropic glutamate receptor (mGluR) agonists represent a novel approach to the treatment of schizophrenia. Inasmuch as the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) activates these receptors, NAAG peptidase inhibitors conceptually represent a parallel path toward development of new antipsychotic drugs. While group II agonists are effective in several animal models of schizophrenia, they are reported to lack efficacy in moderating the effects of phencyclidine (PCP) on prepulse inhibition of acoustic startle in animal models of sensory processing deficits found in this disorder. Objective: The objective of this study was to re-examine the efficacy of a group II metabotropic glutamate agonist and NAAG peptidase inhibitors in prepulse inhibition models of schizophrenia across two strains of mice. Methods: The method used was an assay to det. the efficacy of these drugs in moderating the redn. in prepulse inhibition of acoustic startle in mice treated with PCP and D-amphetamine. Results: The group II agonist LY354740 (5 and 10 mg/kg) moderated the effects of PCP on prepulse inhibition of acoustic startle in DBA/2 but not C57BL/6 mice. In contrast, two NAAG peptidase inhibitors, ZJ43 (150 mg/kg) and 2-PMPA (50, 100, and 150 mg/kg), did not significantly affect the PCP-induced redn. in prepulse inhibition in either strain. Conclusions: These data demonstrate that the efficacy of group II agonists in this model of sensory motor processing is strain-specific in mice. The difference between the effects of the group II agonist and the peptidase inhibitors in the DBA/2 mice may relate to the difference in efficacy of NAAG and the agonist at mGluR2.
- 22Tsai, G.; Dunham, K. S.; Drager, U.; Grier, A.; Anderson, C.; Collura, J.; Coyle, J. T. Early Embryonic Death of Glutamate Carboxypeptidase II (NAALADase) Homozygous Mutants. Synapse 2003, 50 (4), 285– 292, DOI: 10.1002/syn.10263Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVyhtLc%253D&md5=2d05eeef754d6103e4a2e2fdfa25251aEarly embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutantsTsai, G.; Dunham, K. S.; Drager, U.; Grier, A.; Anderson, C.; Collura, J.; Coyle, J. T.Synapse (New York, NY, United States) (2003), 50 (4), 285-292CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss, Inc.)Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 μM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.
- 23Han, L.; Picker, J. D.; Schaevitz, L. R.; Tsai, G.; Feng, J.; Jiang, Z.; Chu, H. C.; Basu, A. C.; Berger-Sweeney, J.; Coyle, J. T. Phenotypic Characterization of Mice Heterozygous for a Null Mutation of Glutamate Carboxypeptidase II. Synapse 2009, 63 (8), 625– 635, DOI: 10.1002/syn.20649Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnslGisL8%253D&md5=2bdc02c54d27d9f4a903c4474ebb0170Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase IIHan, Liqun; Picker, Jonathan D.; Schaevitz, Laura R.; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C.; Basu, Alo C.; Berger-Sweeney, Joanne; Coyle, Joseph T.Synapse (Hoboken, NJ, United States) (2009), 63 (8), 625-635CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss, Inc.)Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of N-methyl-D-aspartate receptors (NMDAR) was implicated in the pathophysiol. of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-α L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate. NAAG is a neuropeptide that is an NMDAR antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDAR activation. To manipulate the expression of GCP II, LoxP sites were inserted flanking exons 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% redn. in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N > 200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm the authors' hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory.
- 24Vorlová, B.; Sedlák, F.; Kašpárek, P.; Šrámková, K.; Malý, M.; Zámečník, J.; Šácha, P.; Konvalinka, J. A Novel PSMA/GCPII-Deficient Mouse Model Shows Enlarged Seminal Vesicles upon Aging. Prostate 2019, 79 (2), 126– 139, DOI: 10.1002/pros.23717Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1Sgu77P&md5=a8219bb4f4f46aaba0d2ef8cb095ddd0A novel PSMA/GCPII-deficient mouse model shows enlarged seminal vesicles upon agingVorlova, Barbora; Sedlak, Frantisek; Kasparek, Petr; Sramkova, Karolina; Maly, Marek; Zamecnik, Josef; Sacha, Pavel; Konvalinka, JanProstate (Hoboken, NJ, United States) (2019), 79 (2), 126-139CODEN: PRSTDS; ISSN:0270-4137. (Wiley-Blackwell)Background : Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is an important diagnostic and therapeutic target in prostate cancer. PSMA/GCPII is also expressed in many healthy tissues, but its function has only been established in the brain and small intestine. Several research groups have attempted to produce PSMA/GCPII-deficient mice to study the physiol. role of PSMA/GCPII in detail. The outcomes of these studies differ dramatically, ranging from embryonic lethality to prodn. of viable PSMA/GCPII-deficient mice without any obvious phenotype. Methods : We produced PSMA/GCPII-deficient mice (hereafter also referred as Folh1-/- mice) by TALEN-mediated mutagenesis on a C57BL/6NCrl background. Using Western blot and an enzyme activity assay, we confirmed the absence of PSMA/GCPII in our Folh1-/- mice. We performed anatomical and histopathol. examn. of selected tissues with a focus on urogenital system. We also examd. the PSMA/GCPII expression profile within the mouse urogenital system using an enzyme activity assay and confirmed the presence of PSMA/GCPII in selected tissues by immunohistochem. Results : Our Folh1-/- mice are viable, breed normally, and do not show any obvious phenotype. Nevertheless, aged Folh1-/- mice of 69-72 wk exhibit seminal vesicle dilation, which is caused by accumulation of luminal fluid. This phenotype was also obsd. in Folh1+/- mice; the overall difference between our three cohorts (Folh1-/-, Folh1+/-, and Folh1+/+) was highly significant (P < 0.002). Of all studied tissues of the mouse urogenital system, only the epididymis appeared to have a physiol. relevant level of PSMA/GCPII expression. Addnl. expts. demonstrated that PSMA/GCPII is also present in the human epididymis. Conclusions : In this study, we provide the first evidence characterizing the reproductive tissue phenotype of PSMA/GCPII-deficient mice. These findings will help lay the groundwork for future studies to reveal PSMA/GCPII function in human reprodn.
- 25Bacich, D. J.; Ramadan, E.; O’Keefe, D. S.; Bukhari, N.; Wegorzewska, I.; Ojeifo, O.; Olszewski, R.; Wrenn, C. C.; Bzdega, T.; Wroblewska, B.; Heston, W. D. W.; Neale, J. H. Deletion of the Glutamate Carboxypeptidase II Gene in Mice Reveals a Second Enzyme Activity That Hydrolyzes N-Acetylaspartylglutamate. J. Neurochem. 2002, 83 (1), 20– 29, DOI: 10.1046/j.1471-4159.2002.01117.xGoogle Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnvVGgsL0%253D&md5=34967d94f7bade511355a412218eacb6Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamateBacich, Dean J.; Ramadan, Epolia; O'Keefe, Denise S.; Bukhari, Noreen; Wegorzewska, Iga; Ojeifo, Olumide; Olszewski, Rafal; Wrenn, Craige C.; Bzdega, Tomasz; Wroblewska, Barbara; Heston, Warren D. W.; Neale, Joseph H.Journal of Neurochemistry (2002), 83 (1), 20-29CODEN: JONRA9; ISSN:0022-3042. (Blackwell Science Ltd.)Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face of glia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. MRNA for GCPII was not detected by northern blotting or RT-PCR anal. of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurol. responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were obsd. among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII null mutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nM and 2494 nM, resp. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nM for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned rat and human GCPII, and 88 nM for the activity in brain membranes of the null mutants.
- 26Gao, Y.; Xu, S.; Cui, Z.; Zhang, M.; Lin, Y.; Cai, L.; Wang, Z.; Luo, X.; Zheng, Y.; Wang, Y.; Luo, Q.; Jiang, J.; Neale, J. H.; Zhong, C. Mice Lacking Glutamate Carboxypeptidase II Develop Normally, but Are Less Susceptible to Traumatic Brain Injury. J. Neurochem. 2015, 134 (2), 340– 353, DOI: 10.1111/jnc.13123Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntFCktb0%253D&md5=6fb001f691d426cf112a265a8aa5a27eMice lacking glutamate carboxypeptidase II develop normally, but are less susceptible to traumatic brain injuryGao, Yang; Xu, Siyi; Cui, Zhenwen; Zhang, Mingkun; Lin, Yingying; Cai, Lei; Wang, Zhugang; Luo, Xingguang; Zheng, Yan; Wang, Yong; Luo, Qizhong; Jiang, Jiyao; Neale, Joseph H.; Zhong, ChunlongJournal of Neurochemistry (2015), 134 (2), 340-353CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia-bound GCPII mediates the hydrolysis of the neurotransmitter N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity assocd. with enhanced glutamate transmission under pathol. conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3-5 of GCPII. This generated a new GCPII KO mice line with no overt differences in std. neurol. behavior compared to their wild-type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addn., GCPII gene KO reduced TBI-induced deficits in long-term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathol. protection with improved long-term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) suppresses glutamate transmission through selective activation of pre-synaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3) after traumatic brain injury (TBI). However, synaptically released NAAG is hydrolyzed to form N-acetylaspartate and glutamate mainly by Glutamate carboxypeptidase II (GCPII), losing neuroprotective effect. In this study, we found that knock out of the GCPII gene is not embryonic lethal and affords histopathol. protection with improved long-term behavioral outcomes after TBI.
- 27Bacich, D. J.; Wozniak, K. M.; Lu, X.-C. M.; O’Keefe, D. S.; Callizot, N.; Heston, W. D. W.; Slusher, B. S. Mice Lacking Glutamate Carboxypeptidase II Are Protected from Peripheral Neuropathy and Ischemic Brain Injury. J. Neurochem. 2005, 95 (2), 314– 323, DOI: 10.1111/j.1471-4159.2005.03361.xGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFegu7rE&md5=5e91a9605c262a68d2468a961abc517cMice lacking glutamate carboxypeptidase II are protected from peripheral neuropathy and ischemic brain injuryBacich, Dean J.; Wozniak, Krystyna M.; Lu, X.-C. May; O'Keefe, Denize S.; Callizot, Noelle; Heston, Warren D. W.; Slusher, Barbara S.Journal of Neurochemistry (2005), 95 (2), 314-323CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)Excessive glutamate release is assocd. with neuronal damage. A new strategy for the treatment of neuronal injury involves inhibition of the neuropeptidase glutamate carboxypeptidase II (GCP II), also known as N-acetylated α-linked acidic dipeptidase. GCP II is believed to mediate the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to glutamate and N-acetyl-aspartate, and inhibition of NAAG peptidase activity (by GCP II and other peptidases) is neuroprotective. Mice were generated in which the Folh1 gene encoding GCP II was disrupted (Folh1-/- mice). No overt behavioral differences were apparent between Folh1-/- mice and wild-type littermates, with respect to their overall performance in locomotion, coordination, pain threshold, cognition and psychiatric behavioral paradigms. Morphol. anal. of peripheral nerves, however, showed significantly smaller axons (reduced myelin sheaths and axon diams.) in sciatic nerves from Folh1-/- mice. Following sciatic nerve crush, Folh1-/- mice suffered less injury and recovered faster than wild-type littermates. In a model of ischemic injury, the Folh1-/- mice exhibited a significant redn. (p < 0.05) in infarct vol. compared with their wild-type littermates when subjected to middle cerebral artery occlusion, a model of stroke. These findings support the hypothesis that GCP II inhibitors may represent a novel treatment for peripheral neuropathies as well as stroke.
- 28Wolf, N. I.; Willemsen, M. A. A. P.; Engelke, U. F.; van der Knaap, M. S.; Pouwels, P. J. W.; Harting, I.; Zschocke, J.; Sistermans, E. A.; Rating, D.; Wevers, R. A. Severe Hypomyelination Associated with Increased Levels of N-Acetylaspartylglutamate in CSF. Neurology 2004, 62 (9), 1503– 1508, DOI: 10.1212/01.WNL.0000123094.13406.20Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjt1ylur8%253D&md5=4563458706ff7d724a544db894089d25Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSFWolf, N. I.; Willemsen, M. A. A. P.; Engelke, U. F.; van der Knaap, M. S.; Pouwels, P. J. W.; Harting, I.; Zschocke, J.; Sistermans, E. A.; Rating, D.; Wevers, R. A.Neurology (2004), 62 (9), 1503-1508CODEN: NEURAI; ISSN:0028-3878. (Lippincott Williams & Wilkins)Two unrelated girls had early onset of nystagmus and epilepsy, absent psychomotor development, and almost complete absence of myelin on cerebral MRI. The clin. features and MR images of both patients resembled the connatal form of Pelizaeus-Merzbacher disease (PMD), which is an X-linked recessive disorder caused by duplications or mutations of the proteolipid protein gene (PLP). Thus, studies were carried out to define a unique neurometabolic disorder with failure of myelination. 1H-NMR of CSF in both girls was performed repeatedly, and both showed highly elevated concns. of N-acetylaspartylglutamate (NAAG). The coding sequence of the gene coding for glutamate carboxypeptidase II, which converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced but revealed no mutations. Even though both patients are girls, the authors sequenced the PLP gene and found no abnormality. Thus, NAAG is an abundant peptide neurotransmitter whose exact role is unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its elevated concn. in CSF may be the biochem. hallmark for a novel neurometabolic disorder. The cause of its accumulation is still unclear.
- 29Sartori, S.; Burlina, A. B.; Salviati, L.; Trevisson, E.; Toldo, I.; Laverda, A. M.; Burlina, A. P. Increased Level of N-Acetylaspartylglutamate (NAAG) in the CSF of a Patient with Pelizaeus-Merzbacher-like Disease due to Mutation in the GJA12 Gene. Eur. J. Paediatr. Neurol. 2008, 12 (4), 348– 350, DOI: 10.1016/j.ejpn.2007.07.011Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1czjt1Gqsg%253D%253D&md5=b6195426fa7491ae67c04a915496b40fIncreased level of N-acetylaspartylglutamate (NAAG) in the CSF of a patient with Pelizaeus-Merzbacher-like disease due to mutation in the GJA12 geneSartori Stefano; Burlina Alberto B; Salviati Leonardo; Trevisson Eva; Toldo Irene; Laverda Anna Maria; Burlina Alessandro PEuropean journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society (2008), 12 (4), 348-50 ISSN:1090-3798.Autosomal recessive Pelizaeus-Merzbacher-like disease 1 (PMLD1) is a hypomyelinating disorder of the central nervous system (CNS) with virtually identical phenotype to Pelizaeus-Merzbacher disease (PMD). PMLD1 is caused by mutations in GJA12 gene, PMD is due to mutations in PLP1 gene. Elevated levels of N-acetylaspartylglutamate (NAAG), the most abundant peptide neuromodulator in the human brain, have been recently reported in cerebral spinal fluid (CSF) of patients with PMD. Using capillary electrophoresis, we analyzed for the first time, the CSF from a girl with PMLD1 and detected high concentrations of NAAG. This finding confirms the hypothesis that NAAG may be involved in myelination-related processes and can be considered as a useful diagnostic marker not only for patients with the PLP1 related disorder, but also in those with Pelizaeus-Merzbacher like hypomyelinating disease due to other defined genetic causes, such as PMLD1.
- 30Mochel, F.; Boildieu, N.; Barritault, J.; Sarret, C.; Eymard-Pierre, E.; Seguin, F.; Schiffmann, R.; Boespflug-Tanguy, O. Elevated CSF N-Acetylaspartylglutamate Suggests Specific Molecular Diagnostic Abnormalities in Patients with White Matter Diseases. Biochim. Biophys. Acta 2010, 1802 (11), 1112– 1117, DOI: 10.1016/j.bbadis.2010.07.005Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFynsbzE&md5=bf840c684afd421eea48d5022e32324bElevated CSF N-acetylaspartylglutamate suggests specific molecular diagnostic abnormalities in patients with white matter diseasesMochel, Fanny; Boildieu, Nadege; Barritault, Julie; Sarret, Catherine; Eymard-Pierre, Eleonore; Seguin, Francois; Schiffmann, Raphael; Boespflug-Tanguy, OdileBiochimica et Biophysica Acta, Molecular Basis of Disease (2010), 1802 (11), 1112-1117CODEN: BBADEX; ISSN:0925-4439. (Elsevier B. V.)Background: In order to identify biomarkers useful for the diagnosis of genetic white matter disorders we compared the metabolic profile of patients with leukodystrophies with a hypomyelinating or a non-hypomyelinating MRI pattern. Methods: We used a non-a priori method of in vitro 1H-NMR spectroscopy on CSF samples of 74 patients with leukodystrophies. Results: We found an elevation of CSF N-acetylaspartylglutamate (NAAG) in patients with Pelizaeus-Merzbacher disease (PMD)-PLP1 gene, Pelizaeus-Merzbacher-like disease-GJC2 gene and Canavan disease-ASPA gene. In the PMD group, NAAG was significantly elevated in the CSF of all patients with PLP1 duplication (19/19) but was strictly normal in 6 out of 7 patients with PLP1 point mutations. Addnl., we previously reported increased CSF NAAG in patients with SLC17A5 mutations. Conclusions: Elevated CSF NAAG is a biomarker that suggests specific mol. diagnostic abnormalities in patients with white matter diseases. Our findings also point to unique pathol. functions of the overexpressed PLP in PMD patients with duplication of this gene.
- 31Francis, J. S.; Wojtas, I.; Markov, V.; Gray, S. J.; McCown, T. J.; Samulski, R. J.; Bilaniuk, L. T.; Wang, D.-J.; De Vivo, D. C.; Janson, C. G.; Leone, P. N-Acetylaspartate Supports the Energetic Demands of Developmental Myelination via Oligodendroglial Aspartoacylase. Neurobiol. Dis. 2016, 96, 323– 334, DOI: 10.1016/j.nbd.2016.10.001Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1GqsbbL&md5=de7a4741302cd62044397511a367a303N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylaseFrancis, Jeremy S.; Wojtas, Ireneusz; Markov, Vladimir; Gray, Steven J.; McCown, Thomas J.; Samulski, R. Jude; Bilaniuk, Larissa T.; Wang, Dah-Jyuu; De Vivo, Darryl C.; Janson, Christopher G.; Leone, PaolaNeurobiology of Disease (2016), 96 (), 323-334CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)Breakdown of neuro-glial N-acetyl-aspartate (NAA) metab. results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathol. progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-assocd. virus vector (AAV-Olig001) to quant. assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathol. and deteriorating motor function. Long-term effects included increased oligodendrocyte nos., a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy obsd. following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile obsd. in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.
- 32Burri, R.; Steffen, C.; Herschkowitz, N. N-Acetyl-L-Aspartate Is a Major Source of Acetyl Groups for Lipid Synthesis during Rat Brain Development. Dev. Neurosci. 1991, 13 (6), 403– 411, DOI: 10.1159/000112191Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhvFWlsrc%253D&md5=aeed7936ea7e3ffc688b3ee058005cdeN-acetyl-L-aspartate is a major source of acetyl groups for lipid synthesis during rat brain developmentBurri, R.; Steffen, C.; Herschkowitz, N.Developmental Neuroscience (Basel, Switzerland) (1991), 13 (6), 403-11CODEN: DENED7; ISSN:0378-5866.The function of N-acetyl-L-aspartate (NAA), a predominant substance in the central nervous system, has not yet been detd. To investigate the possible function of NAA as a lipid precursor [14C]-N-acetyl-L-aspartate (NAA) or [14C]-acetate (AcA) was injected intracerebrally into 8, 15, and 22-day-old rats. These time points were selected because NAA concn. and the activity of the NAA-synthesizing enzyme L-aspartate-N-acetyltransferase (ANAT), were low in 8-day-old rats, intermediate in 15-day-old rats, and high in 22-day-old rats. During an incubation period of 4 h the radioactive acetyl group of NAA is incorporated into the lipid fraction in amts. of 42.9 to 65.7% of recovered total radioactivity, increasing with the age of the rats. In contrast, radioactivity incorporated from AcA is const. for all three ages. With NAA as precursor only 7.2-9.4% of the recovered total radioactivity is incorporated into the protein fraction. With AcA as precursor 27.0-18.1% of recovered radioactivity is incorporated into the protein fraction, the amts. decreasing with age. Taking into account that the in vivo NAA concn. in the brain is much higher than the AcA concn., NAA is clearly the more efficient precursor for lipid synthesis than AcA. Further, the authors compared NAA and AcA as lipid precursors by analyzing the radioactivity in single lipid fractions, expressed as normalized specific incorporation or normalized incorporation. The measured differences between NAA and AcA in normalized specific and normalized incorporation of acetyl groups imply that NAA is not simply degraded to AcA before incorporated into lipids. Apparently, NAA is a major source of acetyl groups for lipid synthesis during rat brain development.
- 33Singhal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.; McDonough, J. The Neuronal Metabolite NAA Regulates Histone H3Methylation in Oligodendrocytes and Myelin Lipid Composition. Exp. Brain Res. 2017, 235 (1), 279– 292, DOI: 10.1007/s00221-016-4789-zGoogle Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1GqsrnE&md5=2e50668708e3d39accb9f3c586da4fd7The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid compositionSinghal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.; McDonough, J.Experimental Brain Research (2017), 235 (1), 279-292CODEN: EXBRAP; ISSN:0014-4819. (Springer)The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metab. of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liq. chromatog.-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatog. (HPTLC) to identify changes in myelin lipid compn. in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amts. of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic anal. of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metab., and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was assocd. with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.
- 34Salji, M. J.; Blomme, A.; Däbritz, J. H. M.; Repiscak, P.; Lilla, S.; Patel, R.; Sumpton, D.; van den Broek, N. J. F.; Daly, R.; Zanivan, S.; Leung, H. Y. Multi-Omics & Pathway Analysis Identify Potential Roles for Tumor N-Acetyl Aspartate Accumulation in Murine Models of Castration-Resistant Prostate Cancer. iScience 2022, 25 (4), 104056 DOI: 10.1016/j.isci.2022.104056Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVOrtbfL&md5=05cd009b1f8ba6285bd9b5f7697e1e71Multi-omics & pathway analysis identify potential roles for tumor N-acetyl aspartate accumulation in murine models of castration-resistant prostate cancerSalji, Mark J.; Blomme, Arnaud; Dabritz, J. Henry M.; Repiscak, Peter; Lilla, Sergio; Patel, Rachana; Sumpton, David; van den Broek, Niels J. F.; Daly, Ronan; Zanivan, Sara; Leung, Hing Y.iScience (2022), 25 (4), 104056CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Castration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge (Oakes and Papa, 2015). Matched untargeted multi-level omic datasets may reveal biol. changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics were performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node, and bone metastasis grown as murine orthografts. Collectively, we tested the feasibility of muti-omics anal. on models of CRPC in revealing pathways of interest for future validation investigation. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-acetylated alpha-linked acidic dipeptidases (FOLH1/NAALADL2). Based on pathway anal. integrating multiple omic levels, we hypothesize that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of acetyl Co-A for upregulated sphingolipid metab. and a pool of glutamate and aspartate for nucleotide synthesis during tumor growth.
