Mechanisms that influence the posttranslational modification of the GluN2B subunit in the aged brain

Memory is essential to everyday life. As we age, deficits in memory become apparent. The toll of age-related cognitive impairment can be devastating to families and costly to society. The NMDA receptor is a molecule in the brain that is instrumental in the formation of memories. The receptor is particularly vulnerable to the effects of aging. Of the fourteen known subunits, the GluN2B subunit has the greatest effect on memory. The subunit also suffers from the greatest loss of expression due to aging. The present study examines the interactions of GluN2B with other proteins and quantifies the changes in posttranslational modification with age. Co-immunoprecipitation was used to measure the interaction of GluN2B with the scaffolding molecules PSD-95 and GIPC in synaptosomes from the frontal cortex of behaviorally characterized mice of three ages. The interaction between GluN2A and GluN2B was also measured. There were more PSD-95 and GluN2A molecules per GluN2B with age. The increased PSD-95/GluN2B relationship in old mice was associated with poorer memory. The GIPC/GluN2B relationship also correlated with spatial reference memory. The GluN2B/GluN2A relationship indicated an increase in triheteromeric receptors in the aged mouse, but it did not correlate with memory declines during aging. These results suggest that an age-related increase in PSD-95/GluN2B is detrimental to memory. Young and old mice were behaviorally characterized and homogenates from the frontal cortex and hippocampus were separated by differential centrifugation followed by lysis in Triton X-100. Western blots containing proteins from each cellular fraction were probed with antibodies for several proteins in the NMDA receptor complex. There was an age-related increase in p1472 in the synaptic fraction from the frontal cortex and an increase in the 115 kDa calpain-mediated cleavage product of GluN2B in the extrasynaptic fraction of old mice with good reference memory. Fyn was increased and p1336 was decreased in the synaptic membranes of poor learners. The percentage of GluN2B, GluN2A, Fyn, and PSD-95 molecules that were palmitoylated increased in an age-dependent manner in the frontal cortex, but not the hippocampus. The thioesterase, APT1, also had an age-related increase in palmitoylation in the frontal cortex. These results suggest that the palmitoylation cycle may be perturbed in the frontal cortex of aged brains and this may influence further processing of the GluN2B subunit. In the last study we attempted to lower levels of protein palmitoylation in aged mice by lowering systemic levels of palmitate. Xanthohumol is a flavonoid from hops that increases beta-oxidation in the liver thereby decreasing systemic levels of fatty acids. Mice were fed a diet supplemented with xanthohumol and behaviorally characterized in the Morris water maze. There was a small treatment effect on cognitive flexibility in the young mice, but this appeared to be a recovery from the negative effects of a phytoestrogen-deficient diet. Treatment with xanthohumol significantly lowered levels of palmitate in the plasma of old mice, but palmitoyl-CoA and protein palmitoylation was unaffected. These results suggest that there may be some promise in using xanthohumol for the treatment of metabolic syndrome, but it may not be suitable for lowering levels of protein palmitoylation in the brain. These results presented in this thesis suggest that an age-related increase in palmitoylation of GluN2B and NMDA receptor effector proteins in the brain may affect the function of neurons in the frontal cortex in two ways. First, increased p1472 enhances clustering of GluN2B-containing NMDA receptors on the synaptic membrane, thereby preserving memory in some old mice. Second, an age-related increase in the calpain-mediated cleavage of GluN2B may eventually lead to increased cell death. Interventions that reduce systemic levels of palmitate may not be effective in treating memory deficits. What is needed is a greater understanding of the mechanisms that govern the palmitoylation cycle in brain in order to design more targeted interventions in the future

Aerosol Mass Measurement and Solution Standard Additions for Quantitation in Laser Ablation-Inductively Coupled Plasma Atomic Emission Spectrometry

