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1.
J Clin Invest ; 125(7): 2772-80, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-26075819

ABSTRACT

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.


Subject(s)
Central Nervous System/enzymology , Superoxide Dismutase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Animals , Carbon Isotopes , Disease Models, Animal , Female , HEK293 Cells , Humans , Isotope Labeling , Kinetics , Male , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/cerebrospinal fluid , Mutant Proteins/genetics , Mutant Proteins/metabolism , Rats , Rats, Transgenic , Recombinant Proteins/cerebrospinal fluid , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Superoxide Dismutase/cerebrospinal fluid , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , Tandem Mass Spectrometry
2.
Ann Neurol ; 78(3): 439-53, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26040676

ABSTRACT

OBJECTIVE: Age is the single greatest risk factor for Alzheimer's disease (AD), with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for AD is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid-beta (Aß) kinetics in the central nervous system (CNS) of humans. METHODS: Aß kinetics were analyzed in 112 participants and compared to the ages of participants and the amount of amyloid deposition. RESULTS: We found a highly significant correlation between increasing age and slowed Aß turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on Aß42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aß42 and a 10-fold higher Aß42 reversible exchange rate. INTERPRETATION: These findings reveal a mechanistic link between human aging and the risk of amyloidosis, which may be owing to a dramatic slowing of Aß turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aß kinetics associated with aging and amyloidosis suggest opportunities for diagnostic and therapeutic strategies. More generally, this study provides an example of how changes in protein turnover kinetics can be used to detect physiological and pathophysiological changes and may be applicable to other proteinopathies.


Subject(s)
Aging/metabolism , Amyloid beta-Peptides/metabolism , Amyloidosis/metabolism , Central Nervous System/metabolism , Peptide Fragments/metabolism , Adult , Aged , Aged, 80 and over , Aging/pathology , Amyloidosis/pathology , Central Nervous System/pathology , Female , Humans , Kinetics , Male , Middle Aged
3.
J Neurosci ; 34(24): 8336-46, 2014 Jun 11.
Article in English | MEDLINE | ID: mdl-24920637

ABSTRACT

BACE, a ß-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aß and soluble APPß (sAPPß), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPß, and Aß in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPß, and Aß were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the ß-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.


Subject(s)
Amyloid Precursor Protein Secretases/cerebrospinal fluid , Amyloid beta-Peptides/cerebrospinal fluid , Amyloid beta-Protein Precursor/cerebrospinal fluid , Central Nervous System/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Carbon Isotopes/metabolism , Cell Line, Tumor , Central Nervous System/drug effects , Cross-Over Studies , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Immunoprecipitation , Leucine/metabolism , Macaca mulatta , Mass Spectrometry , Neuroblastoma , Peptide Fragments , Transfection
4.
Sci Transl Med ; 6(236): 236re4, 2014 May 14.
Article in English | MEDLINE | ID: mdl-24828079

ABSTRACT

Serotonin signaling suppresses generation of amyloid-ß (Aß) in vitro and in animal models of Alzheimer's disease (AD). We show that in an aged transgenic AD mouse model (APP/PS1 plaque-bearing mice), the antidepressant citalopram, a selective serotonin reuptake inhibitor, decreased Aß in brain interstitial fluid in a dose-dependent manner. Growth of individual amyloid plaques was assessed in plaque-bearing mice that were chronically administered citalopram. Citalopram arrested the growth of preexisting plaques and reduced the appearance of new plaques by 78%. In healthy human volunteers, citalopram's effects on Aß production and Aß concentrations in cerebrospinal fluid (CSF) were measured prospectively using stable isotope labeling kinetics, with CSF sampling during acute dosing of citalopram. Aß production in CSF was slowed by 37% in the citalopram group compared to placebo. This change was associated with a 38% decrease in total CSF Aß concentrations in the drug-treated group. The ability to safely decrease Aß concentrations is potentially important as a preventive strategy for AD. This study demonstrates key target engagement for future AD prevention trials.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/cerebrospinal fluid , Antidepressive Agents/chemistry , Citalopram/chemistry , Adult , Animals , Brain/metabolism , Dose-Response Relationship, Drug , Double-Blind Method , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Mice , Mice, Inbred C3H , Mice, Transgenic , Microdialysis , Middle Aged , Plaque, Amyloid/metabolism , Prospective Studies , Serotonin/metabolism , Selective Serotonin Reuptake Inhibitors/chemistry , Signal Transduction , Young Adult
5.
Sci Transl Med ; 5(189): 189ra77, 2013 Jun 12.
Article in English | MEDLINE | ID: mdl-23761040

ABSTRACT

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-ß (Aß) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aß42 compared to Aß40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers. We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related noncarriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aß isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer Pittsburgh compound B (PIB). CNS Aß42 to Aß40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. The fractional turnover rate of soluble Aß42 relative to Aß40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aß42 into plaques, leading to reduced recovery of Aß42 in cerebrospinal fluid (CSF). Reversible exchange of Aß42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aß42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aß42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aß42 concentrations in the CSF.