- 35Jones, S. R.; Carley, S.; Harrison, M. An Introduction to Power and Sample Size Estimation. Emerg. Med. J. 2003, 20 (5), 453– 458, DOI: 10.1136/emj.20.5.453Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3svhsFarug%253D%253D&md5=9bf6b9765fb32466de3c9a5c91d423ceAn introduction to power and sample size estimationJones S R; Carley S; Harrison MEmergency medicine journal : EMJ (2003), 20 (5), 453-8 ISSN:.The importance of power and sample size estimation for study design and analysis.
- 36Rodriguez-Navas, C.; Morselli, E.; Clegg, D. J. Sexually Dimorphic Brain Fatty Acid Composition in Low and High Fat Diet-Fed Mice. Mol. Metab 2016, 5 (8), 680– 689, DOI: 10.1016/j.molmet.2016.06.014Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFent7fO&md5=9e27eff81140e827b19ed7c7d2df4685Sexually dimorphic brain fatty acid composition in low and high fat diet-fed miceRodriguez-Navas, Carlos; Morselli, Eugenia; Clegg, Deborah J.Molecular Metabolism (2016), 5 (8), 680-689CODEN: MMOEAS; ISSN:2212-8778. (Elsevier GmbH)In this study, we analyzed the fatty acid profile of brains and plasma from male and female mice fed chow or a western-style high fat diet (WD) for 16 wk to det. if males and females process fatty acids differently. Based on the differences in fatty acids obsd. in vivo, we performed in vitro expts. on N43 hypothalamic neuronal cells to begin to elucidate how the fatty acid milieu may impact brain inflammation. Using a comprehensive mass spectrometry fatty acid anal., which includes a profile for 52 different fatty acid isomers, we assayed the plasma and brain fatty acid compn. of age-matched male and female mice maintained on chow or a WD. Addnl., using the same techniques, we detd. the fatty acid compn. of N43 hypothalamic cells following exposure to palmitic and linoleic acid, alone or in combination. Our data demonstrate there is a sexual dimorphism in brain fatty acid content both following the consumption of the chow diet, as well as the WD, with males having an increased percentage of satd. fatty acids and redns. in ω6-polyunsatd. fatty acids when compared to females. Interestingly, we did not observe a sexual dimorphism in fatty acid content in the plasma of the same mice. Furthermore, exposure of N43 cells to the ω6-PUFA linoleic acid, which is higher in female brains when compared to males, reduces palmitic acid-induced inflammation. Our data suggest male and female brains, and not plasma, differ in their fatty acid profile. This is the first time, to our knowledge, lipidomic analyses has been used to directly test the hypothesis there is a sexual dimorphism in brain and plasma fatty acid compn. following consumption of the chow diet, as well as following exposure to the WD.
- 37Horoszewicz, J. S.; Kawinski, E.; Murphy, G. P. Monoclonal Antibodies to a New Antigenic Marker in Epithelial Prostatic Cells and Serum of Prostatic Cancer Patients. Anticancer Res. 1987, 7 (5B), 927– 935Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL1c7jt1GmsQ%253D%253D&md5=4672a3d5b6724314037e26dbafbfe743Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patientsHoroszewicz J S; Kawinski E; Murphy G PAnticancer research (1987), 7 (5B), 927-35 ISSN:0250-7005.Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.
- 38Vorlova, B.; Knedlik, T.; Tykvart, J.; Konvalinka, J. GCPII and Its Close Homolog GCPIII: From a Neuropeptidase to a Cancer Marker and beyond. Front. Biosci. 2019, 24, 648– 687, DOI: 10.2741/4742Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFWksL3E&md5=9d2d1e35c14c01dba566ddb9098ea380GCPII and its close homolog GCPIII: from a neuropeptidase to a cancer marker and beyondVorlova, Barbora; Knedlik, Tomas; Tykvart, Jan; Konvalinka, JanFrontiers in Bioscience, Landmark Edition (2019), 24 (4), 648-687CODEN: FRBIF6; ISSN:1093-4715. (Frontiers in Bioscience)Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been assocd. with any specific physiol. function or pathol., and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiol. functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-mol. inhibitors recognizing GCPII/III for diagnosis, imaging and exptl. therapy of human cancers and other pathologies.
- 39Evans, J. C.; Malhotra, M.; Cryan, J. F.; O’Driscoll, C. M. The Therapeutic and Diagnostic Potential of the Prostate Specific Membrane Antigen/glutamate Carboxypeptidase II (PSMA/GCPII) in Cancer and Neurological Disease. Br. J. Pharmacol. 2016, 173 (21), 3041– 3079, DOI: 10.1111/bph.13576Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlehu7zL&md5=e2bd304d8eacbf5390e82751f0c6c98bThe therapeutic and diagnostic potential of the prostate specific membrane antigen/glutamate carboxypeptidase II (PSMA/GCPII) in cancer and neurological diseaseEvans, James C.; Malhotra, Meenakshi; Cryan, John F.; O'Driscoll, Caitriona M.British Journal of Pharmacology (2016), 173 (21), 3041-3079CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)Prostate specific membrane antigen (PSMA) otherwise known as glutamate carboxypeptidase II (GCPII) is a membrane bound protein that has been found to be highly expressed in prostate cancer as well as the neovasculature of a wide variety of tumors including glioblastomas, breast and bladder cancers as well as being implicated in a variety of neurol. diseases including schizophrenia and ALS. In recent years there has been a surge in the development of both diagnostics and therapeutics that take advantage of the expression and activity of PSMA/GCPII. These include gene therapy, immunotherapy, chemotherapy and radiotherapy. In this review, we discuss the biol. roles that PSMA/GCPII plays, both in normal and disease tissues, and the current therapies exploiting its activity that are at the preclin. stage. We conclude by giving an expert opinion on the future direction of PSMA/GCPII based therapies and diagnostics and hurdles that need to be overcome to make them effective and viable.
- 40Olsen, A. S. B.; Færgeman, N. J. Sphingolipids: Membrane Microdomains in Brain Development, Function and Neurological Diseases. Open Biol. 2017, 7 (5), 170069 DOI: 10.1098/rsob.170069Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFartb3K&md5=cf7bb6a75151e4ddf2db6e4e1e4d0216Sphingolipids: membrane microdomains in brain development, function and neurological diseasesOlsen, Anne S. B.; rgeman, Nils J. F.Open Biology (2017), 7 (5), 170069/1-170069/17CODEN: OBPICQ; ISSN:2046-2441. (Royal Society)Sphingolipids are highly enriched in the nervous system where they are pivotal constituents of the plasma membranes and are important for proper brain development and functions. Sphingolipids are not merely structural elements, but are also recognized as regulators of cellular events by their ability to form microdomains in the plasma membrane. The significance of such compartmentalization spans broadly from being involved in differentiation of neurons and synaptic transmission to neuronal- glial interactions and myelin stability. Thus, perturbations of the sphingolipid metab. can lead to rearrangements in the plasma membrane, which has been linked to the development of various neurol. diseases. Studying microdomains and their functions has for a long time been synonymous with studying the role of cholesterol. However, it is becoming increasingly clear that microdomains are very heterogeneous, which among others can be ascribed to the vast no. of sphingolipids. In this review, we discuss the importance of microdomains with emphasis on sphingolipids in brain development and function as well as how disruption of the sphingolipid metab. (and hence microdomains) contributes to the pathogenesis of several neurol. diseases.
- 41Poitelon, Y.; Kopec, A. M.; Belin, S. Myelin Fat Facts: An Overview of Lipids and Fatty Acid Metabolism. Cells 2020, 9 (4), 812, DOI: 10.3390/cells9040812Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1altr7L&md5=1425c867663feaf17db0428527c75a65Myelin fat facts: an overview of lipids and fattyacid metabolismPoitelon, Yannick; Kopec, Ashley M.; Belin, SophieCells (2020), 9 (4), 812CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Myelin is crit. for the proper function of the nervous system and one of the most complex cell-cell interactions of the body. Myelination allows for the rapid conduction of action potentials along axonal fibers and provides phys. and trophic support to neurons. Myelin contains a high content of lipids, and the formation of the myelin sheath requires high levels of fatty acid and lipid synthesis, together with uptake of extracellular fatty acids. Recent studies have further advanced our understanding of the metab. and functions of myelin fatty acids and lipids. In this review, we present an overview of the basic biol. of myelin lipids and recent insights on the regulation of fatty acid metab. and functions in myelinating cells. In addn., this review may serve to provide a foundation for future research characterizing the role of fatty acids and lipids in myelin biol. and metabolic disorders affecting the central and peripheral nervous system.
- 42Aggarwal, S.; Yurlova, L.; Simons, M. Central Nervous System Myelin: Structure, Synthesis and Assembly. Trends Cell Biol. 2011, 21 (10), 585– 593, DOI: 10.1016/j.tcb.2011.06.004Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1antbfN&md5=43dfc209ae4240bca2283d5226a699faCentral nervous system myelin: structure, synthesis and assemblyAggarwal, Shweta; Yurlova, Larisa; Simons, MikaelTrends in Cell Biology (2011), 21 (10), 585-593CODEN: TCBIEK; ISSN:0962-8924. (Elsevier B.V.)A review. The wrapping of multiple layers of myelin membrane sheets around an axon is of fundamental importance for the function of the nervous system. In the central nervous system (CNS) oligodendrocytes synthesize tremendous amts. of cellular membrane to form multiple myelin internodes of highly stable membranes with a specific set of tightly packed lipids and proteins. In recent years, mouse mutants have allowed great advances in our understanding of the functional and structural role of many of the major components of myelin. The challenge now is to extend this knowledge to unravel the mol. machinery and mechanisms required to synthesize, assemble and wrap myelin multiple times around an axon at the appropriate developmental time. Such insight will be essential in designing new therapeutic strategies to promote remyelination in demyelinating disorders such as multiple sclerosis.
- 43Guan, Z.; Wang, Y.; Cairns, N. J.; Lantos, P. L.; Dallner, G.; Sindelar, P. J. Decrease and Structural Modifications of Phosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease. J. Neuropathol. Exp. Neurol. 1999, 58 (7), 740– 747, DOI: 10.1097/00005072-199907000-00008Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltVWhs7g%253D&md5=2d929c20b9acc1bb90cc02d895727b55Decrease and structural modifications of phosphatidylethanolamine plasmalogen in the brain with Alzheimer diseaseGuan, Zhizhong; Wang, Yanan; Cairns, Nigel J.; Lantos, Peter L.; Dallner, Gustav; Sindelar, Pavel J.Journal of Neuropathology and Experimental Neurology (1999), 58 (7), 740-747CODEN: JNENAD; ISSN:0022-3069. (American Association of Neuropathologists, Inc.)Several lipid modifications, some of which were attributed to oxidative stress, have been reported in the brains of patients with Alzheimer disease (AD). To evaluate this possibility, all phospholipids and their ether subclasses from the frontal cortex, hippocampus, and the white matter of AD brain were analyzed by high performance liq. chromatog. and gas chromatog. The total phospholipid in the frontal cortex and hippocampus decreased on a DNA basis by ∼20% and this change was essentially explained by a selective decrease in phosphatidylethanolamine and phosphatidylcholine. The lower content of phosphatidylethanolamine was due to a specific decrease in the plasmalogen subclass. Phosphatidylethanolamine plasmalogen was also the only lipid exhibiting major structural modifications: a significant decrease in polyunsatd. fatty acids and oleic acid as well as a shift of the aldehyde pattern from 18:1 to 18:0. The only modification obsd. in the other phospholipids was a decrease in oleic acid in diacyl-phosphatidylethanolamine and diacyl-phosphatidylcholine. None of these changes were obsd. in the white matter. Both the vinyl ether bond of phosphatidylethanolamine plasmalogen and polyunsatd. fatty acids are major targets in oxidative stress; thus, these specific lipid modifications strongly support the involvement of free radicals in the pathogenesis of AD.
- 44Su, X. Q.; Wang, J.; Sinclair, A. J. Plasmalogens and Alzheimer’s Disease: A Review. Lipids Health Dis. 2019, 18 (1), 100, DOI: 10.1186/s12944-019-1044-1Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M%252FmtFehsw%253D%253D&md5=88bc5e9b3b7575b976eff66125fe2926Plasmalogens and Alzheimer's disease: a reviewSu Xiao Q; Wang Junming; Sinclair Andrew J; Sinclair Andrew JLipids in health and disease (2019), 18 (1), 100 ISSN:.Growing evidence suggests that ethanolamine plasmalogens (PlsEtns), a subtype of phospholipids, have a close association with Alzheimer's disease (AD). Decreased levels of PlsEtns have been commonly found in AD patients, and were correlated with cognition deficit and severity of disease. Limited studies showed positive therapeutic outcomes with plasmalogens interventions in AD subjects and in rodents. The potential mechanisms underlying the beneficial effects of PlsEtns on AD may be related to the reduction of γ-secretase activity, an enzyme that catalyzes the synthesis of β-amyloid (Aβ), a hallmark of AD. Emerging in vitro evidence also showed that PlsEtns prevented neuronal cell death by enhancing phosphorylation of AKT and ERK signaling through the activation of orphan G-protein coupled receptor (GPCR) proteins. In addition, PlsEtns have been found to suppress the death of primary mouse hippocampal neuronal cells through the inhibition of caspase-9 and caspase-3 cleavages. Further in-depth investigations are required to determine the signature molecular species of PlsEtns associated with AD, hence their potential role as biomarkers. Clinical intervention with plasmalogens is still in its infancy but may have the potential to be explored for a novel therapeutic approach to correct AD pathology and neural function.
- 45Fabelo, N.; Martín, V.; Santpere, G.; Marín, R.; Torrent, L.; Ferrer, I.; Díaz, M. Severe Alterations in Lipid Composition of Frontal Cortex Lipid Rafts from Parkinson’s Disease and Incidental Parkinson’s Disease. Mol. Med. 2011, 17 (9–10), 1107– 1118, DOI: 10.2119/molmed.2011.00119Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlCgsbnE&md5=1ce7e15819dda8fb1b416f1d84bb711cSevere alterations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and incidental Parkinson's diseaseFabelo, Noemi; Martin, Virginia; Santpere, Gabriel; Marin, Raquel; Torrent, Laia; Ferrer, Isidre; Diaz, MarioMolecular Medicine (Manhasset, NY, United States) (2011), 17 (9-10), 1107-1118CODEN: MOMEF3; ISSN:1076-1551. (Feinstein Institute for Medical Research)Lipid rafts are cholesterol- and sphingomyelin-enriched microdomains that provide a highly satd. and viscous physicochem. microenvironment to promote protein-lipid and protein-protein interactions. We purified lipid rafts from human frontal cortex from normal, early motor stages of Parkinson's disease (PD) and incidental Parkinson's disease (iPD) subjects and analyzed their lipid compn. We obsd. that lipid rafts from PD and iPD cortices exhibit dramatic redns. in their contents of n-3 and n-6 long-chain polyunsatd. fatty acids, esp. docosahexaenoic acid (22:6-n3) and arachidonic acid (20:4n-6). Also, satd. fatty acids (16:0 and 18:0) were significantly higher than in control brains. Paralleling these findings, unsatn. and peroxidability indexes were considerably reduced in PD and iPD lipid rafts. Lipid classes were also affected in PD and iPD lipid rafts. Thus, phosphatidylserine and phosphatidylinositol were increased in PD and iPD, whereas cerebrosides and sulfatides and plasmalogen levels were considerably diminished. Our data pinpoint a dramatic increase in lipid raft order due to the aberrant biochem. structure in PD and iPD and indicate that these abnormalities of lipid rafts in the frontal cortex occur at early stages of PD pathol. The findings correlate with abnormal lipid raft signaling and cognitive decline obsd. during the development of these neurodegenerative disorders.
- 46Dorninger, F.; Forss-Petter, S.; Berger, J. From Peroxisomal Disorders to Common Neurodegenerative Diseases - the Role of Ether Phospholipids in the Nervous System. FEBS Lett. 2017, 591 (18), 2761– 2788, DOI: 10.1002/1873-3468.12788Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGhtrnO&md5=366e05f52879ad8e06719e250c2bca4aFrom peroxisomal disorders to common neurodegenerative diseases - the role of ether phospholipids in the nervous systemDorninger, Fabian; Forss-Petter, Sonja; Berger, JohannesFEBS Letters (2017), 591 (18), 2761-2788CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)A review. The emerging diverse roles of ether (phospho)lipids in nervous system development and function in health and disease are currently attracting growing interest. Plasmalogens, a subgroup of ether lipids, are important membrane components involved in vesicle fusion and membrane raft compn. They store polyunsatd. fatty acids and may serve as antioxidants. Ether lipid metabolites act as precursors for the formation of glycosyl-phosphatidyl-inositol anchors; others, like platelet-activating factor, are implicated in signaling functions. Consolidating the available information, we attempt to provide mol. explanations for the dramatic neurol. phenotype in ether lipid-deficient human patients and mice by linking individual functional properties of ether lipids with pathol. features. Furthermore, recent publications have identified altered ether lipid levels in the context of many acquired neurol. disorders including Alzheimer's disease (AD) and autism. Finally, current efforts to restore ether lipids in peroxisomal disorders as well as AD are critically reviewed.
- 47Katafuchi, T.; Ifuku, M.; Mawatari, S.; Noda, M.; Miake, K.; Sugiyama, M.; Fujino, T. Effects of Plasmalogens on Systemic Lipopolysaccharide-Induced Glial Activation and β-Amyloid Accumulation in Adult Mice. Ann. N.Y. Acad. Sci. 2012, 1262, 85– 92, DOI: 10.1111/j.1749-6632.2012.06641.xGoogle Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlGrtbfM&md5=6ff33ad3f0fd6a397cc71b0377ba99a3Effects of plasmalogens on systemic lipopolysaccharide-induced glial activation and β-amyloid accumulation in adult miceKatafuchi, Toshihiko; Ifuku, Masataka; Mawatari, Shiro; Noda, Mami; Miake, Kiyotaka; Sugiyama, Masaaki; Fujino, TakehikoAnnals of the New York Academy of Sciences (2012), 1262 (Neuroimmunomodulation in Health and Disease II), 85-92CODEN: ANYAA9; ISSN:0077-8923. (Wiley-Blackwell)Neuroinflammation essentially involves an activation of glial cells as the cause/effect of neurodegenerative diseases such as Alzheimer's disease (AD). Plasmalogens (Pls) are glycerophospholipids constituting cellular membranes and play significant roles in membrane fluidity and cellular processes like vesicular fusion and signal transduction. I.p. (i.p.) injection of lipopolysaccharide (LPS, 250 μg/kg) for 7 days resulted in the morphol. changes and increase in no. of Iba-1+ microglia showing neuroinflammation in the adult mouse hippocampus. The LPS-induced activation of glial cells was significantly attenuated by i.p. pretreatment with Pls dissolved in corn oil. In addn., systemic injection of LPS induced Aβ1-16+ neurons in the hippocampus were also abolished by application of Pls. Finally, contents of Pls in the hippocampus decreased after LPS injection, and the redn. was suppressed by administration of Pls. These findings suggest an antiamyloidogenic effect of Pls, implicating a possible therapeutic application of Pls against AD.
- 48Wallner, S.; Schmitz, G. Plasmalogens the Neglected Regulatory and Scavenging Lipid Species. Chem. Phys. Lipids 2011, 164 (6), 573– 589, DOI: 10.1016/j.chemphyslip.2011.06.008Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpvFCnurc%253D&md5=762062e2e7f960550f689b5fd05952c9Plasmalogens the neglected regulatory and scavenging lipid speciesWallner, Stefan; Schmitz, GerdChemistry and Physics of Lipids (2011), 164 (6), 573-589CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ltd.)A review. Plasmalogens are a class of phospholipids carrying a vinyl ether bond in sn-1 and an ester bond in sn-2 position of the glycerol backbone. Although they are widespread in all tissues and represent up to 18% of the total phospholipid mass in humans, their physiol. function is still poorly understood. The aim of this review is to give an overview over the current knowledge in plasmalogen biol. and pathol. with an emphasis on neglected aspects of their involvement in neurol. and metabolic diseases. Furthermore a better understanding of plasmalogen biol. in health and disease could also lead to the development of better diagnostic and prognostic biomarkers for vascular and metabolic diseases such as obesity and diabetes mellitus, inflammation, neuro-degeneration and cancer.
- 49Luoma, A. M.; Kuo, F.; Cakici, O.; Crowther, M. N.; Denninger, A. R.; Avila, R. L.; Brites, P.; Kirschner, D. A. Plasmalogen Phospholipids Protect Internodal Myelin from Oxidative Damage. Free Radic. Biol. Med. 2015, 84, 296– 310, DOI: 10.1016/j.freeradbiomed.2015.03.012Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvVyrt7w%253D&md5=c86c406514940fec0727481d3f81b2cbPlasmalogen phospholipids protect internodal myelin from oxidative damageLuoma, Adrienne M.; Kuo, Fonghsu; Cakici, Ozgur; Crowther, Michelle N.; Denninger, Andrew R.; Avila, Robin L.; Brites, Pedro; Kirschner, Daniel A.Free Radical Biology & Medicine (2015), 84 (), 296-310CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)Reactive O species (ROS) are implicated in a range of degenerative conditions, including aging, neurodegenerative diseases, and neurol. disorders. Myelin is a lipid-rich multilamellar sheath that facilitates rapid nerve conduction in vertebrates. Given the high energetic demands and low antioxidant capacity of the cells that elaborate the sheaths, myelin is considered intrinsically vulnerable to oxidative damage, raising the question whether addnl. mechanisms prevent structural damage. Here, the authors characterized the structural and biochem. basis of ROS-mediated myelin damage in murine tissues from both the central nervous system (CNS) and the peripheral nervous system (PNS). To det. whether ROS could cause structural damage to the internodal myelin, whole sciatic and optic nerves were incubated ex vivo with a hydroxyl radical-generating system consisting of Cu, H2O2 (I), and o-phenanthroline (II). Quant. assessment of unfixed tissue by x-ray diffraction revealed irreversible compaction of myelin membrane stacking in both sciatic and optic nerves. Incubation in the presence of the hydroxyl radical scavenger, Na formate, prevented this damage, implicating hydroxyl radical species. Myelin membranes were particularly enriched in plasmalogens, a class of ether-linked phospholipids proposed to have antioxidant properties. Myelin in the sciatic nerve from plasmalogen-deficient (Pex7 knockout) mice was significantly more vulnerable to Cu/I/II-mediated ROS-induced compaction than myelin from wild-type mice. The results directly supported the role of plasmalogens as endogenous antioxidants providing a defense that protects ROS-vulnerable myelin.
