Higher levels of phosphorylated Y1472 on GluN2B subunits in the frontal cortex of aged mice are associated with good spatial reference memory, but not cognitive flexibility

Abstract

The N-methyl-D-aspartate receptor (NMDAr) is particularly vulnerable to aging. The GluN2B subunit of the NMDAr, compared to other NMDAr subunits, suffers the greatest losses of expression in the aging brain, especially in the frontal cortex. While expression levels of GluN2B mRNA and protein in the aged brain are well documented, there has been little investigation into agerelated posttranslational modifications of the subunit. In this study, we explored some of the mechanisms that may promote differences in the NMDAr complex in the frontal cortex of aged animals. Two ages of mice, 3 and 24 months, were behaviorally tested in the Morris water maze. The frontal cortex and hippocampus from each mouse were subjected to differential centrifugation followed by solubilization in Triton X-100. Proteins from Triton-insoluble membranes, Tritonsoluble membranes, and intracellular membranes/ cytosol were examined by Western blot. Higher levels of GluN2B tyrosine 1472 phosphorylation in frontal cortex synaptic fractions of old mice were associated with better reference learning but poorer cognitive flexibility. Levels of GluN2B phosphotyrosine 1336 remained steady, but there were greater levels of the calpain-induced 115 kDa GluN2B cleavage product on extrasynaptic membranes in these old good learners. There was an age-related increase in calpain activity, but it was not associated with better learning. These data highlight a unique aging change for aged mice with good spatial learning that might be detrimental to cognitive flexibility. This study also suggests that higher levels of truncated GluN2B on extrasynaptic membranes are not deleterious to spatial memory in aged mice.

Higher Levels of Protein Palmitoylation in the Frontal Cortex across Aging Were Associated with Reference Memory and Executive Function Declines

“…Recent data have indicated that old mice with good reference memory have higher levels of phosphorylation on tyrosine 1472 of the GluN2B subunit on synaptosomal detergent-resistant membrane (TxP) in the frontal cortex (Zamzow et al, 2016). We asked whether the increased p1472 observed in the TxP fraction of frontal cortex of old mice with good reference memory could be attributed to a change in the palmitoylation levels of GluN2B subunits.…”

“…Thirteen mice [7 young (5 months old), 6 old] in Study 1 were fed the defined AIN-93G diet. Subfractionated tissue from a previous study (Zamzow et al, 2016) was also used in Study 2, in which 24 mice [12 young (3 months old), 12 old] were fed a standard chow diet (LabDiet). After the behavioral testing, all animals were killed by exposure to CO 2 and decapitated.…”

“…Biochemical fractionation, following dissection of frontal cortex and hippocampus as described above, was performed on 23 mice (11 young, 12 old; Study 2) as previously described (Dunah and Standaert, 2001; Fan et al, 2012), with a few modifications (Zamzow et al, 2016). Briefly, tissue was homogenized on ice with a Dounce homogenizer in Tris-EDTA buffer (10 m m Tris-HCl, pH 7.4, 1 m m EDTA, 1 m m EGTA) plus 320 m m sucrose and protease inhibitor cocktail (Sigma-Aldrich).…”

“…Subunits of the NMDAR show significant age-related declines of protein expression in the hippocampus and frontal cortex (Magnusson et al, 2002, 2007, 2010; Das and Magnusson, 2011). There is a significant loss of protein expression of the GluN2B subunit in most cellular fractions in the hippocampus of aged mice; however, greater effects due to age are found in the synaptic membrane in the frontal cortex and are related to reference memory declines (Magnusson et al, 2007; Zhao et al, 2009; Zamzow et al, 2016).…”

“…A recent study (Zamzow et al, 2014) found that the number of GluN subunits, Fyn, and PSD-95 proteins that are palmitoylated increases with age in the frontal cortex, but not the hippocampus of mice. Another study (Zamzow et al, 2016), which divided older mice into good and bad reference memory learners based on average place trial performance in the Morris water maze, showed an increase in p1472 on GluN2B in the synaptic membranes from frontal cortex of older good reference learners only, but enhanced Fyn in the same fraction in the poor older learners. It is not known whether a disturbed palmitoylation cycle can explain altered localization and phosphorylation of proteins in the frontal cortices of old mice.…”