Subject(s)
Amyloid beta-Peptides/metabolism , Presenilins/genetics , Adult , Alzheimer Disease/blood , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid/blood , Amyloid/metabolism , Amyloid beta-Peptides/blood , Female , Humans , Male , Middle Aged , Mutation , Positron-Emission Tomography
6.
Am J Clin Nutr ; 97(5): 1029-35, 2013 May.
Article in English | MEDLINE | ID: mdl-23535107

ABSTRACT

BACKGROUND: Gastric cancer is increasingly recognized in Zambia. Although nutritional factors contribute to gastric cancer risk, their effect in Zambia is unknown. OBJECTIVE: The objective was to investigate the association between intake of dietary antioxidants, urinary 8-iso prostaglandin F2α (8-iso PGF2α) as a marker of oxidative stress, and gastric cancer. DESIGN: This was a case-control study at the University Teaching Hospital in Zambia. Gastric cancer cases were compared with age- and sex-matched controls. Urine 8-iso PGF2α was measured primarily by ELISA, and by gas chromatography-mass spectrometry in a subset, expressed as a ratio to creatinine. Blood was collected for Helicobacter pylori, HIV serology, gastrin-17, and pepsinogen 1 and 2 concentrations. Clinical and dietary data were collected by using questionnaires. Food items were broadly classified into 7 major categories (fruit, vegetables, fish, meat, insects, cereals, and starches). RESULTS: Fifty cases with gastric cancer (mean age: 61 y; n = 31 males) and 90 controls (mean age: 54 y; n = 41 males) were enrolled. Median urinary 8-iso PGF2α excretion was higher in cases (0.014; IQR: 0.008-0.021) than in controls (0.011; IQR: 0.006-0.018; P = 0.039). On univariate analysis, habitual fruit intake was lower in cases than in controls during the dry season (P = 0.02). On multivariate analysis, smoking (OR: 7.22; IQR: 1.38-37.9) and gastric atrophy (OR: 2.43; IQR: 1.12-5.13) were independently associated with cancer, and higher fruit intake was protective (OR: 0.44; IQR: 0.20-0.95). Isoprostane excretion was inversely correlated with total fruit intake (ρ = -0.23; n = 140; P = 0.006). CONCLUSION: Urinary 8-iso PGF2α excretion was associated with the risk of gastric cancer, as were smoking and gastric atrophy, but increased fruit intake conferred protection. This trial was registered at www.pactr.org as ISRCTN52971746.


Subject(s)
Antioxidants/administration & dosage , Energy Intake , Feeding Behavior , Isoprostanes/urine , Nutritional Status , Stomach Neoplasms/epidemiology , Adult , Biomarkers/blood , Biomarkers/urine , Case-Control Studies , Creatinine/urine , Dinoprost/analogs & derivatives , Dinoprost/urine , Female , Fruit , Gas Chromatography-Mass Spectrometry , Gastrins/blood , HIV/isolation & purification , Helicobacter pylori/isolation & purification , Humans , Logistic Models , Male , Middle Aged , Multivariate Analysis , Oxidative Stress/drug effects , Pepsinogen A/blood , Pepsinogen C/blood , Prospective Studies , Risk Factors , Smoking/adverse effects , Surveys and Questionnaires , Vegetables , Zambia/epidemiology
7.
Blood ; 120(7): 1422-31, 2012 Aug 16.
Article in English | MEDLINE | ID: mdl-22786879

ABSTRACT

TLR agonists initiate a rapid activation program in dendritic cells (DCs) that requires support from metabolic and bioenergetic resources. We found previously that TLR signaling promotes aerobic glycolysis and a decline in oxidative phosphorylation (OXHPOS) and that glucose restriction prevents activation and leads to premature cell death. However, it remained unclear why the decrease in OXPHOS occurs under these circumstances. Using real-time metabolic flux analysis, in the present study, we show that mitochondrial activity is lost progressively after activation by TLR agonists in inflammatory blood monocyte-derived DCs that express inducible NO synthase. We found that this is because of inhibition of OXPHOS by NO and that the switch to glycolysis is a survival response that serves to maintain ATP levels when OXPHOS is inhibited. Our data identify NO as a profound metabolic regulator in inflammatory monocyte-derived DCs.