- 50Kinoshita, K.; Arai, K.; Kawaura, K.; Hiyoshi, T.; Yamaguchi, J.-I. Development, Validation, and Application of a Surrogate Analyte Method for Determining N-Acetyl-L-Aspartyl-L-Glutamic Acid Levels in Rat Brain, Plasma, and Cerebrospinal Fluid. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015, 1003, 1– 11, DOI: 10.1016/j.jchromb.2015.09.005Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFaqsrvK&md5=798127c101c28ee1116343ed619b49c2Development, validation, and application of a surrogate analyte method for determining N-acetyl-L-aspartyl-L-glutamic acid levels in rat brain, plasma, and cerebrospinal fluidKinoshita, Kohnosuke; Arai, Kotaro; Kawaura, Kazuaki; Hiyoshi, Tetsuaki; Yamaguchi, Jun-ichiJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2015), 1003 (), 1-11CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)A bioanal. strategy for the simple and accurate detn. of endogenous substances in a variety of biol. matrixes using liq. chromatog.-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compds. as a surrogate analyte and an internal std. to construct calibration curves with authentic matrixes that can be applied to det. N-acetyl-L-aspartyl-L-glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extn. and with a short anal. time of 4 min. The validated lower limits of quantification were 1.00 nmol/g for brain and 0.0100 nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naive rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the detn. of NAAG in different matrixes in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biol. matrixes.
- 51Williamson, L. C.; Neale, J. H. Ultrastructural Localization of N-Acetylaspartylglutamate in Synaptic Vesicles of Retinal Neurons. Brain Res. 1988, 456 (2), 375– 381, DOI: 10.1016/0006-8993(88)90243-0Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXkvVKrsb8%253D&md5=79af9ff98dfb4ffd7e141e108e1d83cdUltrastructural localization of N-acetylaspartylglutamate in synaptic vesicles of retinal neuronsWilliamson, Lura C.; Neale, Joseph H.Brain Research (1988), 456 (2), 375-81CODEN: BRREAP; ISSN:0006-8993.In the grass frog (Rana pipiens), N-acetylaspartylglutamate (NAAG) immunoreactivity was localized within vesicles in synaptic endings of presumptive amacrine and bipolar neurons in the inner plexiform layer. Addnl., the peptide was present in vesicles within ribbon synapses in the outer plexiform layer, a result suggestive of release from photoreceptor cells. Apparently, NAAG is secreted at points of synaptic contact between neurons, including retinal amacrine, bipolar, and photoreceptor cells.
- 52Renno, W. M.; Lee, J. H.; Beitz, A. J. Light and Electron Microscopic Immunohistochemical Localization of N-Acetylaspartylglutamate (NAAG) in the Olivocerebellar Pathway of the Rat. Synapse 1997, 26 (2), 140– 154, DOI: 10.1002/(SICI)1098-2396(199706)26:2<140::AID-SYN5>3.0.CO;2-8Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivVGhs7s%253D&md5=45dba8c3f8450d5890dd3d043d5a7f27Light and electron microscopic immunohistochemical localization of N-acetylaspartylglutamate (NAAG) in the olivocerebellar pathway of the ratRenno, Waleed M.; Lee, Jang-Hern; Beitz, Alvin J.Synapse (New York) (1997), 26 (2), 140-154CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss)The aim of the present study was to det. whether N-acetylaspartylglutamate (NAAG) immunoreactivity is present in the inferior olive (IO) and climbing fibers (CF) at the light and electron microscopic levels and to quantitate the amt. of immunogold labeling in olivary neurons and climbing fiber terminals contg. this dipeptide. A polyclonal antisera against NAAG was utilized with a peroxidase-labeled avidin-biotin procedure to demonstrate these immunoreactive neurons in the IO at the light microscopic level. Approx. 45% of olivary neurons display NAAG-like immunoreactivity, and their distribution is unevenly clustered throughout the inferior olive. Using postembedding immunogold electron microscopy in combination with quant. procedures, we found the highest densities of gold particles in the axonal terminals synapsing on olivary neurons (101.0 particles/μ2), in CF terminals (96.3 particles/μm2), and in some mossy fiber terminals (101.0 particles/μm2). Approx. half of the climbing fiber terminals examd. were unlabeled. Moderate labeling occurred in CF axons (70.8 particles/μm2), while IO neuronal perikarya were lightly but significantly labeled (41.6 particles/μm2). The localization of NAAG in the subset of cerebellar climbing fiber terminals provides anatomical support for the hypothesis that NAAG may serve as a neurotransmitter/neuromodulator candidate in the olivocerebellar pathway.
- 53Sácha, P.; Zámecník, J.; Barinka, C.; Hlouchová, K.; Vícha, A.; Mlcochová, P.; Hilgert, I.; Eckschlager, T.; Konvalinka, J. Expression of Glutamate Carboxypeptidase II in Human Brain. Neuroscience 2007, 144 (4), 1361– 1372, DOI: 10.1016/j.neuroscience.2006.10.022Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVartLw%253D&md5=f72f2146d36d083e985403a28a95d0c9Expression of glutamate carboxypeptidase II in human brainSacha, P.; Zamecnik, J.; Barinka, C.; Hlouchova, K.; Vicha, A.; Mlcochova, P.; Hilgert, I.; Eckschlager, T.; Konvalinka, J.Neuroscience (San Diego, CA, United States) (2007), 144 (4), 1361-1372CODEN: NRSCDN; ISSN:0306-4522. (Elsevier)Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a no. of pathol. conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approx. 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochem. anal. revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.
- 54Nagel, J.; Belozertseva, I.; Greco, S.; Kashkin, V.; Malyshkin, A.; Jirgensons, A.; Shekunova, E.; Eilbacher, B.; Bespalov, A.; Danysz, W. Effects of NAAG Peptidase Inhibitor 2-PMPA in Model Chronic Pain - Relation to Brain Concentration. Neuropharmacology 2006, 51 (7–8), 1163– 1171, DOI: 10.1016/j.neuropharm.2006.07.018Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1ertrbE&md5=da6263c8a77cbb46dc9eaf6cee01731dEffects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentrationNagel, Jens; Belozertseva, Irina; Greco, Sergio; Kashkin, Vladimir; Malyshkin, Andrey; Jirgensons, Aigars; Shekunova, Elena; Eilbacher, Bernd; Bespalov, Anton; Danysz, WojciechNeuropharmacology (2006), 51 (7-8), 1163-1171CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concn. in brain ECF under basal conditions and its alteration in relation to the brain ECF concn. of 2-PMPA is unclear. We therefore assessed those brain concns. after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS anal. Administration of 2-PMPA (50 mg/kg) produced a mean peak concn. of 2-PMPA of 29.66±8.1 μM. This concn. is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concn. reaching a max. of 2.89±0.42 μM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were obsd., the NAAG concn. in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the obsd. behavioral effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors.
- 55Lin, S. N.; Slopis, J. M.; Butler, I. J.; Caprioli, R. M. In Vivo Microdialysis and Gas Chromatography/mass Spectrometry for Studies on Release of N-Acetylaspartlyglutamate and N-Acetylaspartate in Rat Brain Hypothalamus. J. Neurosci. Methods 1995, 62 (1–2), 199– 205, DOI: 10.1016/0165-0270(95)00077-1Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK28zlslektw%253D%253D&md5=c1fe594f7e5dec7c7245a801e2badc3fIn vivo microdialysis and gas chromatography/mass spectrometry for studies on release of N-acetylaspartlyglutamate and N-acetylaspartate in rat brain hypothalamusLin S N; Slopis J M; Butler I J; Caprioli R MJournal of neuroscience methods (1995), 62 (1-2), 199-205 ISSN:0165-0270.Microdialysis and gas chromatography/mass spectrometry was used for the measurement of extracellular N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in rat hypothalamus. The sensitivity of the method for each of these compounds was approximately 5 pmol/30 microliters of dialysate. Baseline NAA concentrations in dialysate were estimated to be approximately 25 pmol/36 microliters, while that for NAAG was at or below the detection limit of 5 pmol/ 36 microliters. In vivo and in vitro calibrations of microdialysis probes showed that the recovery for NAA was approximately 10 percent. For NAAG, the in vitro recovery was 6.3%, and in vivo recovery, 11%. Depolarization stimulation using 100 mM KCl in the microdialysis perfusate was employed to measure extracellular NAA and NAAG concentrations. Extracellular NAA was elevated to approximately 70 pmol/36 microliters dialysate following depolarization. No significant elevation of NAAG was observed. By infusing known amounts of stable isotopically labeled NAAG-d3 via the microdialysis probe and measuring the isotopically labeled catabolic product, NAA-d3, in collected microdialysate, we were able to confirm the existence of one or more hydrolytic enzymes active towards NAAG in the hypothalamus. This finding suggest the possible involvement of active metabolic processes in the relationship between NAAG and NAA releases.
- 56Sager, T. N.; Laursen, H.; Hansen, A. J. Changes in N-Acetyl-Aspartate Content during Focal and Global Brain Ischemia of the Rat. J. Cereb. Blood Flow Metab. 1995, 15 (4), 639– 646, DOI: 10.1038/jcbfm.1995.79Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXntVamurY%253D&md5=8a71fb0c93a31ddbcaaf5da814cb2662Changes in N-acetyl-aspartate content during focal and global brain ischemia of the ratSager, Thomas Nikolaj; Laursen, Henning; Hansen, Anker JonJournal of Cerebral Blood Flow and Metabolism (1995), 15 (4), 639-46CODEN: JCBMDN; ISSN:0271-678X.N-Acetyl-aspartate (NAA) is almost exclusively localized in neurons in the mature brain and might be used as a neuronal marker. It has been reported that the NAA content in human brain is decreased in neurodegenerative diseases and in stroke. Since the NAA content can be detd. by NMR techniques, it has potential as a diagnostic and prognostic marker. The objective of this study was to examine the change of NAA content and related substances following cerebral ischemia and compare the results to the damage of the tissue. We used rats to study the changes of NAA, N-acetyl-aspartyl-glutamate (NAAG), glutamate, and aspartate contents over a time course of 24 h in brain regions affected by either permanent middle cerebral artery occlusion (focal ischemia) or decapitation (global ischemia). The decreases of NAA and NAAG contents following global brain ischemia were linear over time but significant only after 4 and 2 h, resp. After 24 h, the levels of NAA and NAAG were 24 and 44% of control values, resp. The concn. of glutamate did not change, whereas the aspartate content increased at a rate comparable with the rate of decrease of NAA content. This is consistent with NAA being preferentially degraded by the enzyme amidohydrolase II in global ischemia. In focal ischemia, there was a rapid decline of NAA within the first 8 h of ischemia followed by a slower rate of redn. The redns. of NAA and NAAG contents in focal ischemia were significant after 4 and 24 h, resp. After 24 h, the NAA and NAAG contents were 33 and 64% of control values, resp. Also, the glutamate and aspartate contents exhibited significant decreases in focal ischemic tissue. Our studies show that NAA decreases during brain ischemia, the initial rate being faster in focal ischemia than in global ischemia. In rat transient focal ischemia, others have shown that a middle cerebral artery occlusion of 2- to 3-h duration is sufficient to produce an infarct that is similar in size to that following permanent occlusion for 24 h. The fact that we obsd. only a 10% decrease of NAA content 2 h after occlusion demonstrates that the NAA content of the tissue does not reflect neuronal viability. Thus, the incompetence with which ischemic/infarcted tissue removes NAA will lead to overestimation of the no. of viable neurons in acute situations. Only when steady state prevails may NAA be used as a marker of viable nerve cells.
- 57Masaharu, M.; Hideo, M.; Mutsuhiko, M.; Yasuo, K. N-Acetyl-L-Aspartic Acid, Acid and β-Citryl-L-Glutamic Acid in Human Urine. Clin. Chim. Acta 1982, 120 (1), 119– 126, DOI: 10.1016/0009-8981(82)90082-1Google ScholarThere is no corresponding record for this reference.
- 58Chigurupati, S.; Son, T. G.; Hyun, D.-H.; Lathia, J. D.; Mughal, M. R.; Savell, J.; Li, S. C.; Nagaraju, G. P. C.; Chan, S. L.; Arumugam, T. V.; Mattson, M. P. Lifelong Running Reduces Oxidative Stress and Degenerative Changes in the Testes of Mice. J. Endocrinol. 2008, 199 (2), 333– 341, DOI: 10.1677/JOE-08-0306Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVClsb%252FM&md5=1883fa72cf8c245e68a227521d1104b1Lifelong running reduces oxidative stress and degenerative changes in the testes of miceChigurupati, Srinivasulu; Son, Tae Gen; Hyun, Dong-Hoon; Lathia, Justin D.; Mughal, Mohamed R.; Savell, Jason; Li, Shuan C.; Nagaraju, G. P. C.; Chan, Sic L.; Arumugam, Thiruma V.; Mattson, Mark P.Journal of Endocrinology (2008), 199 (2), 333-341CODEN: JOENAK; ISSN:0022-0795. (Society for Endocrinology)Regular exercise can counteract the adverse effects of aging on the musculoskeletal and cardiovascular systems. In males, the normal aging process is assocd. with redns. in testosterone prodn. and impaired spermatogenesis, but the underlying mechanisms and their potential modification by exercise are unknown. Here, the authors report that lifelong regular exercise (running) protects the testes against the adverse effects of advancing age, and that this effect of running is assocd. with decreased amts. of oxidative damage to proteins, lipids, and DNA in spermatogenic and Leydig cells. Six-month-old male mice were divided into a sedentary group and a group that ran an av. of 1·75 km/day, until the mice reached the age of 20 mo. Seminiferous tubules of runners exhibited a full complement of cells at different stages of the spermatogenic process and a clear central lumen with large nos. of spermatozoa, in contrast to sedentary mice that exhibited disorganized spermatogenic cells and lacked spermatocytes in a central lumen. Levels of protein carbonyls, nitrotyrosine, lipid peroxidn. products, and oxidatively modified DNA were significantly greater in spermatogenic and Leydig cells of sedentary mice compared with runners. These findings suggest that lifelong regular exercise suppresses aging of testes by a mechanism that involves reduced oxidative damage to spermatogenic and Leydig cells.
- 59Liu, L.; Duff, K. A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse. J. Visualized Exp. 2008, 21, 960, DOI: 10.3791/960-vGoogle ScholarThere is no corresponding record for this reference.
- 60Karlíková, R.; Široká, J.; Friedecký, D.; Faber, E.; Hrdá, M.; Mičová, K.; Fikarová, I.; Gardlo, A.; Janečková, H.; Vrobel, I.; Adam, T. Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients. J. Proteome Res. 2016, 15 (9), 3158– 3166, DOI: 10.1021/acs.jproteome.6b00356Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Gjt73N&md5=e20241ce3956ccd51f791268069419f8Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia PatientsKarlikova, Radana; Siroka, Jitka; Friedecky, David; Faber, Edgar; Hrda, Marcela; Micova, Katerina; Fikarova, Iveta; Gardlo, Alzbeta; Janeckova, Hana; Vrobel, Ivo; Adam, TomasJournal of Proteome Research (2016), 15 (9), 3158-3166CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liq. chromatog. mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metab. We obsd. differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an addnl. tool for the assessment of response/resistance to imatinib.
- 61Xuan, Q.; Hu, C.; Yu, D.; Wang, L.; Zhou, Y.; Zhao, X.; Li, Q.; Hou, X.; Xu, G. Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry. Anal. Chem. 2018, 90 (12), 7608– 7616, DOI: 10.1021/acs.analchem.8b01331Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVeis7fN&md5=f9061a7a1201eea7b20b4340a3370344Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass SpectrometryXuan, Qiuhui; Hu, Chunxiu; Yu, Di; Wang, Lichao; Zhou, Yang; Zhao, Xinjie; Li, Qi; Hou, Xiaoli; Xu, GuowangAnalytical Chemistry (Washington, DC, United States) (2018), 90 (12), 7608-7616CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Lipid coverage is crucial in comprehensive lipidomics studies challenged by high diversity in lipid structures and wide dynamic range in lipid levels. Current state-of-the-art lipidomics technologies are mostly based on mass spectrometry (MS), including direct-infusion MS, chromatog.-MS, and matrix-assisted laser desorption ionization (MALDI) imaging MS, each with its pros and cons. Due to the need or favorability for measurement of isomers and isobars, chromatog.-MS is preferable for lipid profiling. The ultra-HPLC-high resoln. mass spectrometry (UHPLC-HRMS)-based nontargeted lipidomics approach and UHPLC-tandem MS (UHPLC-MS/MS)-based targeted approach are two representative methodol. platforms for chromatog.-MS. The authors developed a high coverage pseudotargeted lipidomics method combining the advantages of nontargeted and targeted lipidomics approaches. The high coverage of lipids was achieved by integration of the detected lipids derived from nontargeted UHPLC-HRMS lipidomics anal. of multiple matrixes (e.g., plasma, cell, and tissue) and the predicted lipids speculated on the basis of the structure and chromatog. retention behavior of the known lipids. A total of 3377 targeted lipid ion pairs with over 7000 lipid mol. structures were defined. The pseudotargeted lipidomics method was well validated with satisfactory anal. characteristics in terms of linearity, precision, reproducibility, and recovery for lipidomics profiling. Importantly, it showed better repeatability and higher coverage of lipids than the nontargeted lipidomics method. The applicability of the developed pseudotargeted lipidomics method was testified in defining differential lipids related to diabetes. The authors believe that comprehensive lipidomics studies will benefit from the developed high coverage pseudotargeted lipidomics approach.
- 62Peng, B.; Kopczynski, D.; Pratt, B. S.; Ejsing, C. S.; Burla, B.; Hermansson, M.; Benke, P. I.; Tan, S. H.; Chan, M. Y.; Torta, F.; Schwudke, D.; Meckelmann, S. W.; Coman, C.; Schmitz, O. J.; MacLean, B.; Manke, M.-C.; Borst, O.; Wenk, M. R.; Hoffmann, N.; Ahrends, R. LipidCreator Workbench to Probe the Lipidomic Landscape. Nat. Commun. 2020, 11 (1), 2057, DOI: 10.1038/s41467-020-15960-zGoogle Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosVOmtrs%253D&md5=d63c13ea857730aea32cbc2327f485abLipidCreator workbench to probe the lipidomic landscapePeng, Bing; Kopczynski, Dominik; Pratt, Brian S.; Ejsing, Christer S.; Burla, Bo; Hermansson, Martin; Benke, Peter Imre; Tan, Sock Hwee; Chan, Mark Y.; Torta, Federico; Schwudke, Dominik; Meckelmann, Sven W.; Coman, Cristina; Schmitz, Oliver J.; MacLean, Brendan; Manke, Mailin-Christin; Borst, Oliver; Wenk, Markus R.; Hoffmann, Nils; Ahrends, RobertNature Communications (2020), 11 (1), 2057CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biol. conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and anal. and prove that it is capable to generate large targeted expts. to analyze blood and to dissect lipid-signaling pathways such as in human platelets.
- 63Drotleff, B.; Roth, S. R.; Henkel, K.; Calderón, C.; Schlotterbeck, J.; Neukamm, M. A.; Lämmerhofer, M. Lipidomic Profiling of Non-Mineralized Dental Plaque and Biofilm by Untargeted UHPLC-QTOF-MS/MS and SWATH Acquisition. Anal. Bioanal. Chem. 2020, 412 (10), 2303– 2314, DOI: 10.1007/s00216-019-02364-2Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivFKhtr4%253D&md5=97cc543d3d468761e0cb63a606fbb668Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisitionDrotleff, Bernhard; Roth, Simon R.; Henkel, Kerstin; Calderon, Carlos; Schlotterbeck, Joerg; Neukamm, Merja A.; Laemmerhofer, MichaelAnalytical and Bioanalytical Chemistry (2020), 412 (10), 2303-2314CODEN: ABCNBP; ISSN:1618-2642. (Springer)Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its compn. may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently studied in the context of oral health and disease. Furthermore, its potential as an alternative matrix for anal. purposes has also been recognized in other disciplines like archeol., food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, however, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for detn. of lipid compns. of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for anal. purposes has been performed. An untargeted lipidomics workflow using a ultra-HPLC (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theor. fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepd. by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-anal. Most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was obsd. in the cultivated biofilms. However, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate are specifically increased in plaque samples.
- 64AlzbetaG. AlzbetaG/Metabol: First Version; Zenodo, 2019, DOI: DOI: 10.5281/ZENODO.3235775 .Google ScholarThere is no corresponding record for this reference.
- 65Dieterle, F.; Ross, A.; Schlotterbeck, G.; Senn, H. Probabilistic Quotient Normalization as Robust Method to Account for Dilution of Complex Biological Mixtures. Application in 1H NMR Metabonomics. Anal. Chem. 2006, 78 (13), 4281– 4290, DOI: 10.1021/ac051632cGoogle Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVCgtro%253D&md5=6eb6377326a9df2a59b6afb2a9c6e47dProbabilistic Quotient Normalization as Robust Method to Account for Dilution of Complex Biological Mixtures. Application in 1H NMR MetabonomicsDieterle, Frank; Ross, Alfred; Schlotterbeck, Goetz; Senn, HansAnalytical Chemistry (2006), 78 (13), 4281-4290CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)For the anal. of the spectra of complex biofluids, preprocessing methods play a crucial role in rendering the subsequent data analyses more robust and accurate. Normalization is a preprocessing method, which accounts for different dilns. of samples by scaling the spectra to the same virtual overall concn. In the field of 1H NMR metabonomics integral normalization, which scales spectra to the same total integral, is the de facto std. In this work, it is shown that integral normalization is a suboptimal method for normalizing spectra from metabonomic studies. Esp. strong metabonomic changes, evident as massive amts. of single metabolites in samples, significantly hamper the integral normalization resulting in incorrectly scaled spectra. The probabilistic quotient normalization is introduced in this work. This method is based on the calcn. of a most probable diln. factor by looking at the distribution of the quotients of the amplitudes of a test spectrum by those of a ref. spectrum. Simulated spectra, spectra of urine samples from a metabonomic study with cyclosporin-A as the active compd., and spectra of more than 4000 samples of control animals demonstrate that the probabilistic quotient normalization is by far more robust and more accurate than the widespread integral normalization and vector length normalization.
- 66Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 2003, 13 (11), 2498– 2504, DOI: 10.1101/gr.1239303Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovFWrtr4%253D&md5=2bcbca9a3bd04717761f0424c0209e43Cytoscape: A software environment for integrated models of biomolecular interaction networksShannon, Paul; Markiel, Andrew; Ozier, Owen; Baliga, Nitin S.; Wang, Jonathan T.; Ramage, Daniel; Amin, Nada; Schwikowski, Benno; Ideker, TreyGenome Research (2003), 13 (11), 2498-2504CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)Cytoscape is an open source software project for integrating biomol. interaction networks with high-throughput expression data and other mol. states into a unified conceptual framework. Although applicable to any system of mol. components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other mol. states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of addnl. computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined phys./functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.
