In summary, our data suggest that β-catenin plays a role in long term memory formation in adults. We have shown that β-catenin is highly expressed in the adult mouse amygdala and is dynamically regulated at both the transcriptional and post-translational levels with fear learning. Pharmacologically enhancing the stability of β-catenin with lithium chloride resulted in an enhancement in learning, while genetic deletion of β-catenin within the amygdala resulted in deficits in learning. By studying the effects of β-catenin deletion in adults, we have been able to identify a novel role for β-catenin in learning and memory, distinct from its role in development.
Our data suggest that β-catenin is required for the consolidation, but not acquisition, of fear memory. However, once the memory has been consolidated, we found that β-catenin is no longer required to express the memory. During this consolidation period, we found that the interaction between β-catenin and cadherin is dynamically regulated. This suggests that β-catenin may be involved in the structural conversion of short-term labile to long-term stable memory traces.
We found that β-catenin mRNA expression was increased in the BLA, but not the somatosensory cortex and striatum, following fear training. Although this is the first study to examine β-catenin in vivo
with learning, this result is consistent with previous in vitro
studies of hippocampal slices showing an increase in nuclear β-catenin with tetanic stimulation12
. In addition, Wnt target genes have also been shown to be up-regulated with long-term potentiation in hippocampal slices, anywhere from 15 min to 120 min following stimulation12
Interestingly, when we measured total β-catenin protein levels, we did not see any alterations with training. It has been shown that depolarization of hippocampal neurons with KCl does not change the total levels of β-catenin at the synapse, but instead, causes a redistribution from dendritic shafts to spines 7
. Thus, it is possible that rapid dynamic changes in breakdown, redistribution, and replacement lead to no apparent change in total protein visualized with immunoblots.
We reported biochemical changes that suggest that both β-catenin's role in cell-cell adhesion and Wnt signaling are affected by fear conditioning. Phosphorylation of β-catenin on tyrosine residue, Y654, has been shown to decrease the affinity of β-catenin for cadherin14,21
. In addition, inhibiting the phosphorylation of Y654 with a point mutation redistributes β-catenin from dendritic shafts to spines, thereby increasing the β-catenin-cadherin interaction7
. In our study, we found that p-Y654 levels were dynamically regulated following training. Our co-immunoprecipitation experiments show a very rapid period of β-catenin-cadherin destabilization, followed by a period of stabilization during consolidation. Overall, these findings suggest that the affinity of β-catenin for cadherin may initially weaken to allow for modifications of the synapse, and then strengthen to stabilize the synapse, which we hypothesize to be a molecular and cellular correlate of memory consolidation.
Such dynamic regulation of p-Y654 on β-catenin has previously been proposed. Brain-derived neurotrophic factor (BDNF) treatment has been shown to induce synaptic vesicle dispersion in hippocampal cultures, which is associated with an increase in β-catenin tyrosine phosphorylation and decrease in β-catenin-cadherin interactions. Soon after this dispersion, the phosphorylation levels decrease, and the β-catenin-cadherin interaction is restored22
. Notably, we have previously shown that BDNF activation of the TrkB receptor is required within the amygdala for consolidation of fear memories23
. Thus, a similar mechanism may be taking place in this in vivo
learning paradigm, such that when new memories are formed, pre-existing synapses must become de-stabilized transiently prior to stabilization of synapses involved in memory formation.
We have also provided both biochemical and behavioral evidence suggesting that increased stabilization of β-catenin, through the inhibition of GSK-3β, may be important for learning and memory. Normally, GSK-3β phosphorylates β-catenin at Ser33/37/Thr41, marking the protein for degradation. However, when GSK-3β is inactivated by phosphorylation at Ser9, β-catenin becomes stabilized10
. In our study, we reported an increase in phosphorylated GSK-3β in the amygdala at 2 hr following fear conditioning. In addition, when we pharmacologically increased the inhibition of GSK-3β with LiCl, we observed decreased β-catenin phosphorylation. Furthermore, acute administration of LiCl 30 min before training resulted in an enhancement in learning measured 48 hr later, without any effect on acquisition. Although administration of LiCl has previously been shown to produce behaviors similar to those displayed by overexpression of β-catenin in the mouse brain24
, LiCl's actions are not necessarily specific to β-catenin.
To more definitively identify the role of β-catenin in long-term memory formation, we used genetic manipulations to delete β-catenin from the adult amygdala. We found that deletion of β-catenin before training does not affect the acquisition or immediate expression of fear, but does produce deficits in learning when measured 48 hr after training. In addition, deletion of β-catenin after consolidation has occurred does not affect the expression of learned fear. These findings provide further support that normal β-catenin expression is necessary to consolidate the newly acquired memory.
One limitation of this study is our inability to specifically inhibit or delete β-catenin
immediately after training. Previous work has elegantly demonstrated, using consolidation of inhibitory avoidance, that post-training manipulations are the gold-standard for demonstrating disruption of fear consolidation33,34
. Although the data on consolidation of amygdala-dependent classical conditioning paradigms has been less clear, this is an important manipulation for interpretation of consolidation effects. Unfortunately, there are no drugs currently available that selectively inhibit β-catenin. Additionally, since a minimum of 7-10 days is required for optimal lentiviral gene expression, we are unable to delete β-catenin
shortly after training. However, we feel that our current powerful method of genetic manipulation is an important approach to specifically examine the role of β-catenin
within the amygdala during learning. Furthermore, we feel that the lack of an effect of β-catenin
deletion on acquisition of fear and expression of fear makes a strong case for its role during the consolidation period.
Based on the results obtained thus far, we propose that during the acquisition of fear and immediately afterwards, synapses may weaken, as demonstrated with decreased β-catenin-cadherin association at 0 and 0.5 hr post-training, thereby alleviating the requirement for β-catenin. Then, once the synapses have been modified during the consolidation process, β-catenin is required to convert that memory trace into long-term memory. Examination of such proposed changes in synaptic strength will need to be further explored. Additional studies are also needed to determine if it is β-catenin's role in cell-cell adhesion, Wnt signaling, or both, that are contributing to its observed effects on learning and memory.
In summary, our results suggest that β-catenin, a ‘hub protein’ involved in both transcriptional regulation and maintaining stability of cell-cell contacts and synaptogenesis, is required for normal consolidation of new memories in adult mice. This finding adds to the body of knowledge describing the role of β-catenin in normal cell functioning, tumor regulation, and development. Although it has been previously implicated with in vitro approaches in synaptogenesis and synaptic plasticity, our results provide definitive support for its function in learning and memory processes. Further understanding its role may provide important insights into the nature of the molecular mechanisms underlying memory consolidation. Additionally in humans, the development of new small molecule specific inhibitors of β-catenin function may eventually provide for a powerful clinical approach to transiently inhibit the consolidation of newly formed trauma memories in the prevention of fear-related disorders, such as post-traumatic stress disorder. Similarly, enhancing β-catenin function may be helpful in disorders of memory such as Alzheimer's disease.