Protein Degradation In The BLA Involved In The Erasure Of Cued Fear Memory

BackgroudWe all know that the Posttraumatic Stress Disorder (PTSD) may develop after a traumatic experience. Many studies have found that there’s a close relation between the occurrence of PTSD and failed extinction of aversive memories. Exposure therapy, which can be regarded as a kind of extinction, is the most effective treatment at present. However, the memory which is seen as "can-be-forgot" returns on the following three conditions, (Ⅰ)renewal (Ⅱ) spontaneous recovery (Ⅲ) reinstatement. The possible explanation for this phenomenon is that the extinction training does not erase the original memory, but produces a new memory.Monfils et al. reported that a retrieval+extinction procedure can permanently erase the fear memory during the course of extinction. However, the mechanism is still unknown.A number of studies have showed that the fear memory formation is required for new protein synthesis in the amygdale, so it is possible that the fear erasure need the elimination of fear-related proteins. And Lee et al. found that postsynaptic proteins are degraded in the hippocampus by polyubiquitination after retrieval of contextual fear memory. Those suggest that stereotaxic protein degradation depending on the ubiquitin-proteasome system (UPS) plays an important part in unstable periods after fear memory retrieval. Consequently, we supposed that the changes of protein degradation in amygdale are vital during memory erasure.To prove our hypothesis and study the possible molecular mechanism, we utilized techniques, such as GST-S5a pull down, protease activity analysis and RNA interference recombinant plasmids.ObjectiveDiscover the role of protein degradation in retrieval+extinction procedure induced memory erasure and research its molecular mechanism.Result:1. Retrieval+extinction procedure induces increased polyubiquitination and proteasome activity in BLAWe utilized GST-S5a pull down polyubiquitinated analysis and proteasome activity detection techniques in vitro. We found that polyubiquitinated protein expression levels in the retrieval+extinction group increased markedly but not in the extinction group (P< 0.01).Compared with the no retrieval+extinction group, the proteasome activity of the retrieval+extinction group had significantly increase (P< 0.01)2. Protein degradation regulates Retrieval+Extinction procedure-induced AFC memory erasureWe first utilized proteasome inhibitors βLac. Compared with the Retrieval+ Vehicle group, the freezing level of the Retrieval+βLac group had markedly increased in renewal, reinstatement and spontaneous recovery test (P< 0.01). What’s more, compared with the no retrieval+Vehicle group, the freezing value of the retrieval+oleuropein group had markedly decreased in renewal, reinstatement and spontaneous recovery test (P<0.01)3. Promoting protein degradation is necessary for memory erasure after extinctionWe found that K48 polyubiquitination level was enhanced in Extinction group 10 minutes after AFC (P<0.05). Blocking protein degradation prevented extinction-induced AFC memory erasure (P< 0.01). Then we used another paradigm, in which memory can’t erase, to further confirm the role of protein degradation in the memory erasure.30 days after training, the rats received retrieval+extinction. We found that K48 polyubiquitinated level was decreased in retrieval+extinction group (P<0.01), and increased protein degradation promoted extinction+induced AFC memory erasure (P<0.01)4. The activation of ubiquitin-proteasome pathway increases degradation of GluAl during memory erasureWe found that retrieval+extinction increased degradation of GluAl (P< 0.01) In the subsequent analysis, We found that 0.5 hour before the extinction, GluAl degradation was rapidly enhanced in rats received bilateral microinfusion of oleuropein (P<0.01)5. After Retrieval+Extinction procedure, increased proteasome activity is mediated by the activation of CaMKIIWe found that retrieval+extinction group exhibited significantly enhanced level of phosphorylation and activity of proteasome (P< 0.01). Then,we blocked CaMKII activity prior to retrieval and the animals showed significantly higher freezing levels (P<0.01) and significant low activity of proteasome (P< 0.01).Results indicated CaMKII activity is essential for GluAl degradation.6. Nedd4-1 ubiquitinates GluAl during memory erasureWe found that retrieval+extinction group showed significantly higher Nedd4-1 level (P< 0.01). Then,30 days before the fear training, rats received bilateral microinfusion of siNedd4-1 then were conducted reinstatement test, which showed siNedd4-1 treatment abolished the recall-induced memory erasure.7. Nitrosation in BLA functions as activator of proteasome during memory erasureWe found that L-NAME led to decreased degradation during recent memory erasure (P< 0.01). Then,rats received L-NAME before Retrieval and we observed the freezing is enhanced (P< 0.01). Furthermore, We found that MOL facilitated remote memory erasure by increasing GluAl degradation (P< 0.05).Finally, we observed the freezing was decreased in rats receiving bilateral microinfusion of MOL before retrieval.Conclusions:AFC memory erasure, induced by retrieval+extinction procedure, depends on ubiquitin-proteasome pathway-mediated protein degradation. Degradation of GluA1 is a specific prerequisite for retrieval+extinction procedure induced AFC memory erasure. The increase of the GluA1 degradation results from activation of CaMKⅡ At the same time, Nedd4-1 mediated GluAl degradation enhances during memory erasure.