A new approach for quantitation in laser ablation-inductively coupled plasma atomic emission spectrometry (LA-ICPAES) is presented. A portion of the laser-ablated sample aerosol is diverted to an aerosol mass monitor to measure variations in the amount of sample ablated and transported to the ICP torch. This provides a normalization for variations in laser ablation efficiency due to changes in laser power and focus at the sample and variations in material transport out of the ablation cell and into the ICP torch. During the laser ablation sampling process, solution standards are nebulized and the aerosol is added to the laser-ablated aerosol to generate a standard addition curve for the analyte being determined. The standard addition procedure corrects for potential plasma-related matrix effects in the ICP emission signal resulting from the ablated sample. The precision of this method, for triplicate analyses for the determination of 16 elements in four glass samples, and the accuracy of this method relative to the nominal glass compositions are both approximately 10%

A continuous sampling air-ICP for metals emission monitoring

An air-inductively coupled plasma (air-ICP) system has been developed for continuous sampling and monitoring of metals as a continuous emission monitor (CEM). The plasma is contained in a metal enclosure to allow reduced-pressure operation. The enclosure and plasma are operated at a pressure slightly less than atmospheric using a Roots blower, so that sample gas is continuously drawn into the plasma. A Teflon sampling chamber, equipped with a sampling pump, is connected to the stack that is to be monitored to isokinetically sample gas from the exhaust line and introduce the sample into the air-ICP. Optical emission from metals in the sampled gas stream is detected and monitored using an acousto-optic tunable filter (AOTF) - echelle spectrometer system. A description of the continuous sampling air-ICP system is given, along with some preliminary laboratory data for continuous monitoring of metals

AOTF-echelle spectrometer for air-ICP-AES continuous emission monitoring of heavy metals and actinides

A spectrometer system consisting of a quartz acousto-optic tunable filter (AOTF) and an echelle grating has been assembled and tested for ICP-AES continuous emission monitoring of heavy metal and actinide elements in stack exhaust offgases introduced into an air plasma. The AOTF is a rapidly tunable bandpass filter that is used to select a small wavelength range (0. 1 to 0.6 am) of optical emission from the air plasma; the echelle grating provides high dispersion, yielding a spectral resolution of approximately 0.004 to 0.008 nm from 200 to 425 nm. The AOTF-echelle spectrometer, equipped with a photodiode array or CCD, provides rapid sequential multielement analysis capabilities. It is much more compact and portable than commercial ICP-AES echelle spectrometers, allowing use ofthe system in field and on-line process monitoring applications. Data will be presented that detail the resolution, detection limits, capabilities, and performance of the AOTFechelle spectrometer for continuous emission monitoring of heavy metals (As, Be, Cd, Cr, Hg, and Pb) and actinides (including U isotopes). The potential use of the AOTF-echelle spectrometer with other emission sources and for other monitoring applications will be discussed

In situ determination of uranium in soil by laser ablation-inductively coupled plasma atomic emission spectrometry

The concentration of uranium in soil has been determined for 80 sites in an area suspected to have uranium contamination by in situ laser ablation- inductively coupled plasma atomic emission spectrometry (LA-ICPAES), utilizing a field-deployable mobile analytical laboratory. For 15 of the 80 sites analyzed, soil samples were collected so that the field LA-ICPAES results could be compared to laboratory-determined values. Uranium concentrations determined in the field by LA-ICPAES for these 15 sites range from \u3c20 parts per million (ppm) by weight to 285 ppm. The uncertainty in the values determined, however, is large relative to the uranium concentrations encountered at this site. The 95% confidence interval (CI) values are approximately 85 ppm. The uranium concentrations determined by laboratory LA- ICPAES analysis range from \u3c20 to 102 ppm (95% CI of approximately 50 ppm); microwave dissolution and subsequent standard addition determination of uranium by solution nebulization ICPAES using an ultrasonic nebulizer yields 19-124 ppm uranium (95% CI of approximately 10 ppm). For 11 of the 15 samples, the field- and laboratory-determined uranium concentrations agree, within the uncertainty of the determined values

