- As the aging population continues to grow worldwide, age-related complications are becoming more apparent within the aging population. One of the first age-related complications to become apparent is age-associated memory impairment and it can make the elderly more dependent on caregivers early on. The N-methyl-D-aspartate (NMDA) receptor is important to learning and memory and appears to be especially vulnerable to the process of aging. The density of NMDA receptors declines with age more than any other ionotropic glutamate receptor. Both the density of NMDA receptors and the mRNA and protein expression of its subunits decline with age. In particular, the GluN2B subunit of the NMDA receptor shows the greatest age-related declines in expression across multiple brain regions, including the frontal lobe (including the prefrontal and frontal cortices), caudate nucleus and hippocampus. These declines are strongly correlated to age-related declines in spatial memory. Specifically, age-related decreases in the protein expression of the GluN2B subunit within crude synaptosomes of the frontal cortex of C57BL/6 mice show a relationship to the declines in performance in a long-term spatial memory task across age groups. However, within the population of aged mice, there was a subpopulation of aged mice in which higher expression of the GluN2B subunit within the synaptic membrane of the hippocampus was associated with poorer performance in the same task. Moreover, transgenic mice designed to express higher
levels of the GluN2B subunit from birth also possess superior memory, including spatial memory, across adulthood to middle-age. Taken together, these data led to the hypothesis that increasing the expression of the GluN2B subunit within the aged brain could potentially alleviate age-related declines in memory. However, increasing its expression regionally was first examined since higher expression of the GluN2B subunit within the hippocampus has been associated with poorer memory in aged animals.
Since age-related decreases in the protein expression of the GluN2B subunit within the frontal cortex show a relationship to impaired memory function, the first study was designed to determine if increasing GluN2B subunit expression in the frontal lobe would improve memory in aged mice. Mice received bilateral injections of either an adenoviral vector, containing cDNA specific for the GluN2B subunit and enhanced Green Fluorescent Protein (eGFP) (GluN2B vector); an adenoviral vector containing only the cDNA for eGFP (control vector); or vehicle into their frontal lobe. Spatial memory, cognitive flexibility and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression in the frontal lobe, exhibited improved long-term spatial memory, comparable to young mice, in the second day of training. Moreover, a higher concentration of the specific GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B subunit expression, as compared to aged controls. The requirement for greater antagonism in aged mice to block memory performance suggests that the number of GluN2B-containing receptors in their frontal lobe was enhanced and contributed to the improved memory. This study provides suggestive evidence that therapies that enhance GluN2B subunit expression within the aged brain could have the potential to ameliorate age-related memory loss.
Since higher expression of the GluN2B subunit within the hippocampus of aged mice is associated with poorer memory, the second study was designed to determine if increasing GluN2B subunit expression in the hippocampus would improve or further impair memory in aged mice. This would help to determine if a therapy aimed at enhancing the GluN2B subunit expression or function of GluN2B-containing receptors throughout the aged brain could help ameliorate age-associated memory loss. Mice were injected bilaterally with either the GluN2B vector, a control vector or vehicle into the hippocampus. Spatial memory, cognitive flexibility and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression in the hippocampus, exhibited improved long-term spatial memory, comparable to young mice, early in training. However, there was a trend for impaired memory later in the long-term spatial memory trials. Still, these data suggest that enhancing GluN2B subunit expression in the aged hippocampus could be more beneficial to memory than harmful. In addition, the results of this study suggest that enhancing GluN2B subunit expression in different brain regions may improve memory at different phases of learning. Therefore, therapies that enhance GluN2B subunit expression throughout the aged brain could help ameliorate age-related memory loss. The first two studies demonstrated that enhancing the expression of the GluN2B subunit within either the frontal lobe or hippocampus of the aged brain has the potential to reduce age-related memory declines. However, the increase was not global nor specific to the synapse. Therefore, a third study was developed with the intent of garnering a more global increase in GluN2B subunit expression that was localized to the synapse. Cyclin dependent kinase 5 (Cdk5) enhances endocytosis of the GluN2B subunit-containing NMDA receptors from the synapse. Previous research has shown that inhibiting Cdk5 increases the number of GluN2B subunits at the synapse and within the whole cell and improves memory in young mice. This
study was designed to determine if using antisense phosphorodiamidate morpholino oligomers (Morpholinos) to decrease the expression of Cdk5 protein within the brain would improve memory in aged mice. Morpholinos were conjugated to a cell penetrating peptide, which enhances cellular uptake, and delivered bilaterally to the lateral ventricles of both young and aged mice via acute stereotaxic injection. Treatments consisted of equivalent volumes and concentrations of either vehicle, control Morpholino or a Morpholino targeting the mRNA of Cdk5 (Cdk5 Morpholino). Memory was evaluated in the Morris water maze and using a novel object recognition task. Aged mice treated with the Cdk5 Morpholino exhibited improved early acquisition and spatial bias in the long-term spatial memory trials, as well as improved performance overall, compared to control Morpholino-treated aged animals. However, aged mice treated with the Cdk5 Morpholino performed similarly to vehicle-treated aged animals. The presence of the peptide-conjugated Morpholinos within the brain may have worsened performance in the Morris water maze task since control Morpholino-treated animals performed significantly worse than vehicle-treated animals. In concurrence, there was significantly greater gliosis in peptide-conjugated Morpholino-treated animals over vehicle-treated brains, suggesting it was neurotoxic. In contrast, young mice treated with the Cdk5 Morpholino showed impaired early acquisition and spatial bias but a trend for improved later learning in the long-term spatial memory task compared to control Morpholino-treated animals. Treatment with the Cdk5 Morpholino had no significant effect on cognitive flexibility, associative memory or novel object recognition for young or aged animals. Immunohistochemistry revealed increased GluN2B subunit expression within cells with characteristics of neurons and astroglia in regions of the frontal lobe, caudate nucleus and hippocampus of aged mice who received the Cdk5 Morpholino compared to control treatments. However, the increased GluN2B subunit expression appeared to be greater within
the hippocampus. These results suggest that inhibiting the translation of Cdk5 using Morpholinos increased GluN2B subunit expression in both young and aged mice and may have contributed to the improved long-term spatial memory observed in aged mice, despite the Morpholino being administered at a presumably toxic concentration. An additional group of mice was used to determine a non-neurotoxic dosage of the peptide conjugated Morpholino. However, future studies are needed to determine the efficacy of the Cdk5 Morpholino at this dosage.
Taken together, the studies presented here suggest that increasing expression of the GluN2B subunit within the aged brain does improve age-associated memory declines. In addition, cell penetrating peptide- conjugated Morpholinos show promise as tools for genetic manipulation within the brain and Cdk5 could prove to be a novel target for enhancing GluN2B subunit expression within the aged brain. Though future studies are needed, the studies presented here do suggest that therapies that enhance GluN2B subunit expression within the aged brain have the potential to help ameliorate memory loss. However, since enhanced GluN2B subunit expression itself can increase the potential for excitotoxicity, an optimal dose of such a therapeutic would need to be determined.