- 67Mitchell, M. W. A Comparison of Aggregate P-Value Methods and Multivariate Statistics for Self-Contained Tests of Metabolic Pathway Analysis. PLoS One 2015, 10 (4), e0125081 DOI: 10.1371/journal.pone.0125081Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisVSiurg%253D&md5=f82e631c472666c6deea9054677f780fA comparison of aggregate P-value methods and multivariate statistics for self-contained tests of metabolic pathway analysisMitchell, Matthew W.PLoS One (2015), 10 (4), e0125081/1-e0125081/17CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)For pathway anal. of genomic data, the most common methods involve combining p-values from individual statistical tests. However, there are several multivariate statistical methods that can be used to test whether a pathway has changed. Because of the large no. of variables and pathway sizes in genomics data, some of these statistics cannot be computed. However, in metabolomics data, the no. of variables and pathway sizes are typically much smaller, making such computations feasible. Of particular interest is being able to detect changes in pathways that may not be detected for the individual variables. We compare the performance of both the p-value methods and multivariate statistics for self-contained tests with an extensive simulation study and a human metabolomics study. Permutation tests, rather than asymptotic results are used to assess the statistical significance of the pathways. Furthermore, both one and two-sided alternatives hypotheses are examd. From the human metabolomic study, many pathways were statistically significant, although the majority of the individual variables in the pathway were not. Overall, the p-value methods perform at least as well as the multivariate statistics for these scenarios.
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- 1Robinson, M. B.; Blakely, R. D.; Couto, R.; Coyle, J. T. Hydrolysis of the Brain Dipeptide N-Acetyl-L-Aspartyl-L-Glutamate. Identification and Characterization of a Novel N-Acetylated Alpha-Linked Acidic Dipeptidase Activity from Rat Brain. J. Biol. Chem. 1987, 262 (30), 14498– 14506, DOI: 10.1016/S0021-9258(18)47823-41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXlvFersrc%253D&md5=27492dd297c2a204212efa2c51d4c8bbHydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated α-linked acidic dipeptidase activity from rat brainRobinson, Michael B.; Blakely, Randy D.; Couto, Renee; Coyle, Joseph T.Journal of Biological Chemistry (1987), 262 (30), 14498-506CODEN: JBCHA3; ISSN:0021-9258.HPLC studies documented the presence of an enzyme activity, N-acetylated α-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatog., which quant. sepd. [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, it was found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain was characterized. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37°. Eadie-Hofstee anal. of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 μM and 1 mM, resp. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by Mn. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated α-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degrdn. of N-acetyl-L-aspartyl-L-glutamate.
- 2Halsted, C. H.; Ling, E. H.; Luthi-Carter, R.; Villanueva, J. A.; Gardner, J. M.; Coyle, J. T. Folylpoly-Gamma-Glutamate Carboxypeptidase from Pig Jejunum. Molecular Characterization and Relation to Glutamate Carboxypeptidase II. J. Biol. Chem. 1998, 273 (32), 20417– 20424, DOI: 10.1074/jbc.273.32.204172https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlsVWgtbs%253D&md5=ec8c60eab7b3348a72060331af48ce1bFolylpoly-γ-glutamate carboxypeptidase from pig jejunum. Molecular characterization and relation to glutamate carboxypeptidase IIHalsted, Charles H.; Ling, Erh-Hsin; Luthi-Carter, Ruth; Villanueva, Jesus A.; Gardner, John M.; Coyle, Joseph T.Journal of Biological Chemistry (1998), 273 (32), 20417-20424CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Jejunal folylpoly-γ-glutamate carboxypeptidase hydrolyzes dietary folates prior to their intestinal absorption. The complete folylpoly-γ-glutamate carboxypeptidase cDNA was isolated from a pig jejunal cDNA library using an amplified homologous probe incorporating primer sequences from prostate-specific membrane antigen, a protein capable of folate hydrolysis. The cDNA encodes a 751-amino acid polypeptide homologous to prostate-specific membrane antigen and rat brain N-acetylated α-linked acidic dipeptidase. PC3 transfectant membranes exhibited activities of folylpoly-γ-carboxypeptidase and N-acetylated α-linked acidic dipeptidase, while immunoblots using monoclonal antibody to native folypoly-γ-glutamate carboxypeptidase identified a glycoprotein at 120 kDa and a polypeptide at 84 kDa. The kinetics of native folylpoly-γ-carboxypeptidase were expressed in membranes of PC3 cells transfected with either pig folylpoly-γ-carboxypeptidase or human prostate-specific membrane antigen. Folylpoly-γ-carboxypeptidase transcripts were identified at 2.8 kilobase pairs in human and pig jejunum, human and rat brain, and human prostate cancer LNCaP cells. Thus, pig folylpoly-γ-carboxypeptidase, rat N-acetylated α-linked acidic dipeptidase, and human prostate-specific membrane antigen appear to represent varied expressions of the same gene in different species and tissues. The discovery of the jejunal folylpoly-γ-carboxypeptidase gene provides a framework for future studies on relationships among these proteins and on the mol. regulation of intestinal folate absorption.
- 3Pangalos, M. N.; Neefs, J. M.; Somers, M.; Verhasselt, P.; Bekkers, M.; van der Helm, L.; Fraiponts, E.; Ashton, D.; Gordon, R. D. Isolation and Expression of Novel Human Glutamate Carboxypeptidases with N-Acetylated Alpha-Linked Acidic Dipeptidase and Dipeptidyl Peptidase IV Activity. J. Biol. Chem. 1999, 274 (13), 8470– 8483, DOI: 10.1074/jbc.274.13.84703https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitFyrs7g%253D&md5=8fbd95de336d72d0d4d8d1440cd18b76Isolation and expression of novel human glutamate carboxypeptidases with N-acetylated α-linked acidic dipeptidase and dipeptidyl peptidase IV activityPangalos, Menelas N.; Neefs, Jean-Marc; Somers, Marijke; Verhasselt, Peter; Bekkers, Mariette; Van der Helm, Liesbet; Fraiponts, Erwin; Ashton, David; Gordon, Robert D.Journal of Biological Chemistry (1999), 274 (13), 8470-8483CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Hydrolysis of the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) by N-acetylated α-linked acidic dipeptidase (NAALADase) to release glutamate may be important in a no. of neurodegenerative disorders in which excitotoxic mechanisms are implicated. The gene coding for human prostate-specific membrane antigen, a marker of prostatic carcinomas, and its rat homolog glutamate carboxypeptidase II have recently been shown to possess such NAALADase activity. In contrast, a closely related member of this gene family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV activity. Here, the authors describe the cloning of human ileal 100-kDa protein, which the authors have called a NAALADase-"like" (NAALADase L) peptidase based on its sequence similarity to other members of this gene family, and its inability to hydrolyze NAAG in transient transfection expts. Furthermore, the authors describe the cloning of a third novel member of this gene family, NAALADase II, which codes for a type II integral membrane protein and which the authors have localized to chromosome 11 by fluorescent in situ hybridization anal. Transient transfection of NAALADase II cDNA confers both NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression studies using reverse transcription-polymerase chain reaction and Northern blot hybridization show that NAALADase II is highly expressed in ovary and testis as well as within discrete brain areas.
- 4Collard, F.; Vertommen, D.; Constantinescu, S.; Buts, L.; Van Schaftingen, E. Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3. J. Biol. Chem. 2011, 286 (44), 38220– 38230, DOI: 10.1074/jbc.M111.2873184https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlyktbrM&md5=fa7065a9e255e4a1639e1fcaa4ccb119Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3Collard, Francois; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, EmileJournal of Biological Chemistry (2011), 286 (44), 38220-38230CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)β-Citrylglutamate (BCG), a compd. present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca2+, the purified enzyme specifically hydrolyzed BCG and did not act on N-acetylaspartylglutamate (NAAG). However, both compds. were hydrolyzed in the presence of Mn2+. This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of NAAG. The mouse tissue distribution of β-citrylglutamate hydrolase was found to be strikingly similar to that of glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed BCG in the presence of Ca2+, and acted on both NAAG and BCG in the presence of Mn2+, whereas recombinant GCP2 only hydrolyzed NAAG and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis expts. revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) was largely responsible for GCP3 being able to hydrolyze BCG. Based on the crystal structure of GCP3 and kinetic anal., the authors propose that GCP3 forms a labile catalytic Zn-Ca cluster that is crit. for its β-citrylglutamate hydrolase activity.
- 5Navrátil, M.; Tykvart, J.; Schimer, J.; Pachl, P.; Navrátil, V.; Rokob, T. A.; Hlouchová, K.; Rulíšek, L.; Konvalinka, J. Comparison of Human Glutamate Carboxypeptidases II and III Reveals Their Divergent Substrate Specificities. FEBS J. 2016, 283 (13), 2528– 2545, DOI: 10.1111/febs.137615https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XptFWgu70%253D&md5=5e6dba4900f70da471495c1319e7c883Comparison of human glutamate carboxypeptidases II and III reveals their divergent substrate specificitiesNavratil, Michal; Tykvart, Jan; Schimer, Jiri; Pachl, Petr; Navratil, Vaclav; Rokob, Tibor Andras; Hlouchova, Klara; Rulisek, Lubomir; Konvalinka, JanFEBS Journal (2016), 283 (13), 2528-2545CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)Glutamate carboxypeptidase III (GCPIII) is best known as a homolog of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurol. disorders and overexpressed in a no. of solid cancers. However, mouse GCPIII was recently shown to cleave β-citryl-L-glutamate (BCG), suggesting that these 2 closely related enzymes have distinct functions. Here, to develop a tool to dissect, evaluate, and quantify the activities of human GCPII and GCPIII, the authors analyzed the catalytic efficiencies of these enzymes toward 3 physiol. substrates (BCG, N-acetyl-L-aspartyl-L-glutamate, and polyglutamated folic acid). The authors obsd. a high efficiency of BCG cleavage by GCPIII but not GCPII. The authors also identified a strong modulation of GCPIII enzymic activity by divalent cations, while they did not observe this effect for GCPII. Addnl., the authors used x-ray crystallog. and computational modeling (QM/MM calcns.) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, resp. Finally, the authors took advantage of the substantial differences in the enzymic efficiencies of GCPII and GCPIII toward their substrates, using enzymic assays for specific detection of these proteins in human tissues. The findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiol. function related to BCG metab. in the human body.
- 6Wroblewska, B.; Santi, M. R.; Neale, J. H. N-Acetylaspartylglutamate Activates Cyclic AMP-Coupled Metabotropic Glutamate Receptors in Cerebellar Astrocytes. Glia 1998, 24 (2), 172– 179, DOI: 10.1002/(SICI)1098-1136(199810)24:2<172::AID-GLIA2>3.0.CO;2-66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK1czps12ltg%253D%253D&md5=2e2c0f07b9e087565a76a407d6b1866eN-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytesWroblewska B; Santi M R; Neale J HGlia (1998), 24 (2), 172-9 ISSN:0894-1491.N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide in the mammalian nervous system. NAAG meets the traditional criteria of a neurotransmitter, including localization in synaptic vesicles, depolarization-induced release, low potency activation of some N-methyl-D-aspartate receptors, and highly selective activation of a cAMP-coupled metabotropic glutamate receptor (mGluR) with potency approaching that of glutamate. The peptide is present in cultured cortical glia in high concentration and is hydrolyzed by cell surface peptidase activity. In the present work, we tested the hypothesis that NAAG selectively activates a subclass of metabotropic receptors on cultured rat cerebellar glia, primarily astrocytes. These glial cells express mRNA for mGluR subtypes 1, 3, 4, 5, 6, 7, and 8. We were not able to detect message for mGluR2 in these cells using the reverse transcriptase-polymerase chain reaction. Cerebellar glia responded to NAAG, glutamate, and trans-ACPD with a decrease in forskolin-stimulated cAMP formation. AP4, an agonist of the group III receptors mGluR4, mGluR6, mGluR7, and mGluR8, had little or no effect on stimulated cAMP levels. Treatment with low micromolar NAAG significantly increased uptake of radioactive thymidine by cultured astrocytes through activation of metabotropic glutamate receptors. Antagonists of group II mGluRs prevented the decrease in cAMP and the increase in uptake of thymidine by NAAG. Cultured cerebellar astrocytes expressed 20 pmol NAAG per mg protein, a value that is at least 30-fold lower than that expressed by mixed glial cultures prepared from mouse cortex. We conclude that cerebellar astrocytes respond to NAAG via the mGluR3 receptor and that the peptide may selectively activate this receptor subtype in astrocytes following release from neurons or glia.
- 7Wroblewska, B. NAAG as a Neurotransmitter. Adv. Exp. Med. Biol. 2006, 576, 317– 325, DOI: 10.1007/0-387-30172-0_237https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtFygtrs%253D&md5=19e8133bd82cc9e117e85bcded7f9b2dNAAG as a neurotransmitterWroblewska, BarbaraAdvances in Experimental Medicine and Biology (2006), 576 (N-Acetylaspartate), 317-325CODEN: AEMBAP; ISSN:0065-2598. (Springer)A review. A review on the role of N-acetylaspartylglutamate (NAAG) as a neurotransmitter. It discusses NAAG in the context of pain models and schizophrenia.
- 8Romei, C.; Raiteri, M.; Raiteri, L. Glycine Release Is Regulated by Metabotropic Glutamate Receptors Sensitive to mGluR2/3 Ligands and Activated by N-Acetylaspartylglutamate (NAAG). Neuropharmacology 2013, 66, 311– 316, DOI: 10.1016/j.neuropharm.2012.05.0308https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XoslGls7o%253D&md5=ab561f27a0d7ffa91fbacb727970ccffGlycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG)Romei, Cristina; Raiteri, Maurizio; Raiteri, LucaNeuropharmacology (2013), 66 (), 311-316CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [3H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY 379268 inhibited the K+-evoked overflow of [3H]glycine in a concn.-dependent manner (EC50 about 0.2 nM). The effect of LY 379268 was prevented by the selective mGluR2/3 antagonist LY 341495 (IC50 about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [3H]glycine overflow with extraordinary potency (EC50 about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of com. NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY 341495 antagonized the NAAG inhibition of [3H]glycine release. The effect of a combination of maximally effective concns. of LY 379268 and NAAG exhibited no additivity. The non-hydrolyzable NAAG analog N-acetylaspartyl-β-linked glutamate (β-NAAG) antagonized NAAG and LY 379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacol.
- 9Ghose, S.; Wroblewska, B.; Corsi, L.; Grayson, D. R.; De Blas, A. L.; Vicini, S.; Neale, J. H. N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABA(A) alpha6 Subunit in Cerebellar Granule Cells. J. Neurochem. 1997, 69 (6), 2326– 2335, DOI: 10.1046/j.1471-4159.1997.69062326.x9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXntlKlsL8%253D&md5=271106d6c26559d0c3d6166eb0c66590N-Acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABAA α6 subunit in cerebellar granule cellGhose, Subroto; Wroblewska, Barbara; Corsi, Lorenzo; Grayson, Dennis R.; De Blas, Angel L.; Vicini, Stefano; Neale, Joseph H.Journal of Neurochemistry (1997), 69 (6), 2326-2335CODEN: JONRA9; ISSN:0022-3042. (Lippincott-Raven Publishers)We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 μM) results in the transient increase in content of GABAA α6 subunit mRNA. Using quant. PCR, this increase was detd. to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)- 2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors neg. linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABAB receptors. Forskolin stimulation of cAMP levels abolished the effect of NAAG. The change in α6 levels induced by 30 μM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concn.-dependent manner, indicating an increase in functional α6-contg. GABAA receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABAA α6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABAA receptor subunit compn.
- 10Thomas, A. G.; Liu, W.; Olkowski, J. L.; Tang, Z.; Lin, Q.; Lu, X. C.; Slusher, B. S. Neuroprotection Mediated by Glutamate Carboxypeptidase II (NAALADase) Inhibition Requires TGF-Beta. Eur. J. Pharmacol. 2001, 430 (1), 33– 40, DOI: 10.1016/S0014-2999(01)01239-010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXotFWiur8%253D&md5=7cc06bc1105eaa01d53d18302f6de90fNeuroprotection mediated by glutamate carboxypeptidase II (NAALADase) inhibition requires TGF-βThomas, Ajit G.; Liu, Weilin; Olkowski, Jennifer L.; Tang, Zhaocheng; Lin, Qian; Lu, Xi-Chun M.; Slusher, Barbara S.European Journal of Pharmacology (2001), 430 (1), 33-40CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier Science B.V.)Inhibition of glutamate carboxypeptidase (GCP) II (EC 3.4.17.21), also termed N-acetylated alpha-linked acidic dipeptidase (NAALADase), has been shown to protect against ischemic injury presumably via decreasing glutamate and increasing N-acetyl-aspartyl-glutamate (NAAG). NAAG is a potent and selective mGlu3 receptor agonist. Activation of glial mGlu3 receptors has been shown to protect against NMDA toxicity by releasing transforming growth factors, TGF-βs. We hypothesized that GCP II inhibition could be neuroprotective also via TGF-βs, due to increased NAAG. To verify this, Enzyme-Linked Immunosorbent Assays (ELISAs) were performed on media from both control and ischemic cultures treated with the GCP II inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). We found that 2-PMPA attenuated ischemia-induced declines in TGF-β. To further assess the role of TGF-βs in 2-PMPA-mediated neuroprotection, a neutralizing antibody to TGF-β (TGF-β Ab) was used. In both in vitro and in vivo models of cerebral ischemia, TGF-β Ab reversed the neuroprotection by 2-PMPA. Antibodies to other growth factors had no effect. Data suggests that neuroprotection by GCP II inhibition may be partially mediated by promoting TGF-β release.
- 11Vornov, J. J.; Wozniak, K.; Lu, M.; Jackson, P.; Tsukamoto, T.; Wang, E.; Slusher, B. Blockade of NAALADase: A Novel Neuroprotective Strategy Based on Limiting Glutamate and Elevating NAAG. Ann. N.Y. Acad. Sci. 1999, 890, 400– 405, DOI: 10.1111/j.1749-6632.1999.tb08019.x11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXnsVGhtQ%253D%253D&md5=93aa2b9a1609c5675a0ba5aea5a0d7eeBlockade of NAALADase: a novel neuroprotective strategy based on limiting glutamate and elevating NAAGVornov, James J.; Wozniak, Krystyna; Lu, May; Jackson, Paul; Tsukamoto, Takashi; Wang, Eric; Slusher, BarbaraAnnals of the New York Academy of Sciences (1999), 890 (Neuroprotective Agents), 400-405CODEN: ANYAA9; ISSN:0077-8923. (New York Academy of Sciences)Excessive glutamate receptor activation is thought to be involved in the neuronal injury caused by stroke. Based on the hypothesis that N-acetyl-aspartyl-glutamate (NAAG) is a modulatory neurotransmitter or storage form of glutamate, the authors have pursued a novel strategy of therapeutic intervention, blockade of N-acetylated alpha-linked acidic dipeptidase (NAALADase), the enzyme that hydrolyzes NAAG to liberate glutamate. Using the suture model of transient middle cerebral artery occlusion (MCAO) in rats, the prototype NAALADase inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) dramatically reduced extracellular glutamate accumulation measured by microdialysis both during a 2-h occlusion and during reperfusion, consistent with an effect on glutamate supply. During reperfusion, the decrease in glutamate was accompanied by an equimolar, reciprocal rise in extracellular NAAG. NAALADase inhibition may prove to be a well tolerated therapy for cerebral ischemia. In addn., NAALADase inhibitors should prove to be important tools in understanding the physiol. role of NAAG in the brain.
- 12Williams, A. J.; Lu, X. M.; Slusher, B.; Tortella, F. C. Electroencephalogram Analysis and Neuroprotective Profile of the N-Acetylated-Alpha-Linked Acidic Dipeptidase Inhibitor, GPI5232, in Normal and Brain-Injured Rats. J. Pharmacol. Exp. Ther. 2001, 299 (1), 48– 5712https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXntFWltro%253D&md5=016872fdcd14c17504959e5b03601b31Electroencephalogram analysis and neuroprotective profile of the N-acetylated-α-linked acidic dipeptidase inhibitor, GPI5232, in normal and brain-injured ratsWilliams, A. J.; Lu, X. M.; Slusher, B.; Tortella, F. C.Journal of Pharmacology and Experimental Therapeutics (2001), 299 (1), 48-57CODEN: JPETAB; ISSN:0022-3565. (American Society for Pharmacology and Experimental Therapeutics)We have evaluated the effects of the N-acetylated-α-linked acidic dipeptidase (NAALADase) inhibitor, GPI5232 [2-([(pentafluorophenylmethyl)hydroxyphosphinyl]methyl)-pentanedioic acid], to not only decrease brain injury but also to alter the inherent electroencephalog. (EEG) changes obsd. in a rat model of transient middle cerebral artery occlusion (MCAo). Continuous i.v. infusion of GPI5232 starting 1 h after injury resulted in more than a 50% redn. in brain infarct vol. caused by 2 h of MCAo. This effect was dose-dependent and significant even when first treatment was delayed for 2 h post-MCAo. At 24 h post-MCAo, EEG spectral anal. of the injured hemisphere revealed functional improvement in GPI5232-treated rats. Significant recovery in high-frequency EEG power (8-30 Hz) was measured in GPI5232-treated animals in both parietal and temporal brain regions but not in vehicle-treated animals. MCAo-injured rats were also predisposed to developing cortical brain seizures, and GPI5232-treated rats had significantly fewer brain seizures than vehicle-treated animals. In sep. expts., acute high doses of GPI5232 in normal rats did not significantly alter EEG brain activity as evaluated by spectral anal. and did not produce any signs of seizure activity or behavioral abnormalities. These results show GPI5232 to be an effective neuroprotective treatment when given postinjury by reducing brain infarction and ameliorating the pathol. EEG assocd. with focal brain ischemia.
