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

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

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

Fiscal Year 2001

Palladium used at Savannah River (SR) for process tritium storage is currently obtained from a commercial source. In order to understand the processes involved in preparing this material, SR is supporting investigations into the chemical reactions used to synthesize this material. The material specifications are shown in Table 1. An improved understanding of the chemical processes should help to guarantee a continued reliable source of Pd in the future. As part of this evaluation, a work-for-others contract between Westinghouse Savannah River Company and Ames Laboratory (AL) was initiated. During FY98, the process for producing Pd powder developed in 1986 by Dan Grove of Mound Applied Technologies, USDOE (the Mound muddy water process) was studied to understand the processing conditions that lead to changes in morphology in the final product. During FY99 and FY00, the process for producing Pd powder that has been used previously at Sandia and Los Alamos National Laboratories (the Sandia/LANL process) was studied to understand the processing conditions that lead to changes in the morphology of the final Pd product. During FY01, scale-up of the process to batch sizes greater than 600 grams of Pd using a 20-gallon Pfaudler reactor was conducted by the Iowa State University (ISU) Chemical Engineering Department. This report summarizes the results of FY99-FY01 Pd processing work done at AL and ISU using the Sandia/LANL process. In the Sandia/LANL process, Pd is dissolved in a mixture of nitric and hydrochloric acids. A number of chemical processing steps are performed to yield an intermediate species, diamminedichloropalladium (Pd(NH{sub 3}){sub 2}Cl{sub 2}, or DADC-Pd), which is isolated. In the final step of the process, the Pd(NH{sub 3}){sub 2}Cl{sub 2} intermediate is subsequently redissolved, and Pd is precipitated by the addition of a reducing agent (RA) mixture of formic acid and sodium formate. It is at this point that the morphology of the Pd product is determined. During FY99 and FY00, a study of how the characteristics of the Pd are affected by changes in processing conditions including the RA/Pd molar ratio, Pd concentration, mole fraction of formic acid (mf-FA) in the RA solution, reaction temperature, and mixing was performed. These parameters all had significant effects on the resulting values of the tap density (TD), BET surface area (SA), and Microtrac particle size (PS) distribution for the Pd samples. These effects were statistically modeled and fit in order to determine ranges of predicted experimental conditions that resulted in material that meets the requirements for the Pd powder to be used at SR. Although not statistically modeled, the method and rate of addition of the RA and the method and duration of stirring were shown to be significant factors affecting the product morphology. Instead of producing an additional statistical fit and due to the likely changes anticipated during scale-up of this processing procedure, these latter conditions were incorporated into a reproducible practical method of synthesis. Palladium powder that met the SR specifications for TD, BET SA, and Microtrac PS was reliably produced at batch sizes ranging from 25-100 grams. In FY01, scale-up of the Sandia/LANL process was investigated by the ISU Chemical Engineering Department for the production of 600-gram batches of Pd. Palladium that meets the SR specifications for TD, BET SA, and Microtrac PS has been produced using the Pfaudler reactor, and additional processing batches will be done during the remainder of FY01 to investigate the range of conditions that can be used to produce Pd powder within specifications. Palladium product samples were analyzed at AL and SR to determine TD and at SR to determine BET SA, Microtrac PS distribution, and Pd nodule size and morphology by scanning electron microscopy (SEM)