Abstract
Numerous studies have suggested that memories “destabilize” and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin–proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin–proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was “upstream” of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.
Introduction
The formation of long-term fear memories requires de novo gene transcription and protein translation in neurons during memory consolidation (Johansen et al., 2011, McGaugh, 2000). While once thought to be permanent, it is now widely supported that upon retrieval once consolidated memories “destabilize” and require new protein synthesis in order to “restabilize”, a process referred to as memory reconsolidation (Alberini and Ledoux, 2013, Nader et al., 2000, Tronson and Taylor, 2007). This reconsolidation process is thought to be dynamic, allowing modification of previously formed memories. Consistent with this, numerous studies have shown that reconsolidation can strengthen, weaken or change the specific content of a memory (De Oliveira Alvares et al., 2013, Inda et al., 2011, Lee, 2008, Lee, 2010, Monfils et al., 2009, Schiller et al., 2010, Sierra et al., 2013), which highlights the therapeutic potential of the reconsolidation process in alleviating fear associated with traumatic memories.
While most studies have focused on the mechanisms that regulate the restabilization or protein synthesis-dependent phase of the reconsolidation process, few have examined the mechanisms that regulate memory trace destabilization. NMDA receptor activation appears to initiate the destabilization process as inhibition of NMDA receptor activity in the amygdala prior to retrieval prevents the memory impairments that result from post-retrieval administration of the protein synthesis inhibitor anisomycin (Ben Mamou et al., 2006, Lopez et al., 2015, Wang et al., 2009). Downstream of NMDA receptors, ubiquitin–proteasome mediated protein degradation has been consistently implicated as a critical regulator of memory trace destabilization since blocking functional proteasome activity prevents memories from undergoing reconsolidation and can attenuate reconsolidation-dependent memory modification (Jarome et al., 2011, Lee, 2008, Lee et al., 2008). However, though NMDA receptor activity can result in increased proteasome activity in vitro and in vivo (Bingol and Schuman, 2006, Jarome et al., 2011), it is hypothesized that this occurs through a second messenger and not as a direct result of calcium influx (Jarome & Helmstetter, 2013). To date, the molecule(s) that links NMDA receptor activation to protein degradation during the destabilization process remains equivocal.
One molecule that is directly activated by increased intracellular calcium levels is the calcium–calmodulin dependent protein kinase II (CaMKII), which has well described roles in the memory consolidation process (Bejar et al., 2002, Mayford et al., 1996, Rodrigues et al., 2004, Yasuda and Mayford, 2006). Interestingly, the role of CaMKII in the reconsolidation of fear memories has never been examined. Additionally, studies examining the role of CaMKII in the reconsolidation of memory for other behavioral tasks have found mixed results, with some indicating normal memory retention following post-retrieval inhibition of CaMKII signaling (Arguello et al., 2014, Da Silva et al., 2013, Sakurai et al., 2007). One intriguing explanation for these mixed results is that CaMKII regulates protein degradation upstream of its potential (but not proven) regulation of protein synthesis during the reconsolidation process (Jarome & Helmstetter, 2013). Consistent with this, CaMKII can regulate proteasome activity and phosphorylation in vitro and in vivo (Bingol et al., 2010, Djakovic et al., 2009, Djakovic et al., 2012, Hamilton et al., 2012, Jarome et al., 2013), though this relationship has never been examined during memory reconsolidation. Here, using a combination of biochemical, pharmacological and behavioral approaches, we directly tested whether CaMKII controls memory trace destabilization through its regulation of the proteasome.