- 13Cai, Z.; Lin, S.; Rhodes, P. G. Neuroprotective Effects of N-Acetylaspartylglutamate in a Neonatal Rat Model of Hypoxia-Ischemia. Eur. J. Pharmacol. 2002, 437 (3), 139– 145, DOI: 10.1016/S0014-2999(02)01289-X13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhvVWrt7o%253D&md5=571c0efc81a95837323203319f9f9065Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemiaCai, Zhengwei; Lin, Shuying; Rhodes, Philip G.European Journal of Pharmacology (2002), 437 (3), 139-145CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier Science B.V.)Neuroprotective effects of N-acetylaspartylglutamate (NAAG), the precursor of glutamate and a selective agonist at the Group II metabotropic glutamate (mGlu) receptor, against hypoxic-ischemic brain injury were examd. in a neonatal rat model of cerebral hypoxia-ischemia. The neonatal hypoxia-ischemia procedure (unilateral carotid artery ligation followed by exposure to an 8% oxygen hypoxic condition for 1.5 h) was performed in 7-day-old rat pups. Following unilateral carotid artery ligation, NAAG (0.5 to 20 mg/kg, i.p.) was administered before or after the hypoxic exposure. Brain injury was examd. 1-wk later by wt. redn. in the ipsilateral brain and by neuron d. in the hippocampal CA1 area. In the saline-treated rat, neonatal hypoxia-ischemia resulted in severe brain injury as indicated by a 24% redn. in the ipsilateral brain wt. Low doses of NAAG (2-10 mg/kg, but not 0.5 mg/kg), administered before or even if 1 h after the hypoxic exposure, greatly reduced hypoxia-ischemia-induced brain injury (3.8-14.2% redn. in the ipsilateral brain wt.). A high dose of NAAG (20 mg/kg) was ineffective. While l(+)-2-Amino-4-phosphonobutyric acid (L-AP4) and trans-[1S,3R]-1-Amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) were unable to provide protection against hypoxic-ischemic brain injury, 2-(phosphonomethyl) pentanedioic acid (2-PMPA), an inhibitor of N-acetylated alpha-linked acidic dipeptidase (NAALADase), which hydrolyzes endogenous NAAG into N-acetyl-aspartate and glutamate, significantly reduced neonatal hypoxia-ischemia-induced brain injury. (αS)-α-Amino-α-[(1S, 2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), a selective antagonist at the mGlu2/3 receptor, prevented the neuroprotective effect of NAAG. Neuron d. data measured in the hippocampal CA1 area confirmed that ipsilateral brain wt. redn. was a valid measure for hypoxic-ischemic brain injury. Neonatal hypoxia-ischemia stimulated an elevation of cAMP concn. in the saline-treated rat brain. NAAG, L-AP4 and t-ACPD all significantly decreased hypoxia-ischemia-induced elevation of cAMP. LY341495 blocked the effect of NAAG, but not of L-AP4 or t-ACPD, on hypoxia-ischemia-stimulated cAMP elevation. The overall results suggest that the neuroprotective effect of NAAG is largely assocd. with activation of mGlu2/3 receptor.
- 14Zhong, C.; Zhao, X.; Sarva, J.; Kozikowski, A.; Neale, J. H.; Lyeth, B. G. NAAG Peptidase Inhibitor Reduces Acute Neuronal Degeneration and Astrocyte Damage Following Lateral Fluid Percussion TBI in Rats. J. Neurotrauma 2005, 22 (2), 266– 276, DOI: 10.1089/neu.2005.22.26614https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2M%252Fos12gtw%253D%253D&md5=9213155428f974705aa703a2fe2871cfNAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in ratsZhong Chunlong; Zhao Xueren; Sarva Jayaprakash; Kozikowski Alan; Neale Joseph H; Lyeth Bruce GJournal of neurotrauma (2005), 22 (2), 266-76 ISSN:0897-7151.Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate associated with excitotoxicity and secondary brain pathology. The peptide neurotransmitter Nacetylaspartylglutamate (NAAG) suppresses glutamate transmission through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Thus, inhibition of NAAG peptidase activity and the prolong presence of synaptic NAAG were hypothesized to have significant potential for cellular protection following TBI. In the present study, a novel NAAG peptidase inhibitor, ZJ-43, was used in four different doses (0, 50, 100, or 150 mg/kg). Each dose was repeatedly administered i.p. (n=5/group) by multiple injections at three times (0 time, 8 h, 16 h) after moderate lateral fluid percussion TBI in the rat. An additional group was co-administered ZJ-43 (150 mg/kg) and the Group II mGluR antagonist, LY341495 (1 mg/kg), which was predicted to abolish any protective effects of ZJ-43. Rats were euthanized at 24 h after TBI, and brains were processed with a selective marker for degenerating neurons (Fluoro-Jade B) and a marker for astrocytes (GFAP). Ipsilateral neuronal degeneration and bilateral astrocyte loss in the CA2/3 regions of the hippocampus were quantified using stereological techniques. Compared with vehicle, ZJ-43 significantly reduced the number of the ipsilateral degenerating neurons (p<0.01) with the greatest neuroprotection at the 50 mg/kg dose. Moreover, LY341495 successfully abolished the protective effects of ZJ-43. 50 mg/kg of ZJ-43 also significantly reduced the ipsilateral astrocyte loss (p<0.05). We conclude that the NAAG peptidase inhibitor ZJ-43 is a potential novel strategy to reduce both neuronal and astrocyte damage associated with the glutamate excitotoxicity after TBI.
- 15Ghadge, G. D.; Slusher, B. S.; Bodner, A.; Canto, M. D.; Wozniak, K.; Thomas, A. G.; Rojas, C.; Tsukamoto, T.; Majer, P.; Miller, R. J.; Monti, A. L.; Roos, R. P. Glutamate Carboxypeptidase II Inhibition Protects Motor Neurons from Death in Familial Amyotrophic Lateral Sclerosis Models. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (16), 9554– 9559, DOI: 10.1073/pnas.153016810015https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmtlyku7k%253D&md5=9142ca962162bd94ee0d25e0bf3f1516Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis modelsGhadge, Ghanashyam D.; Slusher, Barbara S.; Bodner, Amos; Dal Canto, Mauro; Wozniak, Krystyna; Thomas, Ajit G.; Rojas, Camilo; Tsukamoto, Takashi; Majer, Pavel; Miller, Richard J.; Monti, Anna Liza; Roos, Raymond P.Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (16), 9554-9559CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Approx. 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and ≈ 20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.
- 16Yamamoto, T.; Kozikowski, A.; Zhou, J.; Neale, J. H. Intracerebroventricular Administration of N-Acetylaspartylglutamate (NAAG) Peptidase Inhibitors Is Analgesic in Inflammatory Pain. Mol. Pain 2008, 4, 31, DOI: 10.1186/1744-8069-4-3116https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1crit1Cqug%253D%253D&md5=d4b7128c2e8ad234ac923de798c3da4cIntracerebroventricular administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory painYamamoto Tatsuo; Kozikowski Alan; Zhou Jia; Neale Joseph HMolecular pain (2008), 4 (), 31 ISSN:.BACKGROUND: The peptide neurotransmitter N-Acetylaspartylglutamate (NAAG) is the third most prevalent transmitter in the mammalian central nervous system. Local, intrathecal and systemic administration of inhibitors of enzymes that inactivate NAAG decrease responses to inflammatory pain in rat models. Consistent with NAAG's activation of group II metabotropic glutamate receptors, this analgesia is blocked by a group II antagonist. RESULTS: This research aimed at determining if analgesia obtained following systemic administration of NAAG peptidase inhibitors is due to NAAG activation of group II mGluRs in brain circuits that mediate perception of inflammatory pain. NAAG and NAAG peptidase inhibitors, ZJ43 and 2-PMPA, were microinjected into a lateral ventricle prior to injection of formalin in the rat footpad. Each treatment reduced the early and late phases of the formalin-induced inflammatory pain response in a dose-dependent manner. The group II mGluR antagonist reversed these analgesic effects consistent with the conclusion that analgesia was mediated by increasing NAAG levels and the peptide's activation of group II receptors. CONCLUSION: These data contribute to proof of the concept that NAAG peptidase inhibition is a novel therapeutic approach to inflammatory pain and that these inhibitors achieve analgesia by elevating synaptic levels of NAAG within pain processing circuits in brain.
- 17Yamamoto, T.; Saito, O.; Aoe, T.; Bartolozzi, A.; Sarva, J.; Zhou, J.; Kozikowski, A.; Wroblewska, B.; Bzdega, T.; Neale, J. H. Local Administration of N-Acetylaspartylglutamate (NAAG) Peptidase Inhibitors Is Analgesic in Peripheral Pain in Rats. Eur. J. Neurosci. 2007, 25 (1), 147– 158, DOI: 10.1111/j.1460-9568.2006.05272.x17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2s%252Fkt1Skug%253D%253D&md5=0dc9a94990e727813453ae3aba93dae7Local administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in ratsYamamoto Tatsuo; Saito Osamu; Aoe Tomohiko; Bartolozzi Alessandra; Sarva Jayaprakash; Zhou Jia; Kozikowski Alan; Wroblewska Barbara; Bzdega Tomasz; Neale Joseph HThe European journal of neuroscience (2007), 25 (1), 147-58 ISSN:0953-816X.The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) selectively activates group II metabotropic glutamate receptors (mGluRs). Systemic administration of inhibitors of the enzymes that inactivate NAAG results in decreased pain responses in rat models of inflammatory and neuropathic pain. These effects are blocked by a group II mGluR antagonist. This research tested the hypothesis that some analgesic effects of NAAG peptidase inhibition are mediated by NAAG acting on sensory neurite mGluRs at the site of inflammation. Group II mGluR agonists, SLx-3095-1, NAAG and APDC, or NAAG peptidase inhibitors, ZJ-43 and 2-PMPA, injected into the rat footpad reduced pain responses in carrageenan or formalin models. The analgesic effects of SLx-3095-1, APDC, ZJ-43, 2-PMPA and NAAG were blocked by co-injection of LY341495, a selective group II mGluR antagonist. Injection of group II mGluR agonists, NAAG or the peptidase inhibitors into the contralateral rat footpad had no effect on pain perception in the injected paw. At 10-100 microm ZJ-43 and 2-PMPA demonstrated no consistent agonist activity at mGluR2 or mGluR3. Consistent with the conclusion that peripherally administered NAAG peptidase inhibitors increase the activation of mGluR3 by NAAG that is released from peripheral sensory neurites, we found that the tissue average concentration of NAAG in the unstimulated rat hind paw was about 6 microm. These data extend our understanding of the role of this peptide in sensory neurons and reveal the potential for treatment of inflammatory pain via local application of NAAG peptidase inhibitors at doses that may have little or no central nervous system effects.
- 18Zhang, W.; Murakawa, Y.; Wozniak, K. M.; Slusher, B.; Sima, A. A. F. The Preventive and Therapeutic Effects of GCPII (NAALADase) Inhibition on Painful and Sensory Diabetic Neuropathy. J. Neurol. Sci. 2006, 247 (2), 217– 223, DOI: 10.1016/j.jns.2006.05.05218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XpsVKksrY%253D&md5=31c8dadecc4fb2c34346f4fbc1c38e7fThe preventive and therapeutic effects of GCPII (NAALADase) inhibition on painful and sensory diabetic neuropathyZhang, W.; Murakawa, Y.; Wozniak, K. M.; Slusher, B.; Sima, A. A. F.Journal of the Neurological Sciences (2006), 247 (2), 217-223CODEN: JNSCAG; ISSN:0022-510X. (Elsevier B.V.)Excitotoxic glutamate release occurs in several neurol. disorders. One source is derived from the hydrolysis of the neuropeptide N-acetyl aspartyl glutamate (NAAG) by glutamate carboxypeptidase II (GCPII, also known as NAALADase). Drugs that attenuate glutamate transmission have been shown to relieve neuropathic pain, however side effects have limited their clin. use. It appears that GCPII is exclusively recruited to provide a glutamate source in hyperglutamatergic, excitotoxic conditions and therefore would be devoid of such side effects. Here we report on the therapeutic effects of an orally bio-available GCP II inhibitor on established painful and sensory neuropathy in the spontaneously diabetic BB/Wor rat. It significantly improved hyperalgesia, nerve conduction velocity and underlying myelinated fiber atrophy. The data suggest that GCP II inhibition may provide a meaningful and effective approach to the treatment of painful diabetic neuropathy.
- 19Wozniak, K. M.; Wu, Y.; Vornov, J. J.; Lapidus, R.; Rais, R.; Rojas, C.; Tsukamoto, T.; Slusher, B. S. The Orally Active Glutamate Carboxypeptidase II Inhibitor E2072 Exhibits Sustained Nerve Exposure and Attenuates Peripheral Neuropathy. J. Pharmacol. Exp. Ther. 2012, 343 (3), 746– 754, DOI: 10.1124/jpet.112.19766519https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhslyjs7zN&md5=eb9a44606e863472a46b5b6ce9eca88dThe orally active glutamate carboxypeptidase II inhibitor E2072 exhibits sustained nerve exposure and attenuates peripheral neuropathyWozniak, Krystyna M.; Wu, Ying; Vornov, James J.; Lapidus, Rena; Rais, Rana; Rojas, Camilo; Tsukamoto, Takashi; Slusher, Barbara S.Journal of Pharmacology and Experimental Therapeutics (2012), 343 (3), 746-754CODEN: JPETAB; ISSN:1521-0103. (American Society for Pharmacology and Experimental Therapeutics)Peripheral neuropathy from nerve trauma is a significant problem in the human population and often constitutes a dose-limiting toxicity in patients receiving chemotherapy. (3-2-Mercaptoethyl)biphenyl-2,3-dicarboxylic acid (E2072) is a potent (Ki = 10 nM), selective, and orally available inhibitor of glutamate carboxypeptidase II (GCPII). Here, we report that E2072 attenuates hyperalgesia and nerve conduction velocity deficits in preclin. rodent models of neuropathic pain and oxaliplatin-induced neuropathy. In the chronic constrictive injury model, orally administered E2072 reversed pre-existing thermal hyperalgesia in rats in a dose-dependent fashion with a minimally ED of 0.1 mg/kg/day. It is noteworthy that multiple days of dosing of E2072 were required before analgesia was realized even though GCPII inhibitory exposures were achieved on the 1st day of dosing. In addn., analgesia was found to persist for ≤ 7 days after cessation of dosing, consistent with E2072's pharmacokinetic profile and sustained exposure. Furthermore, in a chronic oxaliplatin-induced neuropathy model (6 mg/kg i.p. oxaliplatin twice weekly for 4 wk), female BALB/c mice receiving daily oral E2072 at 1.0 and 0.1 mg/kg displayed no deficits in either caudal or digital velocity compared with significant deficits obsd. in mice treated with oxaliplatin alone (12 and 9%, resp.). Similar findings were seen with oxaliplatin-induced digital and caudal amplitude deficits. It is noteworthy that E2072 showed no interference with the antineoplastic efficacy of oxaliplatin in mice bearing leukemia (L1210), even at doses 100 times its neuroprotective/analgesic dose, indicating a selective effect on neuropathy. These data support the therapeutic utility of GCPII inhibitors in neuropathy and neuropathic pain.
- 20Olszewski, R. T.; Bukhari, N.; Zhou, J.; Kozikowski, A. P.; Wroblewski, J. T.; Shamimi-Noori, S.; Wroblewska, B.; Bzdega, T.; Vicini, S.; Barton, F. B.; Neale, J. H. NAAG Peptidase Inhibition Reduces Locomotor Activity and Some Stereotypes in the PCP Model of Schizophrenia via Group II mGluR. J. Neurochem. 2004, 89 (4), 876– 885, DOI: 10.1111/j.1471-4159.2004.02358.x20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXksVyksLg%253D&md5=4ca607a68135b77ae0cd997eed5d2566NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluROlszewski, Rafal T.; Bukhari, Noreen; Zhou, Jia; Kozikowski, Alan P.; Wroblewski, Jarda T.; Shamimi-Noori, Susan; Wroblewska, Barbara; Bzdega, Tomasz; Vicini, Stefano; Barton, Franca B.; Neale, Joseph H.Journal of Neurochemistry (2004), 89 (4), 876-885CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)Phencyclidine (PCP) administration elicits pos. and neg. symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochem. effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. The authors synthesized and characterized a urea-based NAAG analog, ZJ43. This novel compd. is a potent inhibitor of enzymes, glutamate carboxypeptidase II (Ki = 0.8 nM) and III (Ki = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 μM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Addnl., LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.
- 21Profaci, C. P.; Krolikowski, K. A.; Olszewski, R. T.; Neale, J. H. Group II mGluR Agonist LY354740 and NAAG Peptidase Inhibitor Effects on Prepulse Inhibition in PCP and D-Amphetamine Models of Schizophrenia. Psychopharmacology 2011, 216 (2), 235– 243, DOI: 10.1007/s00213-011-2200-021https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhvFKltLo%253D&md5=486c472ac2b6bd6a95da455150ef7576Group II mGluR agonist LY354740 and NAAG peptidase inhibitor effects on prepulse inhibition in PCP and D-amphetamine models of schizophreniaProfaci, Caterina P.; Krolikowski, Kristyn A.; Olszewski, Rafal T.; Neale, Joseph H.Psychopharmacology (Heidelberg, Germany) (2011), 216 (2), 235-243CODEN: PSCHDL; ISSN:0033-3158. (Springer)Rationale: Group II metabotropic glutamate receptor (mGluR) agonists represent a novel approach to the treatment of schizophrenia. Inasmuch as the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) activates these receptors, NAAG peptidase inhibitors conceptually represent a parallel path toward development of new antipsychotic drugs. While group II agonists are effective in several animal models of schizophrenia, they are reported to lack efficacy in moderating the effects of phencyclidine (PCP) on prepulse inhibition of acoustic startle in animal models of sensory processing deficits found in this disorder. Objective: The objective of this study was to re-examine the efficacy of a group II metabotropic glutamate agonist and NAAG peptidase inhibitors in prepulse inhibition models of schizophrenia across two strains of mice. Methods: The method used was an assay to det. the efficacy of these drugs in moderating the redn. in prepulse inhibition of acoustic startle in mice treated with PCP and D-amphetamine. Results: The group II agonist LY354740 (5 and 10 mg/kg) moderated the effects of PCP on prepulse inhibition of acoustic startle in DBA/2 but not C57BL/6 mice. In contrast, two NAAG peptidase inhibitors, ZJ43 (150 mg/kg) and 2-PMPA (50, 100, and 150 mg/kg), did not significantly affect the PCP-induced redn. in prepulse inhibition in either strain. Conclusions: These data demonstrate that the efficacy of group II agonists in this model of sensory motor processing is strain-specific in mice. The difference between the effects of the group II agonist and the peptidase inhibitors in the DBA/2 mice may relate to the difference in efficacy of NAAG and the agonist at mGluR2.
- 22Tsai, G.; Dunham, K. S.; Drager, U.; Grier, A.; Anderson, C.; Collura, J.; Coyle, J. T. Early Embryonic Death of Glutamate Carboxypeptidase II (NAALADase) Homozygous Mutants. Synapse 2003, 50 (4), 285– 292, DOI: 10.1002/syn.1026322https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovVyhtLc%253D&md5=2d05eeef754d6103e4a2e2fdfa25251aEarly embryonic death of glutamate carboxypeptidase II (NAALADase) homozygous mutantsTsai, G.; Dunham, K. S.; Drager, U.; Grier, A.; Anderson, C.; Collura, J.; Coyle, J. T.Synapse (New York, NY, United States) (2003), 50 (4), 285-292CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss, Inc.)Glutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 μM) of the neuropeptide N-acetylaspartylglutamate to yield N-acetylaspartate and glutamate and also serves as a high-affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N-acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N-acetylaspartylglutamate. The PGK-Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild-type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild-type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis.
- 23Han, L.; Picker, J. D.; Schaevitz, L. R.; Tsai, G.; Feng, J.; Jiang, Z.; Chu, H. C.; Basu, A. C.; Berger-Sweeney, J.; Coyle, J. T. Phenotypic Characterization of Mice Heterozygous for a Null Mutation of Glutamate Carboxypeptidase II. Synapse 2009, 63 (8), 625– 635, DOI: 10.1002/syn.2064923https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnslGisL8%253D&md5=2bdc02c54d27d9f4a903c4474ebb0170Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase IIHan, Liqun; Picker, Jonathan D.; Schaevitz, Laura R.; Tsai, Guochuan; Feng, Jiamin; Jiang, Zhichun; Chu, Hillary C.; Basu, Alo C.; Berger-Sweeney, Joanne; Coyle, Joseph T.Synapse (Hoboken, NJ, United States) (2009), 63 (8), 625-635CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss, Inc.)Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of N-methyl-D-aspartate receptors (NMDAR) was implicated in the pathophysiol. of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N-acetyl-α L-aspartyl-L-glutamate (NAAG) into glutamate and N-acetyl-aspartate. NAAG is a neuropeptide that is an NMDAR antagonist as well as an agonist for the metabotropic glutamate receptor-3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDAR activation. To manipulate the expression of GCP II, LoxP sites were inserted flanking exons 1 and 2, which were excised by crossing with a Cre-expressing mouse. The mice heterozygous for this deletion showed a 50% redn. in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N > 200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm the authors' hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory.
- 24Vorlová, B.; Sedlák, F.; Kašpárek, P.; Šrámková, K.; Malý, M.; Zámečník, J.; Šácha, P.; Konvalinka, J. A Novel PSMA/GCPII-Deficient Mouse Model Shows Enlarged Seminal Vesicles upon Aging. Prostate 2019, 79 (2), 126– 139, DOI: 10.1002/pros.2371724https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1Sgu77P&md5=a8219bb4f4f46aaba0d2ef8cb095ddd0A novel PSMA/GCPII-deficient mouse model shows enlarged seminal vesicles upon agingVorlova, Barbora; Sedlak, Frantisek; Kasparek, Petr; Sramkova, Karolina; Maly, Marek; Zamecnik, Josef; Sacha, Pavel; Konvalinka, JanProstate (Hoboken, NJ, United States) (2019), 79 (2), 126-139CODEN: PRSTDS; ISSN:0270-4137. (Wiley-Blackwell)Background : Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is an important diagnostic and therapeutic target in prostate cancer. PSMA/GCPII is also expressed in many healthy tissues, but its function has only been established in the brain and small intestine. Several research groups have attempted to produce PSMA/GCPII-deficient mice to study the physiol. role of PSMA/GCPII in detail. The outcomes of these studies differ dramatically, ranging from embryonic lethality to prodn. of viable PSMA/GCPII-deficient mice without any obvious phenotype. Methods : We produced PSMA/GCPII-deficient mice (hereafter also referred as Folh1-/- mice) by TALEN-mediated mutagenesis on a C57BL/6NCrl background. Using Western blot and an enzyme activity assay, we confirmed the absence of PSMA/GCPII in our Folh1-/- mice. We performed anatomical and histopathol. examn. of selected tissues with a focus on urogenital system. We also examd. the PSMA/GCPII expression profile within the mouse urogenital system using an enzyme activity assay and confirmed the presence of PSMA/GCPII in selected tissues by immunohistochem. Results : Our Folh1-/- mice are viable, breed normally, and do not show any obvious phenotype. Nevertheless, aged Folh1-/- mice of 69-72 wk exhibit seminal vesicle dilation, which is caused by accumulation of luminal fluid. This phenotype was also obsd. in Folh1+/- mice; the overall difference between our three cohorts (Folh1-/-, Folh1+/-, and Folh1+/+) was highly significant (P < 0.002). Of all studied tissues of the mouse urogenital system, only the epididymis appeared to have a physiol. relevant level of PSMA/GCPII expression. Addnl. expts. demonstrated that PSMA/GCPII is also present in the human epididymis. Conclusions : In this study, we provide the first evidence characterizing the reproductive tissue phenotype of PSMA/GCPII-deficient mice. These findings will help lay the groundwork for future studies to reveal PSMA/GCPII function in human reprodn.
