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

Graduation date: 2015Memory 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.\ud 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.\ud 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.\ud 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.\ud 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