Subject(s)
Dendritic Cells/metabolism , Dendritic Cells/pathology , Glycolysis , Inflammation/pathology , Nitric Oxide/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Cell Death/drug effects , Cell Respiration/drug effects , Cell Survival/drug effects , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Glycolysis/drug effects , Inflammation/enzymology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Mitochondria/metabolism , Models, Immunological , Monocytes/pathology , Nitric Oxide Synthase Type II/metabolism , Time Factors , Toll-Like Receptors/metabolism
8.
J Comb Chem ; 11(3): 469-80, 2009.
Article in English | MEDLINE | ID: mdl-19296668

ABSTRACT

A solid-phase organic synthesis method has been developed for the preparation of trisubstituted pyrimidin-6-one carboxylic acids 12, which allows elaboration to a 3-dimensional combinatorial library. Three substituents are introduced by initial Knoevenagel condensation of an aldehyde and malonate ester resin 7 to give resin bound 1. Cyclization of 1 with an N-substituted amidine 10, oxidation, and cleavage afforded pyrimidinone 12. The initial solid-phase reaction sequence was followed by gel-phase (19)FNMR and direct-cleavage (1)H NMR of intermediate resins to determine the optimal conditions. The scope of the method for library production was determined by investigation of a 3 x 4 pilot library of twelve compounds. Cyclocondensation of N-methylamidines and 7 followed by CAN oxidation gave mixtures of the resin bound pyrimidin-6-one 11 and the regioisomeric pyrimidin-4-one 15, which after cleavage from the resin afforded a nearly 1:1 mixture of pyrimidin-6-one and pyrimidin-4-one carboxylic acids 12 and 16, respectively. The regiochemical assignment was confirmed by ROESY1D and gHMBC NMR experiments. A library was prepared using 8 aldehydes, 3 nitriles, and 4 amines to give a full combinatorial set of 96 pyrimidinones 12. Confirmation of structural identity and purity was carried out by LCMS using coupled ELS detection and by high-throughput flow (1)H NMR.


Subject(s)
Combinatorial Chemistry Techniques/methods , Pyrimidinones/chemical synthesis , Small Molecule Libraries/chemical synthesis , Aldehydes/chemical synthesis , Aldehydes/chemistry , Amines/chemical synthesis , Amines/chemistry , Isomerism , Magnetic Resonance Spectroscopy , Nitriles/chemical synthesis , Nitriles/chemistry , Pyrimidinones/chemistry , Small Molecule Libraries/chemistry
9.
Diab Vasc Dis Res ; 4(2): 117-23, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17654445

ABSTRACT

We evaluated the effects of two thiazolidinediones (TZDs), the potent PPARgamma agonist rosiglitazone currently being used to treat diabetes, and a structurally similar experimental compound that is a poor PPARgamma agonist, in a non-diabetic, established hypertension model with continuous measurement of blood pressure by telemetry. Hypertension was induced in male Dahl salt-sensitive rats by a three-week pre-treatment with 4% salt before initiation of treatment. Fasting blood samples were taken for analysis of a biomarker panel to assess metabolic, anti-inflammatory and antioxidant activity of the treatments. Both TZDs significantly reduced both systolic and diastolic blood pressure. When used at the maximally effective doses established for metabolic improvement, both compounds produced equivalent reduction in lipids and elevation of adiponectin, yet the poorer PPARgamma agonist produced significantly greater reductions in blood pressure. Neither compound had a significant effect on circulating glucose or insulin in this animal model. The data demonstrate that these TZDs lower blood pressure significantly in Dahl rats and that this cardiovascular pharmacology is not directly correlated with the metabolic actions or with the magnitude of PPARgamma activation. These data suggest that it may be possible to find insulin-sensitising agents that have beneficial cardiovascular pharmacology with broad applications for disease prevention.


Subject(s)
Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Hypertension/drug therapy , Pyridines/pharmacology , Thiazolidinediones/pharmacology , Adiponectin/blood , Animals , Antihypertensive Agents/pharmacokinetics , Antihypertensive Agents/therapeutic use , Blood Glucose/drug effects , Disease Models, Animal , Disease Progression , Heart Rate/drug effects , Hypertension/chemically induced , Hypertension/metabolism , Hypertension/physiopathology , Insulin/blood , Lipids/blood , Male , PPAR gamma/agonists , PPAR gamma/metabolism , Pyridines/pharmacokinetics , Pyridines/therapeutic use , Rats , Rats, Inbred Dahl , Rosiglitazone , Sodium Chloride, Dietary , Thiazolidinediones/pharmacokinetics , Thiazolidinediones/therapeutic use
10.
Bioorg Med Chem Lett ; 12(13): 1743-6, 2002 Jul 08.
Article in English | MEDLINE | ID: mdl-12067551

ABSTRACT

Imidazole glycerol phosphate dehydratase (IGPD) has become an attractive target for herbicide discovery since it is present in plants and not in mammals. Currently no knowledge is available on the 3-D structure of the IGPD active site. Therefore, we used a pharmacophore model based on known inhibitors and 3-D database searches to identify new active compounds. In vitro testing of compounds from the database searches led to the identification of a class of pyrrole aldehydes as novel inhibitors of IGPD.


Subject(s)
Enzyme Inhibitors/chemistry , Hydro-Lyases/antagonists & inhibitors , Hydro-Lyases/chemistry , Pyrroles/chemistry , Triazoles/chemistry , Aldehydes/analysis , Aldehydes/chemistry , Binding Sites , Databases, Factual , Herbicides/chemistry , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , Pyrroles/analysis
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