- 25Bacich, D. J.; Ramadan, E.; O’Keefe, D. S.; Bukhari, N.; Wegorzewska, I.; Ojeifo, O.; Olszewski, R.; Wrenn, C. C.; Bzdega, T.; Wroblewska, B.; Heston, W. D. W.; Neale, J. H. Deletion of the Glutamate Carboxypeptidase II Gene in Mice Reveals a Second Enzyme Activity That Hydrolyzes N-Acetylaspartylglutamate. J. Neurochem. 2002, 83 (1), 20– 29, DOI: 10.1046/j.1471-4159.2002.01117.x25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XnvVGgsL0%253D&md5=34967d94f7bade511355a412218eacb6Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamateBacich, Dean J.; Ramadan, Epolia; O'Keefe, Denise S.; Bukhari, Noreen; Wegorzewska, Iga; Ojeifo, Olumide; Olszewski, Rafal; Wrenn, Craige C.; Bzdega, Tomasz; Wroblewska, Barbara; Heston, Warren D. W.; Neale, Joseph H.Journal of Neurochemistry (2002), 83 (1), 20-29CODEN: JONRA9; ISSN:0022-3042. (Blackwell Science Ltd.)Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face of glia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. MRNA for GCPII was not detected by northern blotting or RT-PCR anal. of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurol. responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were obsd. among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII null mutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nM and 2494 nM, resp. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nM for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned rat and human GCPII, and 88 nM for the activity in brain membranes of the null mutants.
- 26Gao, Y.; Xu, S.; Cui, Z.; Zhang, M.; Lin, Y.; Cai, L.; Wang, Z.; Luo, X.; Zheng, Y.; Wang, Y.; Luo, Q.; Jiang, J.; Neale, J. H.; Zhong, C. Mice Lacking Glutamate Carboxypeptidase II Develop Normally, but Are Less Susceptible to Traumatic Brain Injury. J. Neurochem. 2015, 134 (2), 340– 353, DOI: 10.1111/jnc.1312326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXntFCktb0%253D&md5=6fb001f691d426cf112a265a8aa5a27eMice lacking glutamate carboxypeptidase II develop normally, but are less susceptible to traumatic brain injuryGao, Yang; Xu, Siyi; Cui, Zhenwen; Zhang, Mingkun; Lin, Yingying; Cai, Lei; Wang, Zhugang; Luo, Xingguang; Zheng, Yan; Wang, Yong; Luo, Qizhong; Jiang, Jiyao; Neale, Joseph H.; Zhong, ChunlongJournal of Neurochemistry (2015), 134 (2), 340-353CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)Glutamate carboxypeptidase II (GCPII) is a transmembrane zinc metallopeptidase found mainly in the nervous system, prostate and small intestine. In the nervous system, glia-bound GCPII mediates the hydrolysis of the neurotransmitter N-acetylaspartylglutamate (NAAG) into glutamate and N-acetylaspartate. Inhibition of GCPII has been shown to attenuate excitotoxicity assocd. with enhanced glutamate transmission under pathol. conditions. However, different strains of mice lacking the GCPII gene are reported to exhibit striking phenotypic differences. In this study, a GCPII gene knockout (KO) strategy involved removing exons 3-5 of GCPII. This generated a new GCPII KO mice line with no overt differences in std. neurol. behavior compared to their wild-type (WT) littermates. However, GCPII KO mice were significantly less susceptible to moderate traumatic brain injury (TBI). GCPII gene KO significantly lessened neuronal degeneration and astrocyte damage in the CA2 and CA3 regions of the hippocampus 24 h after moderate TBI. In addn., GCPII gene KO reduced TBI-induced deficits in long-term spatial learning/memory tested in the Morris water maze and motor balance tested via beam walking. Knockout of the GCPII gene is not embryonic lethal and affords histopathol. protection with improved long-term behavioral outcomes after TBI, a result that further validates GCPII as a target for drug development consistent with results from studies using GCPII peptidase inhibitors. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) suppresses glutamate transmission through selective activation of pre-synaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3) after traumatic brain injury (TBI). However, synaptically released NAAG is hydrolyzed to form N-acetylaspartate and glutamate mainly by Glutamate carboxypeptidase II (GCPII), losing neuroprotective effect. In this study, we found that knock out of the GCPII gene is not embryonic lethal and affords histopathol. protection with improved long-term behavioral outcomes after TBI.
- 27Bacich, D. J.; Wozniak, K. M.; Lu, X.-C. M.; O’Keefe, D. S.; Callizot, N.; Heston, W. D. W.; Slusher, B. S. Mice Lacking Glutamate Carboxypeptidase II Are Protected from Peripheral Neuropathy and Ischemic Brain Injury. J. Neurochem. 2005, 95 (2), 314– 323, DOI: 10.1111/j.1471-4159.2005.03361.x27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFegu7rE&md5=5e91a9605c262a68d2468a961abc517cMice lacking glutamate carboxypeptidase II are protected from peripheral neuropathy and ischemic brain injuryBacich, Dean J.; Wozniak, Krystyna M.; Lu, X.-C. May; O'Keefe, Denize S.; Callizot, Noelle; Heston, Warren D. W.; Slusher, Barbara S.Journal of Neurochemistry (2005), 95 (2), 314-323CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)Excessive glutamate release is assocd. with neuronal damage. A new strategy for the treatment of neuronal injury involves inhibition of the neuropeptidase glutamate carboxypeptidase II (GCP II), also known as N-acetylated α-linked acidic dipeptidase. GCP II is believed to mediate the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to glutamate and N-acetyl-aspartate, and inhibition of NAAG peptidase activity (by GCP II and other peptidases) is neuroprotective. Mice were generated in which the Folh1 gene encoding GCP II was disrupted (Folh1-/- mice). No overt behavioral differences were apparent between Folh1-/- mice and wild-type littermates, with respect to their overall performance in locomotion, coordination, pain threshold, cognition and psychiatric behavioral paradigms. Morphol. anal. of peripheral nerves, however, showed significantly smaller axons (reduced myelin sheaths and axon diams.) in sciatic nerves from Folh1-/- mice. Following sciatic nerve crush, Folh1-/- mice suffered less injury and recovered faster than wild-type littermates. In a model of ischemic injury, the Folh1-/- mice exhibited a significant redn. (p < 0.05) in infarct vol. compared with their wild-type littermates when subjected to middle cerebral artery occlusion, a model of stroke. These findings support the hypothesis that GCP II inhibitors may represent a novel treatment for peripheral neuropathies as well as stroke.
- 28Wolf, N. I.; Willemsen, M. A. A. P.; Engelke, U. F.; van der Knaap, M. S.; Pouwels, P. J. W.; Harting, I.; Zschocke, J.; Sistermans, E. A.; Rating, D.; Wevers, R. A. Severe Hypomyelination Associated with Increased Levels of N-Acetylaspartylglutamate in CSF. Neurology 2004, 62 (9), 1503– 1508, DOI: 10.1212/01.WNL.0000123094.13406.2028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXjt1ylur8%253D&md5=4563458706ff7d724a544db894089d25Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSFWolf, N. I.; Willemsen, M. A. A. P.; Engelke, U. F.; van der Knaap, M. S.; Pouwels, P. J. W.; Harting, I.; Zschocke, J.; Sistermans, E. A.; Rating, D.; Wevers, R. A.Neurology (2004), 62 (9), 1503-1508CODEN: NEURAI; ISSN:0028-3878. (Lippincott Williams & Wilkins)Two unrelated girls had early onset of nystagmus and epilepsy, absent psychomotor development, and almost complete absence of myelin on cerebral MRI. The clin. features and MR images of both patients resembled the connatal form of Pelizaeus-Merzbacher disease (PMD), which is an X-linked recessive disorder caused by duplications or mutations of the proteolipid protein gene (PLP). Thus, studies were carried out to define a unique neurometabolic disorder with failure of myelination. 1H-NMR of CSF in both girls was performed repeatedly, and both showed highly elevated concns. of N-acetylaspartylglutamate (NAAG). The coding sequence of the gene coding for glutamate carboxypeptidase II, which converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced but revealed no mutations. Even though both patients are girls, the authors sequenced the PLP gene and found no abnormality. Thus, NAAG is an abundant peptide neurotransmitter whose exact role is unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its elevated concn. in CSF may be the biochem. hallmark for a novel neurometabolic disorder. The cause of its accumulation is still unclear.
- 29Sartori, S.; Burlina, A. B.; Salviati, L.; Trevisson, E.; Toldo, I.; Laverda, A. M.; Burlina, A. P. Increased Level of N-Acetylaspartylglutamate (NAAG) in the CSF of a Patient with Pelizaeus-Merzbacher-like Disease due to Mutation in the GJA12 Gene. Eur. J. Paediatr. Neurol. 2008, 12 (4), 348– 350, DOI: 10.1016/j.ejpn.2007.07.01129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1czjt1Gqsg%253D%253D&md5=b6195426fa7491ae67c04a915496b40fIncreased level of N-acetylaspartylglutamate (NAAG) in the CSF of a patient with Pelizaeus-Merzbacher-like disease due to mutation in the GJA12 geneSartori Stefano; Burlina Alberto B; Salviati Leonardo; Trevisson Eva; Toldo Irene; Laverda Anna Maria; Burlina Alessandro PEuropean journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society (2008), 12 (4), 348-50 ISSN:1090-3798.Autosomal recessive Pelizaeus-Merzbacher-like disease 1 (PMLD1) is a hypomyelinating disorder of the central nervous system (CNS) with virtually identical phenotype to Pelizaeus-Merzbacher disease (PMD). PMLD1 is caused by mutations in GJA12 gene, PMD is due to mutations in PLP1 gene. Elevated levels of N-acetylaspartylglutamate (NAAG), the most abundant peptide neuromodulator in the human brain, have been recently reported in cerebral spinal fluid (CSF) of patients with PMD. Using capillary electrophoresis, we analyzed for the first time, the CSF from a girl with PMLD1 and detected high concentrations of NAAG. This finding confirms the hypothesis that NAAG may be involved in myelination-related processes and can be considered as a useful diagnostic marker not only for patients with the PLP1 related disorder, but also in those with Pelizaeus-Merzbacher like hypomyelinating disease due to other defined genetic causes, such as PMLD1.
- 30Mochel, F.; Boildieu, N.; Barritault, J.; Sarret, C.; Eymard-Pierre, E.; Seguin, F.; Schiffmann, R.; Boespflug-Tanguy, O. Elevated CSF N-Acetylaspartylglutamate Suggests Specific Molecular Diagnostic Abnormalities in Patients with White Matter Diseases. Biochim. Biophys. Acta 2010, 1802 (11), 1112– 1117, DOI: 10.1016/j.bbadis.2010.07.00530https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtFynsbzE&md5=bf840c684afd421eea48d5022e32324bElevated CSF N-acetylaspartylglutamate suggests specific molecular diagnostic abnormalities in patients with white matter diseasesMochel, Fanny; Boildieu, Nadege; Barritault, Julie; Sarret, Catherine; Eymard-Pierre, Eleonore; Seguin, Francois; Schiffmann, Raphael; Boespflug-Tanguy, OdileBiochimica et Biophysica Acta, Molecular Basis of Disease (2010), 1802 (11), 1112-1117CODEN: BBADEX; ISSN:0925-4439. (Elsevier B. V.)Background: In order to identify biomarkers useful for the diagnosis of genetic white matter disorders we compared the metabolic profile of patients with leukodystrophies with a hypomyelinating or a non-hypomyelinating MRI pattern. Methods: We used a non-a priori method of in vitro 1H-NMR spectroscopy on CSF samples of 74 patients with leukodystrophies. Results: We found an elevation of CSF N-acetylaspartylglutamate (NAAG) in patients with Pelizaeus-Merzbacher disease (PMD)-PLP1 gene, Pelizaeus-Merzbacher-like disease-GJC2 gene and Canavan disease-ASPA gene. In the PMD group, NAAG was significantly elevated in the CSF of all patients with PLP1 duplication (19/19) but was strictly normal in 6 out of 7 patients with PLP1 point mutations. Addnl., we previously reported increased CSF NAAG in patients with SLC17A5 mutations. Conclusions: Elevated CSF NAAG is a biomarker that suggests specific mol. diagnostic abnormalities in patients with white matter diseases. Our findings also point to unique pathol. functions of the overexpressed PLP in PMD patients with duplication of this gene.
- 31Francis, J. S.; Wojtas, I.; Markov, V.; Gray, S. J.; McCown, T. J.; Samulski, R. J.; Bilaniuk, L. T.; Wang, D.-J.; De Vivo, D. C.; Janson, C. G.; Leone, P. N-Acetylaspartate Supports the Energetic Demands of Developmental Myelination via Oligodendroglial Aspartoacylase. Neurobiol. Dis. 2016, 96, 323– 334, DOI: 10.1016/j.nbd.2016.10.00131https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1GqsbbL&md5=de7a4741302cd62044397511a367a303N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylaseFrancis, Jeremy S.; Wojtas, Ireneusz; Markov, Vladimir; Gray, Steven J.; McCown, Thomas J.; Samulski, R. Jude; Bilaniuk, Larissa T.; Wang, Dah-Jyuu; De Vivo, Darryl C.; Janson, Christopher G.; Leone, PaolaNeurobiology of Disease (2016), 96 (), 323-334CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)Breakdown of neuro-glial N-acetyl-aspartate (NAA) metab. results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathol. progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-assocd. virus vector (AAV-Olig001) to quant. assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathol. and deteriorating motor function. Long-term effects included increased oligodendrocyte nos., a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy obsd. following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile obsd. in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase.
- 32Burri, R.; Steffen, C.; Herschkowitz, N. N-Acetyl-L-Aspartate Is a Major Source of Acetyl Groups for Lipid Synthesis during Rat Brain Development. Dev. Neurosci. 1991, 13 (6), 403– 411, DOI: 10.1159/00011219132https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhvFWlsrc%253D&md5=aeed7936ea7e3ffc688b3ee058005cdeN-acetyl-L-aspartate is a major source of acetyl groups for lipid synthesis during rat brain developmentBurri, R.; Steffen, C.; Herschkowitz, N.Developmental Neuroscience (Basel, Switzerland) (1991), 13 (6), 403-11CODEN: DENED7; ISSN:0378-5866.The function of N-acetyl-L-aspartate (NAA), a predominant substance in the central nervous system, has not yet been detd. To investigate the possible function of NAA as a lipid precursor [14C]-N-acetyl-L-aspartate (NAA) or [14C]-acetate (AcA) was injected intracerebrally into 8, 15, and 22-day-old rats. These time points were selected because NAA concn. and the activity of the NAA-synthesizing enzyme L-aspartate-N-acetyltransferase (ANAT), were low in 8-day-old rats, intermediate in 15-day-old rats, and high in 22-day-old rats. During an incubation period of 4 h the radioactive acetyl group of NAA is incorporated into the lipid fraction in amts. of 42.9 to 65.7% of recovered total radioactivity, increasing with the age of the rats. In contrast, radioactivity incorporated from AcA is const. for all three ages. With NAA as precursor only 7.2-9.4% of the recovered total radioactivity is incorporated into the protein fraction. With AcA as precursor 27.0-18.1% of recovered radioactivity is incorporated into the protein fraction, the amts. decreasing with age. Taking into account that the in vivo NAA concn. in the brain is much higher than the AcA concn., NAA is clearly the more efficient precursor for lipid synthesis than AcA. Further, the authors compared NAA and AcA as lipid precursors by analyzing the radioactivity in single lipid fractions, expressed as normalized specific incorporation or normalized incorporation. The measured differences between NAA and AcA in normalized specific and normalized incorporation of acetyl groups imply that NAA is not simply degraded to AcA before incorporated into lipids. Apparently, NAA is a major source of acetyl groups for lipid synthesis during rat brain development.
- 33Singhal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.; McDonough, J. The Neuronal Metabolite NAA Regulates Histone H3Methylation in Oligodendrocytes and Myelin Lipid Composition. Exp. Brain Res. 2017, 235 (1), 279– 292, DOI: 10.1007/s00221-016-4789-z33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1GqsrnE&md5=2e50668708e3d39accb9f3c586da4fd7The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid compositionSinghal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.; McDonough, J.Experimental Brain Research (2017), 235 (1), 279-292CODEN: EXBRAP; ISSN:0014-4819. (Springer)The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metab. of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liq. chromatog.-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatog. (HPTLC) to identify changes in myelin lipid compn. in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amts. of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic anal. of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metab., and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was assocd. with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.
- 34Salji, M. J.; Blomme, A.; Däbritz, J. H. M.; Repiscak, P.; Lilla, S.; Patel, R.; Sumpton, D.; van den Broek, N. J. F.; Daly, R.; Zanivan, S.; Leung, H. Y. Multi-Omics & Pathway Analysis Identify Potential Roles for Tumor N-Acetyl Aspartate Accumulation in Murine Models of Castration-Resistant Prostate Cancer. iScience 2022, 25 (4), 104056 DOI: 10.1016/j.isci.2022.10405634https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVOrtbfL&md5=05cd009b1f8ba6285bd9b5f7697e1e71Multi-omics & pathway analysis identify potential roles for tumor N-acetyl aspartate accumulation in murine models of castration-resistant prostate cancerSalji, Mark J.; Blomme, Arnaud; Dabritz, J. Henry M.; Repiscak, Peter; Lilla, Sergio; Patel, Rachana; Sumpton, David; van den Broek, Niels J. F.; Daly, Ronan; Zanivan, Sara; Leung, Hing Y.iScience (2022), 25 (4), 104056CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Castration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge (Oakes and Papa, 2015). Matched untargeted multi-level omic datasets may reveal biol. changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics were performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node, and bone metastasis grown as murine orthografts. Collectively, we tested the feasibility of muti-omics anal. on models of CRPC in revealing pathways of interest for future validation investigation. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-acetylated alpha-linked acidic dipeptidases (FOLH1/NAALADL2). Based on pathway anal. integrating multiple omic levels, we hypothesize that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of acetyl Co-A for upregulated sphingolipid metab. and a pool of glutamate and aspartate for nucleotide synthesis during tumor growth.
- 35Jones, S. R.; Carley, S.; Harrison, M. An Introduction to Power and Sample Size Estimation. Emerg. Med. J. 2003, 20 (5), 453– 458, DOI: 10.1136/emj.20.5.45335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3svhsFarug%253D%253D&md5=9bf6b9765fb32466de3c9a5c91d423ceAn introduction to power and sample size estimationJones S R; Carley S; Harrison MEmergency medicine journal : EMJ (2003), 20 (5), 453-8 ISSN:.The importance of power and sample size estimation for study design and analysis.
- 36Rodriguez-Navas, C.; Morselli, E.; Clegg, D. J. Sexually Dimorphic Brain Fatty Acid Composition in Low and High Fat Diet-Fed Mice. Mol. Metab 2016, 5 (8), 680– 689, DOI: 10.1016/j.molmet.2016.06.01436https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFent7fO&md5=9e27eff81140e827b19ed7c7d2df4685Sexually dimorphic brain fatty acid composition in low and high fat diet-fed miceRodriguez-Navas, Carlos; Morselli, Eugenia; Clegg, Deborah J.Molecular Metabolism (2016), 5 (8), 680-689CODEN: MMOEAS; ISSN:2212-8778. (Elsevier GmbH)In this study, we analyzed the fatty acid profile of brains and plasma from male and female mice fed chow or a western-style high fat diet (WD) for 16 wk to det. if males and females process fatty acids differently. Based on the differences in fatty acids obsd. in vivo, we performed in vitro expts. on N43 hypothalamic neuronal cells to begin to elucidate how the fatty acid milieu may impact brain inflammation. Using a comprehensive mass spectrometry fatty acid anal., which includes a profile for 52 different fatty acid isomers, we assayed the plasma and brain fatty acid compn. of age-matched male and female mice maintained on chow or a WD. Addnl., using the same techniques, we detd. the fatty acid compn. of N43 hypothalamic cells following exposure to palmitic and linoleic acid, alone or in combination. Our data demonstrate there is a sexual dimorphism in brain fatty acid content both following the consumption of the chow diet, as well as the WD, with males having an increased percentage of satd. fatty acids and redns. in ω6-polyunsatd. fatty acids when compared to females. Interestingly, we did not observe a sexual dimorphism in fatty acid content in the plasma of the same mice. Furthermore, exposure of N43 cells to the ω6-PUFA linoleic acid, which is higher in female brains when compared to males, reduces palmitic acid-induced inflammation. Our data suggest male and female brains, and not plasma, differ in their fatty acid profile. This is the first time, to our knowledge, lipidomic analyses has been used to directly test the hypothesis there is a sexual dimorphism in brain and plasma fatty acid compn. following consumption of the chow diet, as well as following exposure to the WD.
- 37Horoszewicz, J. S.; Kawinski, E.; Murphy, G. P. Monoclonal Antibodies to a New Antigenic Marker in Epithelial Prostatic Cells and Serum of Prostatic Cancer Patients. Anticancer Res. 1987, 7 (5B), 927– 93537https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL1c7jt1GmsQ%253D%253D&md5=4672a3d5b6724314037e26dbafbfe743Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patientsHoroszewicz J S; Kawinski E; Murphy G PAnticancer research (1987), 7 (5B), 927-35 ISSN:0250-7005.Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.
- 38Vorlova, B.; Knedlik, T.; Tykvart, J.; Konvalinka, J. GCPII and Its Close Homolog GCPIII: From a Neuropeptidase to a Cancer Marker and beyond. Front. Biosci. 2019, 24, 648– 687, DOI: 10.2741/474238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitFWksL3E&md5=9d2d1e35c14c01dba566ddb9098ea380GCPII and its close homolog GCPIII: from a neuropeptidase to a cancer marker and beyondVorlova, Barbora; Knedlik, Tomas; Tykvart, Jan; Konvalinka, JanFrontiers in Bioscience, Landmark Edition (2019), 24 (4), 648-687CODEN: FRBIF6; ISSN:1093-4715. (Frontiers in Bioscience)Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been assocd. with any specific physiol. function or pathol., and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiol. functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-mol. inhibitors recognizing GCPII/III for diagnosis, imaging and exptl. therapy of human cancers and other pathologies.