Real-time atomic absorption mercury continuous emission monitor

A continuous emission monitor (CEM) for mercury(Hg) in combustor flue gas streams has been designed and tested for the detection of Hg by optical absorption. A sampling system that allows continuous introduction of stack gas is incorporated into the CEM, for the sequential analysis of elemental and total Hg. A heated pyrolysis tube is used in the system to convert oxidizedHg compounds to elemental Hg for analysis of total Hg; the pyrolysis tube is bypassed to determine the elemental Hg concentration in the gas stream. A key component of the CEM is a laboratory-designed and -assembled echelle spectrometer that provides simultaneous detection of all of the emission lines from a Hg pen lamp, which is used as the light source for the optical absorption measurement. This feature allows for on-line spectroscopic correction for interferent gases such as sulfur dioxide and nitrogen dioxide, typically present in combustion stack gas streams, that also absorb at the Hg detection wavelength (253.65 nm). This article provides a detailed description of the CEM system, the characteristics and performance of the CEM, and the results of field tests performed at the Environmental Protection Agency-Rotary Kiln at Research Triangle Park, NC

An Increase in the Association of GluN2B Containing NMDA Receptors with Membrane Scaffolding Proteins Was Related to Memory Declines during Aging

The NMDA receptor is an important component of spatial working and reference memory. The receptor is a heterotetramer composed of a family of related subunits. The GluN2B subunit of the NMDA receptor appears to be essential for some forms of memory and is particularly vulnerable to change with age in both the hippocampus and cerebral cortex. GluN2B expression is particularly reduced in frontal cortex synaptic membranes. The current study examined the relationship between spatial cognition and protein-protein interactions of GluN2B-containing NMDA receptors in frontal cortex crude synaptosome from 3, 12, and 26-month-old C57BL/6 mice. Aged mice showed a significant decline in spatial reference memory and reversal learning from both young and middle-aged mice. Coimmunoprecipitation of GluN2B subunits revealed an age-related increase in the ratio of both postsynaptic density-95 (PSD-95) and the GluN2A subunit to the GluN2B subunit. Higher ratios of PSD-95/GluN2B and GAIP-interacting protein C-terminus (GIPC)/GluN2B were associated with poorer learning index scores across all ages. There was a significant correlation between GIPC/GluN2B and PSD-95/GluN2B ratios, but PSD-95/GluN2B and GluN2A/GluN2B ratios did not show a relationship. These results suggest that there were more triheteromeric (GluN2B/GluN2A/GluN1) NMDA receptors in older mice than in young adults, but this did not appear to impact spatial reference memory. Instead, an increased association of GluN2B-containing NMDA receptors with synaptic scaffolding proteins in aged animals may have contributed to the age-related memory declines.This is the publisher’s final pdf. The published article is copyrighted by Society for Neuroscience and can be found at: http://www.jneurosci.org/.Keywords: Mice, Long term potentiation, Binding, Expression, Subunits, Determinants, Performance, Glutamate receptors, PSD-95, traffickin

Elemental and Isotopic Analysis of Uranium Oxide an NIST Glass Standards by FEMTOSECOND-LA-ICP-MIC-MS

The objective of this work was to test and demonstrate the analytical figures of merit of a femtosecond-laser ablation (fs-LA) system coupled with an inductively coupled plasma-multi-ion collector-mass spectrometer (ICP-MIC-MS). The mobile fs-LA sampling system was designed and assembled at Ames Laboratory and shipped to Oak Ridge National Laboratory (ORNL), where it was integrated with an ICP-MIC-MS. The test period of the integrated systems was February 2-6, 2009. Spatially-resolved analysis of particulate samples is accomplished by 100-shot laser ablation using a fs-pulsewidth laser and monitoring selected isotopes in the resulting ICP-MS transient signal. The capability of performing high sensitivity, spatially resolved, isotopic analyses with high accuracy and precision and with virtually no sample preparation makes fs-LA-ICP-MIC-MS valuable for the measurement of actinide isotopes at low concentrations in very small samples for nonproliferation purposes. Femtosecond-LA has been shown to generate particles from the sample that are more representative of the bulk composition, thereby minimizing weaknesses encountered in previous work using nanosecond-LA (ns-LA). The improvement of fs- over ns-LA sampling arises from the different mechanisms for transfer of energy into the sample in these two laser pulse-length regimes. The shorter duration fs-LA pulses induce less heating and cause less damage to the sample than the longer ns pulses. This results in better stoichiometric sampling (i.e., a closer correlation between the composition of the ablated particles and that of the original solid sample), which improves accuracy for both intra- and inter-elemental analysis. The primary samples analyzed in this work are (a) solid uranium oxide powdered samples having different {sup 235}U to {sup 238}U concentration ratios, and (b) glass reference materials (NIST 610, 612, 614, and 616). Solid uranium oxide samples containing {sup 235}U in depleted, natural, and enriched abundances were analyzed as particle aggregates immobilized in a collodion substrate. The uranium oxide samples were nuclear reference materials (CRMs U0002, U005-A, 129-A, U015, U030-A, and U050) obtained from New Brunswick Laboratory-USDOE