- 39Evans, J. C.; Malhotra, M.; Cryan, J. F.; O’Driscoll, C. M. The Therapeutic and Diagnostic Potential of the Prostate Specific Membrane Antigen/glutamate Carboxypeptidase II (PSMA/GCPII) in Cancer and Neurological Disease. Br. J. Pharmacol. 2016, 173 (21), 3041– 3079, DOI: 10.1111/bph.1357639https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhtlehu7zL&md5=e2bd304d8eacbf5390e82751f0c6c98bThe therapeutic and diagnostic potential of the prostate specific membrane antigen/glutamate carboxypeptidase II (PSMA/GCPII) in cancer and neurological diseaseEvans, James C.; Malhotra, Meenakshi; Cryan, John F.; O'Driscoll, Caitriona M.British Journal of Pharmacology (2016), 173 (21), 3041-3079CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)Prostate specific membrane antigen (PSMA) otherwise known as glutamate carboxypeptidase II (GCPII) is a membrane bound protein that has been found to be highly expressed in prostate cancer as well as the neovasculature of a wide variety of tumors including glioblastomas, breast and bladder cancers as well as being implicated in a variety of neurol. diseases including schizophrenia and ALS. In recent years there has been a surge in the development of both diagnostics and therapeutics that take advantage of the expression and activity of PSMA/GCPII. These include gene therapy, immunotherapy, chemotherapy and radiotherapy. In this review, we discuss the biol. roles that PSMA/GCPII plays, both in normal and disease tissues, and the current therapies exploiting its activity that are at the preclin. stage. We conclude by giving an expert opinion on the future direction of PSMA/GCPII based therapies and diagnostics and hurdles that need to be overcome to make them effective and viable.
- 40Olsen, A. S. B.; Færgeman, N. J. Sphingolipids: Membrane Microdomains in Brain Development, Function and Neurological Diseases. Open Biol. 2017, 7 (5), 170069 DOI: 10.1098/rsob.17006940https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFartb3K&md5=cf7bb6a75151e4ddf2db6e4e1e4d0216Sphingolipids: membrane microdomains in brain development, function and neurological diseasesOlsen, Anne S. B.; rgeman, Nils J. F.Open Biology (2017), 7 (5), 170069/1-170069/17CODEN: OBPICQ; ISSN:2046-2441. (Royal Society)Sphingolipids are highly enriched in the nervous system where they are pivotal constituents of the plasma membranes and are important for proper brain development and functions. Sphingolipids are not merely structural elements, but are also recognized as regulators of cellular events by their ability to form microdomains in the plasma membrane. The significance of such compartmentalization spans broadly from being involved in differentiation of neurons and synaptic transmission to neuronal- glial interactions and myelin stability. Thus, perturbations of the sphingolipid metab. can lead to rearrangements in the plasma membrane, which has been linked to the development of various neurol. diseases. Studying microdomains and their functions has for a long time been synonymous with studying the role of cholesterol. However, it is becoming increasingly clear that microdomains are very heterogeneous, which among others can be ascribed to the vast no. of sphingolipids. In this review, we discuss the importance of microdomains with emphasis on sphingolipids in brain development and function as well as how disruption of the sphingolipid metab. (and hence microdomains) contributes to the pathogenesis of several neurol. diseases.
- 41Poitelon, Y.; Kopec, A. M.; Belin, S. Myelin Fat Facts: An Overview of Lipids and Fatty Acid Metabolism. Cells 2020, 9 (4), 812, DOI: 10.3390/cells904081241https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1altr7L&md5=1425c867663feaf17db0428527c75a65Myelin fat facts: an overview of lipids and fattyacid metabolismPoitelon, Yannick; Kopec, Ashley M.; Belin, SophieCells (2020), 9 (4), 812CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)Myelin is crit. for the proper function of the nervous system and one of the most complex cell-cell interactions of the body. Myelination allows for the rapid conduction of action potentials along axonal fibers and provides phys. and trophic support to neurons. Myelin contains a high content of lipids, and the formation of the myelin sheath requires high levels of fatty acid and lipid synthesis, together with uptake of extracellular fatty acids. Recent studies have further advanced our understanding of the metab. and functions of myelin fatty acids and lipids. In this review, we present an overview of the basic biol. of myelin lipids and recent insights on the regulation of fatty acid metab. and functions in myelinating cells. In addn., this review may serve to provide a foundation for future research characterizing the role of fatty acids and lipids in myelin biol. and metabolic disorders affecting the central and peripheral nervous system.
- 42Aggarwal, S.; Yurlova, L.; Simons, M. Central Nervous System Myelin: Structure, Synthesis and Assembly. Trends Cell Biol. 2011, 21 (10), 585– 593, DOI: 10.1016/j.tcb.2011.06.00442https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1antbfN&md5=43dfc209ae4240bca2283d5226a699faCentral nervous system myelin: structure, synthesis and assemblyAggarwal, Shweta; Yurlova, Larisa; Simons, MikaelTrends in Cell Biology (2011), 21 (10), 585-593CODEN: TCBIEK; ISSN:0962-8924. (Elsevier B.V.)A review. The wrapping of multiple layers of myelin membrane sheets around an axon is of fundamental importance for the function of the nervous system. In the central nervous system (CNS) oligodendrocytes synthesize tremendous amts. of cellular membrane to form multiple myelin internodes of highly stable membranes with a specific set of tightly packed lipids and proteins. In recent years, mouse mutants have allowed great advances in our understanding of the functional and structural role of many of the major components of myelin. The challenge now is to extend this knowledge to unravel the mol. machinery and mechanisms required to synthesize, assemble and wrap myelin multiple times around an axon at the appropriate developmental time. Such insight will be essential in designing new therapeutic strategies to promote remyelination in demyelinating disorders such as multiple sclerosis.
- 43Guan, Z.; Wang, Y.; Cairns, N. J.; Lantos, P. L.; Dallner, G.; Sindelar, P. J. Decrease and Structural Modifications of Phosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease. J. Neuropathol. Exp. Neurol. 1999, 58 (7), 740– 747, DOI: 10.1097/00005072-199907000-0000843https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXltVWhs7g%253D&md5=2d929c20b9acc1bb90cc02d895727b55Decrease and structural modifications of phosphatidylethanolamine plasmalogen in the brain with Alzheimer diseaseGuan, Zhizhong; Wang, Yanan; Cairns, Nigel J.; Lantos, Peter L.; Dallner, Gustav; Sindelar, Pavel J.Journal of Neuropathology and Experimental Neurology (1999), 58 (7), 740-747CODEN: JNENAD; ISSN:0022-3069. (American Association of Neuropathologists, Inc.)Several lipid modifications, some of which were attributed to oxidative stress, have been reported in the brains of patients with Alzheimer disease (AD). To evaluate this possibility, all phospholipids and their ether subclasses from the frontal cortex, hippocampus, and the white matter of AD brain were analyzed by high performance liq. chromatog. and gas chromatog. The total phospholipid in the frontal cortex and hippocampus decreased on a DNA basis by ∼20% and this change was essentially explained by a selective decrease in phosphatidylethanolamine and phosphatidylcholine. The lower content of phosphatidylethanolamine was due to a specific decrease in the plasmalogen subclass. Phosphatidylethanolamine plasmalogen was also the only lipid exhibiting major structural modifications: a significant decrease in polyunsatd. fatty acids and oleic acid as well as a shift of the aldehyde pattern from 18:1 to 18:0. The only modification obsd. in the other phospholipids was a decrease in oleic acid in diacyl-phosphatidylethanolamine and diacyl-phosphatidylcholine. None of these changes were obsd. in the white matter. Both the vinyl ether bond of phosphatidylethanolamine plasmalogen and polyunsatd. fatty acids are major targets in oxidative stress; thus, these specific lipid modifications strongly support the involvement of free radicals in the pathogenesis of AD.
- 44Su, X. Q.; Wang, J.; Sinclair, A. J. Plasmalogens and Alzheimer’s Disease: A Review. Lipids Health Dis. 2019, 18 (1), 100, DOI: 10.1186/s12944-019-1044-144https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M%252FmtFehsw%253D%253D&md5=88bc5e9b3b7575b976eff66125fe2926Plasmalogens and Alzheimer's disease: a reviewSu Xiao Q; Wang Junming; Sinclair Andrew J; Sinclair Andrew JLipids in health and disease (2019), 18 (1), 100 ISSN:.Growing evidence suggests that ethanolamine plasmalogens (PlsEtns), a subtype of phospholipids, have a close association with Alzheimer's disease (AD). Decreased levels of PlsEtns have been commonly found in AD patients, and were correlated with cognition deficit and severity of disease. Limited studies showed positive therapeutic outcomes with plasmalogens interventions in AD subjects and in rodents. The potential mechanisms underlying the beneficial effects of PlsEtns on AD may be related to the reduction of γ-secretase activity, an enzyme that catalyzes the synthesis of β-amyloid (Aβ), a hallmark of AD. Emerging in vitro evidence also showed that PlsEtns prevented neuronal cell death by enhancing phosphorylation of AKT and ERK signaling through the activation of orphan G-protein coupled receptor (GPCR) proteins. In addition, PlsEtns have been found to suppress the death of primary mouse hippocampal neuronal cells through the inhibition of caspase-9 and caspase-3 cleavages. Further in-depth investigations are required to determine the signature molecular species of PlsEtns associated with AD, hence their potential role as biomarkers. Clinical intervention with plasmalogens is still in its infancy but may have the potential to be explored for a novel therapeutic approach to correct AD pathology and neural function.
- 45Fabelo, N.; Martín, V.; Santpere, G.; Marín, R.; Torrent, L.; Ferrer, I.; Díaz, M. Severe Alterations in Lipid Composition of Frontal Cortex Lipid Rafts from Parkinson’s Disease and Incidental Parkinson’s Disease. Mol. Med. 2011, 17 (9–10), 1107– 1118, DOI: 10.2119/molmed.2011.0011945https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlCgsbnE&md5=1ce7e15819dda8fb1b416f1d84bb711cSevere alterations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and incidental Parkinson's diseaseFabelo, Noemi; Martin, Virginia; Santpere, Gabriel; Marin, Raquel; Torrent, Laia; Ferrer, Isidre; Diaz, MarioMolecular Medicine (Manhasset, NY, United States) (2011), 17 (9-10), 1107-1118CODEN: MOMEF3; ISSN:1076-1551. (Feinstein Institute for Medical Research)Lipid rafts are cholesterol- and sphingomyelin-enriched microdomains that provide a highly satd. and viscous physicochem. microenvironment to promote protein-lipid and protein-protein interactions. We purified lipid rafts from human frontal cortex from normal, early motor stages of Parkinson's disease (PD) and incidental Parkinson's disease (iPD) subjects and analyzed their lipid compn. We obsd. that lipid rafts from PD and iPD cortices exhibit dramatic redns. in their contents of n-3 and n-6 long-chain polyunsatd. fatty acids, esp. docosahexaenoic acid (22:6-n3) and arachidonic acid (20:4n-6). Also, satd. fatty acids (16:0 and 18:0) were significantly higher than in control brains. Paralleling these findings, unsatn. and peroxidability indexes were considerably reduced in PD and iPD lipid rafts. Lipid classes were also affected in PD and iPD lipid rafts. Thus, phosphatidylserine and phosphatidylinositol were increased in PD and iPD, whereas cerebrosides and sulfatides and plasmalogen levels were considerably diminished. Our data pinpoint a dramatic increase in lipid raft order due to the aberrant biochem. structure in PD and iPD and indicate that these abnormalities of lipid rafts in the frontal cortex occur at early stages of PD pathol. The findings correlate with abnormal lipid raft signaling and cognitive decline obsd. during the development of these neurodegenerative disorders.
- 46Dorninger, F.; Forss-Petter, S.; Berger, J. From Peroxisomal Disorders to Common Neurodegenerative Diseases - the Role of Ether Phospholipids in the Nervous System. FEBS Lett. 2017, 591 (18), 2761– 2788, DOI: 10.1002/1873-3468.1278846https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVGhtrnO&md5=366e05f52879ad8e06719e250c2bca4aFrom peroxisomal disorders to common neurodegenerative diseases - the role of ether phospholipids in the nervous systemDorninger, Fabian; Forss-Petter, Sonja; Berger, JohannesFEBS Letters (2017), 591 (18), 2761-2788CODEN: FEBLAL; ISSN:0014-5793. (Wiley-Blackwell)A review. The emerging diverse roles of ether (phospho)lipids in nervous system development and function in health and disease are currently attracting growing interest. Plasmalogens, a subgroup of ether lipids, are important membrane components involved in vesicle fusion and membrane raft compn. They store polyunsatd. fatty acids and may serve as antioxidants. Ether lipid metabolites act as precursors for the formation of glycosyl-phosphatidyl-inositol anchors; others, like platelet-activating factor, are implicated in signaling functions. Consolidating the available information, we attempt to provide mol. explanations for the dramatic neurol. phenotype in ether lipid-deficient human patients and mice by linking individual functional properties of ether lipids with pathol. features. Furthermore, recent publications have identified altered ether lipid levels in the context of many acquired neurol. disorders including Alzheimer's disease (AD) and autism. Finally, current efforts to restore ether lipids in peroxisomal disorders as well as AD are critically reviewed.
- 47Katafuchi, T.; Ifuku, M.; Mawatari, S.; Noda, M.; Miake, K.; Sugiyama, M.; Fujino, T. Effects of Plasmalogens on Systemic Lipopolysaccharide-Induced Glial Activation and β-Amyloid Accumulation in Adult Mice. Ann. N.Y. Acad. Sci. 2012, 1262, 85– 92, DOI: 10.1111/j.1749-6632.2012.06641.x47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlGrtbfM&md5=6ff33ad3f0fd6a397cc71b0377ba99a3Effects of plasmalogens on systemic lipopolysaccharide-induced glial activation and β-amyloid accumulation in adult miceKatafuchi, Toshihiko; Ifuku, Masataka; Mawatari, Shiro; Noda, Mami; Miake, Kiyotaka; Sugiyama, Masaaki; Fujino, TakehikoAnnals of the New York Academy of Sciences (2012), 1262 (Neuroimmunomodulation in Health and Disease II), 85-92CODEN: ANYAA9; ISSN:0077-8923. (Wiley-Blackwell)Neuroinflammation essentially involves an activation of glial cells as the cause/effect of neurodegenerative diseases such as Alzheimer's disease (AD). Plasmalogens (Pls) are glycerophospholipids constituting cellular membranes and play significant roles in membrane fluidity and cellular processes like vesicular fusion and signal transduction. I.p. (i.p.) injection of lipopolysaccharide (LPS, 250 μg/kg) for 7 days resulted in the morphol. changes and increase in no. of Iba-1+ microglia showing neuroinflammation in the adult mouse hippocampus. The LPS-induced activation of glial cells was significantly attenuated by i.p. pretreatment with Pls dissolved in corn oil. In addn., systemic injection of LPS induced Aβ1-16+ neurons in the hippocampus were also abolished by application of Pls. Finally, contents of Pls in the hippocampus decreased after LPS injection, and the redn. was suppressed by administration of Pls. These findings suggest an antiamyloidogenic effect of Pls, implicating a possible therapeutic application of Pls against AD.
- 48Wallner, S.; Schmitz, G. Plasmalogens the Neglected Regulatory and Scavenging Lipid Species. Chem. Phys. Lipids 2011, 164 (6), 573– 589, DOI: 10.1016/j.chemphyslip.2011.06.00848https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXpvFCnurc%253D&md5=762062e2e7f960550f689b5fd05952c9Plasmalogens the neglected regulatory and scavenging lipid speciesWallner, Stefan; Schmitz, GerdChemistry and Physics of Lipids (2011), 164 (6), 573-589CODEN: CPLIA4; ISSN:0009-3084. (Elsevier Ltd.)A review. Plasmalogens are a class of phospholipids carrying a vinyl ether bond in sn-1 and an ester bond in sn-2 position of the glycerol backbone. Although they are widespread in all tissues and represent up to 18% of the total phospholipid mass in humans, their physiol. function is still poorly understood. The aim of this review is to give an overview over the current knowledge in plasmalogen biol. and pathol. with an emphasis on neglected aspects of their involvement in neurol. and metabolic diseases. Furthermore a better understanding of plasmalogen biol. in health and disease could also lead to the development of better diagnostic and prognostic biomarkers for vascular and metabolic diseases such as obesity and diabetes mellitus, inflammation, neuro-degeneration and cancer.
- 49Luoma, A. M.; Kuo, F.; Cakici, O.; Crowther, M. N.; Denninger, A. R.; Avila, R. L.; Brites, P.; Kirschner, D. A. Plasmalogen Phospholipids Protect Internodal Myelin from Oxidative Damage. Free Radic. Biol. Med. 2015, 84, 296– 310, DOI: 10.1016/j.freeradbiomed.2015.03.01249https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmvVyrt7w%253D&md5=c86c406514940fec0727481d3f81b2cbPlasmalogen phospholipids protect internodal myelin from oxidative damageLuoma, Adrienne M.; Kuo, Fonghsu; Cakici, Ozgur; Crowther, Michelle N.; Denninger, Andrew R.; Avila, Robin L.; Brites, Pedro; Kirschner, Daniel A.Free Radical Biology & Medicine (2015), 84 (), 296-310CODEN: FRBMEH; ISSN:0891-5849. (Elsevier B.V.)Reactive O species (ROS) are implicated in a range of degenerative conditions, including aging, neurodegenerative diseases, and neurol. disorders. Myelin is a lipid-rich multilamellar sheath that facilitates rapid nerve conduction in vertebrates. Given the high energetic demands and low antioxidant capacity of the cells that elaborate the sheaths, myelin is considered intrinsically vulnerable to oxidative damage, raising the question whether addnl. mechanisms prevent structural damage. Here, the authors characterized the structural and biochem. basis of ROS-mediated myelin damage in murine tissues from both the central nervous system (CNS) and the peripheral nervous system (PNS). To det. whether ROS could cause structural damage to the internodal myelin, whole sciatic and optic nerves were incubated ex vivo with a hydroxyl radical-generating system consisting of Cu, H2O2 (I), and o-phenanthroline (II). Quant. assessment of unfixed tissue by x-ray diffraction revealed irreversible compaction of myelin membrane stacking in both sciatic and optic nerves. Incubation in the presence of the hydroxyl radical scavenger, Na formate, prevented this damage, implicating hydroxyl radical species. Myelin membranes were particularly enriched in plasmalogens, a class of ether-linked phospholipids proposed to have antioxidant properties. Myelin in the sciatic nerve from plasmalogen-deficient (Pex7 knockout) mice was significantly more vulnerable to Cu/I/II-mediated ROS-induced compaction than myelin from wild-type mice. The results directly supported the role of plasmalogens as endogenous antioxidants providing a defense that protects ROS-vulnerable myelin.
- 50Kinoshita, K.; Arai, K.; Kawaura, K.; Hiyoshi, T.; Yamaguchi, J.-I. Development, Validation, and Application of a Surrogate Analyte Method for Determining N-Acetyl-L-Aspartyl-L-Glutamic Acid Levels in Rat Brain, Plasma, and Cerebrospinal Fluid. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2015, 1003, 1– 11, DOI: 10.1016/j.jchromb.2015.09.00550https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFaqsrvK&md5=798127c101c28ee1116343ed619b49c2Development, validation, and application of a surrogate analyte method for determining N-acetyl-L-aspartyl-L-glutamic acid levels in rat brain, plasma, and cerebrospinal fluidKinoshita, Kohnosuke; Arai, Kotaro; Kawaura, Kazuaki; Hiyoshi, Tetsuaki; Yamaguchi, Jun-ichiJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2015), 1003 (), 1-11CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)A bioanal. strategy for the simple and accurate detn. of endogenous substances in a variety of biol. matrixes using liq. chromatog.-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compds. as a surrogate analyte and an internal std. to construct calibration curves with authentic matrixes that can be applied to det. N-acetyl-L-aspartyl-L-glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extn. and with a short anal. time of 4 min. The validated lower limits of quantification were 1.00 nmol/g for brain and 0.0100 nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naive rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the detn. of NAAG in different matrixes in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biol. matrixes.
- 51Williamson, L. C.; Neale, J. H. Ultrastructural Localization of N-Acetylaspartylglutamate in Synaptic Vesicles of Retinal Neurons. Brain Res. 1988, 456 (2), 375– 381, DOI: 10.1016/0006-8993(88)90243-051https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXkvVKrsb8%253D&md5=79af9ff98dfb4ffd7e141e108e1d83cdUltrastructural localization of N-acetylaspartylglutamate in synaptic vesicles of retinal neuronsWilliamson, Lura C.; Neale, Joseph H.Brain Research (1988), 456 (2), 375-81CODEN: BRREAP; ISSN:0006-8993.In the grass frog (Rana pipiens), N-acetylaspartylglutamate (NAAG) immunoreactivity was localized within vesicles in synaptic endings of presumptive amacrine and bipolar neurons in the inner plexiform layer. Addnl., the peptide was present in vesicles within ribbon synapses in the outer plexiform layer, a result suggestive of release from photoreceptor cells. Apparently, NAAG is secreted at points of synaptic contact between neurons, including retinal amacrine, bipolar, and photoreceptor cells.
- 52Renno, W. M.; Lee, J. H.; Beitz, A. J. Light and Electron Microscopic Immunohistochemical Localization of N-Acetylaspartylglutamate (NAAG) in the Olivocerebellar Pathway of the Rat. Synapse 1997, 26 (2), 140– 154, DOI: 10.1002/(SICI)1098-2396(199706)26:2<140::AID-SYN5>3.0.CO;2-852https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivVGhs7s%253D&md5=45dba8c3f8450d5890dd3d043d5a7f27Light and electron microscopic immunohistochemical localization of N-acetylaspartylglutamate (NAAG) in the olivocerebellar pathway of the ratRenno, Waleed M.; Lee, Jang-Hern; Beitz, Alvin J.Synapse (New York) (1997), 26 (2), 140-154CODEN: SYNAET; ISSN:0887-4476. (Wiley-Liss)The aim of the present study was to det. whether N-acetylaspartylglutamate (NAAG) immunoreactivity is present in the inferior olive (IO) and climbing fibers (CF) at the light and electron microscopic levels and to quantitate the amt. of immunogold labeling in olivary neurons and climbing fiber terminals contg. this dipeptide. A polyclonal antisera against NAAG was utilized with a peroxidase-labeled avidin-biotin procedure to demonstrate these immunoreactive neurons in the IO at the light microscopic level. Approx. 45% of olivary neurons display NAAG-like immunoreactivity, and their distribution is unevenly clustered throughout the inferior olive. Using postembedding immunogold electron microscopy in combination with quant. procedures, we found the highest densities of gold particles in the axonal terminals synapsing on olivary neurons (101.0 particles/μ2), in CF terminals (96.3 particles/μm2), and in some mossy fiber terminals (101.0 particles/μm2). Approx. half of the climbing fiber terminals examd. were unlabeled. Moderate labeling occurred in CF axons (70.8 particles/μm2), while IO neuronal perikarya were lightly but significantly labeled (41.6 particles/μm2). The localization of NAAG in the subset of cerebellar climbing fiber terminals provides anatomical support for the hypothesis that NAAG may serve as a neurotransmitter/neuromodulator candidate in the olivocerebellar pathway.
- 53Sácha, P.; Zámecník, J.; Barinka, C.; Hlouchová, K.; Vícha, A.; Mlcochová, P.; Hilgert, I.; Eckschlager, T.; Konvalinka, J. Expression of Glutamate Carboxypeptidase II in Human Brain. Neuroscience 2007, 144 (4), 1361– 1372, DOI: 10.1016/j.neuroscience.2006.10.02253https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVartLw%253D&md5=f72f2146d36d083e985403a28a95d0c9Expression of glutamate carboxypeptidase II in human brainSacha, P.; Zamecnik, J.; Barinka, C.; Hlouchova, K.; Vicha, A.; Mlcochova, P.; Hilgert, I.; Eckschlager, T.; Konvalinka, J.Neuroscience (San Diego, CA, United States) (2007), 144 (4), 1361-1372CODEN: NRSCDN; ISSN:0306-4522. (Elsevier)Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a no. of pathol. conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approx. 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochem. anal. revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.
- 54Nagel, J.; Belozertseva, I.; Greco, S.; Kashkin, V.; Malyshkin, A.; Jirgensons, A.; Shekunova, E.; Eilbacher, B.; Bespalov, A.; Danysz, W. Effects of NAAG Peptidase Inhibitor 2-PMPA in Model Chronic Pain - Relation to Brain Concentration. Neuropharmacology 2006, 51 (7–8), 1163– 1171, DOI: 10.1016/j.neuropharm.2006.07.01854https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1ertrbE&md5=da6263c8a77cbb46dc9eaf6cee01731dEffects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentrationNagel, Jens; Belozertseva, Irina; Greco, Sergio; Kashkin, Vladimir; Malyshkin, Andrey; Jirgensons, Aigars; Shekunova, Elena; Eilbacher, Bernd; Bespalov, Anton; Danysz, WojciechNeuropharmacology (2006), 51 (7-8), 1163-1171CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concn. in brain ECF under basal conditions and its alteration in relation to the brain ECF concn. of 2-PMPA is unclear. We therefore assessed those brain concns. after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS anal. Administration of 2-PMPA (50 mg/kg) produced a mean peak concn. of 2-PMPA of 29.66±8.1 μM. This concn. is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concn. reaching a max. of 2.89±0.42 μM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were obsd., the NAAG concn. in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the obsd. behavioral effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors.
- 55Lin, S. N.; Slopis, J. M.; Butler, I. J.; Caprioli, R. M. In Vivo Microdialysis and Gas Chromatography/mass Spectrometry for Studies on Release of N-Acetylaspartlyglutamate and N-Acetylaspartate in Rat Brain Hypothalamus. J. Neurosci. Methods 1995, 62 (1–2), 199– 205, DOI: 10.1016/0165-0270(95)00077-155https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK28zlslektw%253D%253D&md5=c1fe594f7e5dec7c7245a801e2badc3fIn vivo microdialysis and gas chromatography/mass spectrometry for studies on release of N-acetylaspartlyglutamate and N-acetylaspartate in rat brain hypothalamusLin S N; Slopis J M; Butler I J; Caprioli R MJournal of neuroscience methods (1995), 62 (1-2), 199-205 ISSN:0165-0270.Microdialysis and gas chromatography/mass spectrometry was used for the measurement of extracellular N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in rat hypothalamus. The sensitivity of the method for each of these compounds was approximately 5 pmol/30 microliters of dialysate. Baseline NAA concentrations in dialysate were estimated to be approximately 25 pmol/36 microliters, while that for NAAG was at or below the detection limit of 5 pmol/ 36 microliters. In vivo and in vitro calibrations of microdialysis probes showed that the recovery for NAA was approximately 10 percent. For NAAG, the in vitro recovery was 6.3%, and in vivo recovery, 11%. Depolarization stimulation using 100 mM KCl in the microdialysis perfusate was employed to measure extracellular NAA and NAAG concentrations. Extracellular NAA was elevated to approximately 70 pmol/36 microliters dialysate following depolarization. No significant elevation of NAAG was observed. By infusing known amounts of stable isotopically labeled NAAG-d3 via the microdialysis probe and measuring the isotopically labeled catabolic product, NAA-d3, in collected microdialysate, we were able to confirm the existence of one or more hydrolytic enzymes active towards NAAG in the hypothalamus. This finding suggest the possible involvement of active metabolic processes in the relationship between NAAG and NAA releases.
- 56Sager, T. N.; Laursen, H.; Hansen, A. J. Changes in N-Acetyl-Aspartate Content during Focal and Global Brain Ischemia of the Rat. J. Cereb. Blood Flow Metab. 1995, 15 (4), 639– 646, DOI: 10.1038/jcbfm.1995.7956https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXntVamurY%253D&md5=8a71fb0c93a31ddbcaaf5da814cb2662Changes in N-acetyl-aspartate content during focal and global brain ischemia of the ratSager, Thomas Nikolaj; Laursen, Henning; Hansen, Anker JonJournal of Cerebral Blood Flow and Metabolism (1995), 15 (4), 639-46CODEN: JCBMDN; ISSN:0271-678X.N-Acetyl-aspartate (NAA) is almost exclusively localized in neurons in the mature brain and might be used as a neuronal marker. It has been reported that the NAA content in human brain is decreased in neurodegenerative diseases and in stroke. Since the NAA content can be detd. by NMR techniques, it has potential as a diagnostic and prognostic marker. The objective of this study was to examine the change of NAA content and related substances following cerebral ischemia and compare the results to the damage of the tissue. We used rats to study the changes of NAA, N-acetyl-aspartyl-glutamate (NAAG), glutamate, and aspartate contents over a time course of 24 h in brain regions affected by either permanent middle cerebral artery occlusion (focal ischemia) or decapitation (global ischemia). The decreases of NAA and NAAG contents following global brain ischemia were linear over time but significant only after 4 and 2 h, resp. After 24 h, the levels of NAA and NAAG were 24 and 44% of control values, resp. The concn. of glutamate did not change, whereas the aspartate content increased at a rate comparable with the rate of decrease of NAA content. This is consistent with NAA being preferentially degraded by the enzyme amidohydrolase II in global ischemia. In focal ischemia, there was a rapid decline of NAA within the first 8 h of ischemia followed by a slower rate of redn. The redns. of NAA and NAAG contents in focal ischemia were significant after 4 and 24 h, resp. After 24 h, the NAA and NAAG contents were 33 and 64% of control values, resp. Also, the glutamate and aspartate contents exhibited significant decreases in focal ischemic tissue. Our studies show that NAA decreases during brain ischemia, the initial rate being faster in focal ischemia than in global ischemia. In rat transient focal ischemia, others have shown that a middle cerebral artery occlusion of 2- to 3-h duration is sufficient to produce an infarct that is similar in size to that following permanent occlusion for 24 h. The fact that we obsd. only a 10% decrease of NAA content 2 h after occlusion demonstrates that the NAA content of the tissue does not reflect neuronal viability. Thus, the incompetence with which ischemic/infarcted tissue removes NAA will lead to overestimation of the no. of viable neurons in acute situations. Only when steady state prevails may NAA be used as a marker of viable nerve cells.
- 57Masaharu, M.; Hideo, M.; Mutsuhiko, M.; Yasuo, K. N-Acetyl-L-Aspartic Acid, Acid and β-Citryl-L-Glutamic Acid in Human Urine. Clin. Chim. Acta 1982, 120 (1), 119– 126, DOI: 10.1016/0009-8981(82)90082-1There is no corresponding record for this reference.
- 58Chigurupati, S.; Son, T. G.; Hyun, D.-H.; Lathia, J. D.; Mughal, M. R.; Savell, J.; Li, S. C.; Nagaraju, G. P. C.; Chan, S. L.; Arumugam, T. V.; Mattson, M. P. Lifelong Running Reduces Oxidative Stress and Degenerative Changes in the Testes of Mice. J. Endocrinol. 2008, 199 (2), 333– 341, DOI: 10.1677/JOE-08-030658https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVClsb%252FM&md5=1883fa72cf8c245e68a227521d1104b1Lifelong running reduces oxidative stress and degenerative changes in the testes of miceChigurupati, Srinivasulu; Son, Tae Gen; Hyun, Dong-Hoon; Lathia, Justin D.; Mughal, Mohamed R.; Savell, Jason; Li, Shuan C.; Nagaraju, G. P. C.; Chan, Sic L.; Arumugam, Thiruma V.; Mattson, Mark P.Journal of Endocrinology (2008), 199 (2), 333-341CODEN: JOENAK; ISSN:0022-0795. (Society for Endocrinology)Regular exercise can counteract the adverse effects of aging on the musculoskeletal and cardiovascular systems. In males, the normal aging process is assocd. with redns. in testosterone prodn. and impaired spermatogenesis, but the underlying mechanisms and their potential modification by exercise are unknown. Here, the authors report that lifelong regular exercise (running) protects the testes against the adverse effects of advancing age, and that this effect of running is assocd. with decreased amts. of oxidative damage to proteins, lipids, and DNA in spermatogenic and Leydig cells. Six-month-old male mice were divided into a sedentary group and a group that ran an av. of 1·75 km/day, until the mice reached the age of 20 mo. Seminiferous tubules of runners exhibited a full complement of cells at different stages of the spermatogenic process and a clear central lumen with large nos. of spermatozoa, in contrast to sedentary mice that exhibited disorganized spermatogenic cells and lacked spermatocytes in a central lumen. Levels of protein carbonyls, nitrotyrosine, lipid peroxidn. products, and oxidatively modified DNA were significantly greater in spermatogenic and Leydig cells of sedentary mice compared with runners. These findings suggest that lifelong regular exercise suppresses aging of testes by a mechanism that involves reduced oxidative damage to spermatogenic and Leydig cells.
- 59Liu, L.; Duff, K. A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse. J. Visualized Exp. 2008, 21, 960, DOI: 10.3791/960-vThere is no corresponding record for this reference.
- 60Karlíková, R.; Široká, J.; Friedecký, D.; Faber, E.; Hrdá, M.; Mičová, K.; Fikarová, I.; Gardlo, A.; Janečková, H.; Vrobel, I.; Adam, T. Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia Patients. J. Proteome Res. 2016, 15 (9), 3158– 3166, DOI: 10.1021/acs.jproteome.6b0035660https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xht1Gjt73N&md5=e20241ce3956ccd51f791268069419f8Metabolite Profiling of the Plasma and Leukocytes of Chronic Myeloid Leukemia PatientsKarlikova, Radana; Siroka, Jitka; Friedecky, David; Faber, Edgar; Hrda, Marcela; Micova, Katerina; Fikarova, Iveta; Gardlo, Alzbeta; Janeckova, Hana; Vrobel, Ivo; Adam, TomasJournal of Proteome Research (2016), 15 (9), 3158-3166CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)The discovery of tyrosine kinase inhibitors (TKIs) brought a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). Pathogenetic CML events are closely linked with the Bcr-Abl protein with tyrosine kinase activity. TKIs block the ATP-binding site; therefore, the signal pathways leading to malignant transformation are no longer active. However, there is limited information about the impact of TKI treatment on the metabolome of CML patients. Using liq. chromatog. mass spectrometric metabolite profiling and multivariate statistical methods, we analyzed plasma and leukocyte samples of patients newly diagnosed with CML, patients treated with hydroxyurea and TKIs (imatinib, dasatinib, nilotinib), and healthy controls. The global metabolic profiles clearly distinguished the newly diagnosed CML patients and the patients treated with hydroxyurea from those treated with TKIs and the healthy controls. The major changes were found in glycolysis, the citric acid cycle, and amino acid metab. We obsd. differences in the levels of amino acids and acylcarnitines between those patients responding to imatinib treatment and those who were resistant to it. According to our findings, the metabolic profiling may be potentially used as an addnl. tool for the assessment of response/resistance to imatinib.
- 61Xuan, Q.; Hu, C.; Yu, D.; Wang, L.; Zhou, Y.; Zhao, X.; Li, Q.; Hou, X.; Xu, G. Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass Spectrometry. Anal. Chem. 2018, 90 (12), 7608– 7616, DOI: 10.1021/acs.analchem.8b0133161https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVeis7fN&md5=f9061a7a1201eea7b20b4340a3370344Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography-Mass SpectrometryXuan, Qiuhui; Hu, Chunxiu; Yu, Di; Wang, Lichao; Zhou, Yang; Zhao, Xinjie; Li, Qi; Hou, Xiaoli; Xu, GuowangAnalytical Chemistry (Washington, DC, United States) (2018), 90 (12), 7608-7616CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Lipid coverage is crucial in comprehensive lipidomics studies challenged by high diversity in lipid structures and wide dynamic range in lipid levels. Current state-of-the-art lipidomics technologies are mostly based on mass spectrometry (MS), including direct-infusion MS, chromatog.-MS, and matrix-assisted laser desorption ionization (MALDI) imaging MS, each with its pros and cons. Due to the need or favorability for measurement of isomers and isobars, chromatog.-MS is preferable for lipid profiling. The ultra-HPLC-high resoln. mass spectrometry (UHPLC-HRMS)-based nontargeted lipidomics approach and UHPLC-tandem MS (UHPLC-MS/MS)-based targeted approach are two representative methodol. platforms for chromatog.-MS. The authors developed a high coverage pseudotargeted lipidomics method combining the advantages of nontargeted and targeted lipidomics approaches. The high coverage of lipids was achieved by integration of the detected lipids derived from nontargeted UHPLC-HRMS lipidomics anal. of multiple matrixes (e.g., plasma, cell, and tissue) and the predicted lipids speculated on the basis of the structure and chromatog. retention behavior of the known lipids. A total of 3377 targeted lipid ion pairs with over 7000 lipid mol. structures were defined. The pseudotargeted lipidomics method was well validated with satisfactory anal. characteristics in terms of linearity, precision, reproducibility, and recovery for lipidomics profiling. Importantly, it showed better repeatability and higher coverage of lipids than the nontargeted lipidomics method. The applicability of the developed pseudotargeted lipidomics method was testified in defining differential lipids related to diabetes. The authors believe that comprehensive lipidomics studies will benefit from the developed high coverage pseudotargeted lipidomics approach.
- 62Peng, B.; Kopczynski, D.; Pratt, B. S.; Ejsing, C. S.; Burla, B.; Hermansson, M.; Benke, P. I.; Tan, S. H.; Chan, M. Y.; Torta, F.; Schwudke, D.; Meckelmann, S. W.; Coman, C.; Schmitz, O. J.; MacLean, B.; Manke, M.-C.; Borst, O.; Wenk, M. R.; Hoffmann, N.; Ahrends, R. LipidCreator Workbench to Probe the Lipidomic Landscape. Nat. Commun. 2020, 11 (1), 2057, DOI: 10.1038/s41467-020-15960-z62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosVOmtrs%253D&md5=d63c13ea857730aea32cbc2327f485abLipidCreator workbench to probe the lipidomic landscapePeng, Bing; Kopczynski, Dominik; Pratt, Brian S.; Ejsing, Christer S.; Burla, Bo; Hermansson, Martin; Benke, Peter Imre; Tan, Sock Hwee; Chan, Mark Y.; Torta, Federico; Schwudke, Dominik; Meckelmann, Sven W.; Coman, Cristina; Schmitz, Oliver J.; MacLean, Brendan; Manke, Mailin-Christin; Borst, Oliver; Wenk, Markus R.; Hoffmann, Nils; Ahrends, RobertNature Communications (2020), 11 (1), 2057CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biol. conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and anal. and prove that it is capable to generate large targeted expts. to analyze blood and to dissect lipid-signaling pathways such as in human platelets.
- 63Drotleff, B.; Roth, S. R.; Henkel, K.; Calderón, C.; Schlotterbeck, J.; Neukamm, M. A.; Lämmerhofer, M. Lipidomic Profiling of Non-Mineralized Dental Plaque and Biofilm by Untargeted UHPLC-QTOF-MS/MS and SWATH Acquisition. Anal. Bioanal. Chem. 2020, 412 (10), 2303– 2314, DOI: 10.1007/s00216-019-02364-263https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXivFKhtr4%253D&md5=97cc543d3d468761e0cb63a606fbb668Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisitionDrotleff, Bernhard; Roth, Simon R.; Henkel, Kerstin; Calderon, Carlos; Schlotterbeck, Joerg; Neukamm, Merja A.; Laemmerhofer, MichaelAnalytical and Bioanalytical Chemistry (2020), 412 (10), 2303-2314CODEN: ABCNBP; ISSN:1618-2642. (Springer)Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its compn. may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently studied in the context of oral health and disease. Furthermore, its potential as an alternative matrix for anal. purposes has also been recognized in other disciplines like archeol., food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, however, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for detn. of lipid compns. of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for anal. purposes has been performed. An untargeted lipidomics workflow using a ultra-HPLC (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theor. fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepd. by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-anal. Most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was obsd. in the cultivated biofilms. However, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate are specifically increased in plaque samples.
- 64AlzbetaG. AlzbetaG/Metabol: First Version; Zenodo, 2019, DOI: DOI: 10.5281/ZENODO.3235775 .There is no corresponding record for this reference.
- 65Dieterle, F.; Ross, A.; Schlotterbeck, G.; Senn, H. Probabilistic Quotient Normalization as Robust Method to Account for Dilution of Complex Biological Mixtures. Application in 1H NMR Metabonomics. Anal. Chem. 2006, 78 (13), 4281– 4290, DOI: 10.1021/ac051632c65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltVCgtro%253D&md5=6eb6377326a9df2a59b6afb2a9c6e47dProbabilistic Quotient Normalization as Robust Method to Account for Dilution of Complex Biological Mixtures. Application in 1H NMR MetabonomicsDieterle, Frank; Ross, Alfred; Schlotterbeck, Goetz; Senn, HansAnalytical Chemistry (2006), 78 (13), 4281-4290CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)For the anal. of the spectra of complex biofluids, preprocessing methods play a crucial role in rendering the subsequent data analyses more robust and accurate. Normalization is a preprocessing method, which accounts for different dilns. of samples by scaling the spectra to the same virtual overall concn. In the field of 1H NMR metabonomics integral normalization, which scales spectra to the same total integral, is the de facto std. In this work, it is shown that integral normalization is a suboptimal method for normalizing spectra from metabonomic studies. Esp. strong metabonomic changes, evident as massive amts. of single metabolites in samples, significantly hamper the integral normalization resulting in incorrectly scaled spectra. The probabilistic quotient normalization is introduced in this work. This method is based on the calcn. of a most probable diln. factor by looking at the distribution of the quotients of the amplitudes of a test spectrum by those of a ref. spectrum. Simulated spectra, spectra of urine samples from a metabonomic study with cyclosporin-A as the active compd., and spectra of more than 4000 samples of control animals demonstrate that the probabilistic quotient normalization is by far more robust and more accurate than the widespread integral normalization and vector length normalization.
- 66Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 2003, 13 (11), 2498– 2504, DOI: 10.1101/gr.123930366https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXovFWrtr4%253D&md5=2bcbca9a3bd04717761f0424c0209e43Cytoscape: A software environment for integrated models of biomolecular interaction networksShannon, Paul; Markiel, Andrew; Ozier, Owen; Baliga, Nitin S.; Wang, Jonathan T.; Ramage, Daniel; Amin, Nada; Schwikowski, Benno; Ideker, TreyGenome Research (2003), 13 (11), 2498-2504CODEN: GEREFS; ISSN:1088-9051. (Cold Spring Harbor Laboratory Press)Cytoscape is an open source software project for integrating biomol. interaction networks with high-throughput expression data and other mol. states into a unified conceptual framework. Although applicable to any system of mol. components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other mol. states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of addnl. computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined phys./functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.
- 67Mitchell, M. W. A Comparison of Aggregate P-Value Methods and Multivariate Statistics for Self-Contained Tests of Metabolic Pathway Analysis. PLoS One 2015, 10 (4), e0125081 DOI: 10.1371/journal.pone.012508167https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XisVSiurg%253D&md5=f82e631c472666c6deea9054677f780fA comparison of aggregate P-value methods and multivariate statistics for self-contained tests of metabolic pathway analysisMitchell, Matthew W.PLoS One (2015), 10 (4), e0125081/1-e0125081/17CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)For pathway anal. of genomic data, the most common methods involve combining p-values from individual statistical tests. However, there are several multivariate statistical methods that can be used to test whether a pathway has changed. Because of the large no. of variables and pathway sizes in genomics data, some of these statistics cannot be computed. However, in metabolomics data, the no. of variables and pathway sizes are typically much smaller, making such computations feasible. Of particular interest is being able to detect changes in pathways that may not be detected for the individual variables. We compare the performance of both the p-value methods and multivariate statistics for self-contained tests with an extensive simulation study and a human metabolomics study. Permutation tests, rather than asymptotic results are used to assess the statistical significance of the pathways. Furthermore, both one and two-sided alternatives hypotheses are examd. From the human metabolomic study, many pathways were statistically significant, although the majority of the individual variables in the pathway were not. Overall, the p-value methods perform at least as well as the multivariate statistics for these scenarios.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.3c00494.
Lipid retention pattern plots, overview of changes in the brain and CSF lipidome across all lipid classes as comparison between female (+/+♀Y) and male (+/+♂Y) data sets and comparison of the (−/–♂Y/+/+♂Y) and (−/–♀Y/+/+♀Y), quantitative analysis of NAAG and BCG in the CSF and brain from mice of different NAALAD2 genotypes (PDF)
MS parameters, retention time, and data from metabolomic and lipidomic analysis of plasma, urine, CSF, and brain tissue after processing, normalization, and transformation (XLSX)
Normality testing (Shapiro–Wilk) of the data from metabolomic and lipidomic analysis of plasma, urine, CSF, and brain tissue after processing, normalization, and transformation (XLSX)
Results of univariate statistical analysis (t test and fold change) for metabolomic and lipidomic data, calculation of the cumulative p value (using Fisher’s method) to evaluate systematic changes in lipid classes in the brain tissue and CSF (XLSX)
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