Xanthohumol improved cognitive flexibility in young mice

The protein palmitoylation cycle has been shown to be important for protein signaling and synaptic plasticity. Data from our lab showed a change in the palmitoylation status of certain proteins with age. A greater percentage of the NMDA receptor subunits GluN2A and GluN2B, along with Fyn and PSD95 proteins, were palmitoylated in the old mice. The higher level of protein palmitoylation was also associated with poorer learning scores. Xanthohumol is a prenylated flavonoid that has been shown to increase beta-oxidation in the livers of rodents, decreasing circulating free fatty acids in the serum. What is not known is whether the application of xanthohumol could influence the palmitoylation status of proteins. In this study, young and old mice were fed a diet supplemented with xanthohumol for 8 weeks. Spatial memory was assessed with the Morris water maze and protein palmitoylation quantified. The young xanthohumol-treated mice showed a significant improvement in cognitive flexibility. However, this appeared to be associated with the young control mice, on a defined, phytoestrogen-deficient diet, performing as poorly as the old mice and xanthohumol reversing this effect. The old mice receiving xanthohumol did not significantly improve their learning scores. Xanthohumol treatment was unable to affect the palmitoylation of NMDA receptor subunits and associated proteins assessed in this study. This evidence suggests that xanthohumol may play a role in improving cognitive flexibility in young animals, but it appears to be ineffective in adjusting the palmitoylation status of neuronal proteins in aged individuals.Keywords: Palmitoylation, Reversal trials, Aging, Memory, PalmitateKeywords: Palmitoylation, Reversal trials, Aging, Memory, Palmitat

Reducing expression of GluN1OXX subunit splice variants of the NMDA receptor interferes with spatial reference memory

The GluN1 subunit of the NMDA receptor shows age-related changes in its expression pattern, some of which correlate with spatial memory performance in mice. Aged C57BL/6 mice show an age-related increase in mRNA expression of GluN1 subunit splice variants that lack the N terminal splice cassette, GluN1ₒₓₓ (GluN1-a). This increase in expression is associated with good performance in reference and working memory tasks. The present study was undertaken to determine if GluN1ₒₓₓ splice variants are required for good performance in reference memory tasks in young mice. Mice were bilaterally injected with either siRNA specific for GluN1ₒₓₓ splice variants, control siRNA or vehicle alone into ventro-lateral orbital cortices. A fourth group of mice did not receive any injections. Starting five days post-injection, mice were tested for their performance in spatial reference memory, associative memory and cognitive flexibility tasks over 4 days in the Morris water maze. There was a 10 -19% reduction in mRNA expression for GluN1ₒₓₓ splice variants within the ventro-lateral orbital cortices in mice following GluN1ₒₓₓ siRNA treatment. Declines in performance within the first half of reference memory testing were seen in the mice receiving siRNA against the GluN1ₒₓₓ splice variants, as compared to the mice injected with control siRNA, vehicle and/or no treatment. These results suggest a role for the GluN1ₒₓₓ splice variants in orbital regions for early acquisition and/or consolidation of spatial reference memory.Keywords: NMDA receptor, siRNA, Memory, NR1, Splice variant, Zeta