| Background:Memory consolidation is the gradual process of converting acquired short-term memory into the long-term memory. Previous studies have shown that the activating of gene and protein signaling pathway participates in memory consolidation process.Studies have found that the regulation of both gene expression and protein synthesis after memory acquisition is crucial for the memory consolidation. In recent studies,the epigenetic regulation of gene expression has been extensively investigated in terms of learning and memory. The epigenetic regulation of gene expression has received considerable attention within the realm of neuropsychiatric disorders. Among these epigenetic mechanisms, microRNA (miRNA)-mediated regulation of gene expression has been implicated in spatial memory, trace conditioning, extinction memory, and fear memory consolidation. miRNA achieve post-transcriptional repression by directly binding to the 3' UTR of target mRNAs and highly expressed in the brain, and their expression are dynamically regulated during plasticity, both cellular and behavioral levels. miRNA mediated regulation of gene expression has been implicated in synapse plasticity in neurons and long-term memory formations in behavior. However, thousands of miRNA have been found in the brain, the functions of most miRNA in learning and memory remain unknown. Whether these miRNA have same functions in memory consolidation or could they have different functions in memoy process is still unclear.Object:We aim to find out the changed miRNA after CFC training and indicate whether abolishing or overexpressing these miRNA could affect the CFC training and testing process in mice. Moreover, we wish to indicate its targeted proteins that miRNA could regulate to participate in learning and memory to enhance our understanding of the functions of miRNA in learning and memory and find out the potentially important therapeutic targets that merits further study.Methods:1. Find out the changed miRNA in hippocampus after CFC training.1.1 Find out the changed miRNA using an array based approach.8 weeks old adult C57BL/6J mice were used to CFC training and a microarray was performed on miRNA extracted from hippocampus of adult mice 1 h after fear conditioning. The hippocampus of adult mice 1 h after shock alone was a control.Hundreds of miRNA were read by the microarray. miRNA with signal more than 500 were chosen for further analyses to find out the signaificantly changed miRNA after CFC training.1.2 Using qPCR to quantify the changed miRNA after CFC training.We would use qPCR to quantify the levels of these miRNA 1 h and 6 h after contextual fear conditioning training and found miRNA including miR-181a, miR-126,miR-222, miR-143 and miR-151a were significantly increased and miR-125, miR-690 were decreased.1.3 Whether blocking above miRNA could affect hippocampal memory.We used different antagomirs to abolish targeted miRNA. We stereotactically injected miR-143 or miR-181 a antagomirs into the dentate gyrus (DG) of adult mice to abolish miR-143 or miR-181 a levels. We then evaluated the effect of miR-143 or miR-181a knockdown on hippocampus-dependent memory. Our results exhibited that blocking miR-143 had no effect on CFC memory training and consolidation.miR-181a abolition had no effect on contextual fear memory acquisition but impaired the memory consolidation. These results showed that miR-181 a but not miR-143 was sufficient for CFC memory consolidation. Next, we would inject the antagomirs of other miRNA to research the functions of other miRNA which changed after CFC training in CFC memory process.1.4 Whether overexpressing miRNA could affect hippocampal memory.miR-overexpression lentivirus, which simultaneously expressed GFP protein,was injected into mice DG to overexpress the miRNA. Four weeks later, we could perform the CFC testing to found out whether miR-181a or other miRNA could affect the CFC memory.2. Find out the mechanisms of miRNA participating in CFC memory2.1 Using luciferase reporter assay to identify whetehr miR-181a could target PRKAA1 and REDD1.Our above data showed that miR-181a could take part in CFC consolidation.Next we aim to determine miR-181a-regulated targets participating in hippocampal memory by scanning the 3' UTR of mRNAs for potential miR-181 a binding sites using different commonly mined algorithms (Targetscan, miR-22,miRDB,microcosm). These algorithms generate many putative targets. To limit the scope,KEGG analysis was performed on the predicted targets to examine miR-181a-regulated molecular pathways. Dozens of miR-181a-regulated signaling pathways had been predicted. Since our focus is on the miR-181a function in learning and memory, we filtered out pathways not involved or important in learning and memory. As a result, only a few pathways were selected for the next round of screening. Then we took intersection elements from molecular pathways of different algorithm-predicted targets. We noticed that the mTOR pathway was predicted by all algorithms used, suggesting that miR-181a could play important roles in mTOR pathway regulation. PRKAA1 and REDD1 were two proteins in the mTOR pathway signaling predicted to be regulated by miR-181a. We then performed luciferase reporter assay to determine whether PRKAA1 and REDD1 are indeed the molecular targets of miR-181a.2.2 Protein levels of PRKAA1 and REDD1 decreased after CFC training is induced by miR-181a.The above results show that PRKAA1 and REDD1 were the targets of miR-181 a in vitro. We then investigated whether these targets were regulated by miR-181a after CFC training. To this end, we first examined PRKAA1 and REDD1 protein levels after CFC training. Then we used antagomir-blocked miR-181 a and gave mice CFC training to find out whether PRKAA1 and REDD1 could be regulated by miR-181a after CFC training.2.3 The effect of miR-181a on contextual fear memory consolidation depends on PRKAA1 and REDD1.Data above revealed that miR-181a could decrease PRKAA1 and REDD1 protein levels after CFC training. We then wanted to determine whether miR-181a's effect on fear memory consolidation depends on PRKAA1 and REDD 1. To this end,we aim to design two experiments:2.3.1 The functions of PRKAA1 and REDD1 in CFC memory consolidation.To indicate the functions of PRKAA1 and REDD 1 in memory consolidation, ,the specific PRKAA1 activator, AICAR, and inhibitor, Compound 9,were used to activate or inhibit PRKAA1 in adult mice. We aim to inject C9 or AICAR to mice hippocampus and found out whether abolishing or activing PRAKK1 could affect the memory consolidation. At the same time, the siREDD1 lentivirus which simultaneously express GFP protein was used to knockdown REDD1 levels in DG, while the REDD 1-overexpression lentivirus encode REDD1-GFP, a fusion protein of REDD 1 and GFP, was used to overexpress the REDD1 protein. We then aim to inject the lentivirus to mice hippocampus to reseach whether REDD1 could participate in CFC memory consolidation.2.3.2 The effect of miR-181a on contextual fear memory consolidation depends on PRKAA1 and REDD1.We then asked whether miR-181a's effect on contextual fear memory consolidation depends on PRKAA1 and REDD1. We divided mice into four groups,GFP+veh,REDD1-OE+AICAR, 181-OE+veh and 181a-OE+REDD1-OE+AICAR.We then tested the four group mice and observe the mice performance in CFC training and testing to find out whether the effect of miR-181a on contextual fear memory consolidation depends on PRKAA1 and REDD1..3. Whether miR-181a could regulate the mTOR pathway.Previous results have shown that PRKAA1 and REDD1 could inhibit mTOR activity. Therefore, we examined mTOR activity after CFC training. We next want to find out whether miR-181a could reduce PRKA A1 and REDD1 levels to enhance mTOR activity after CFC training.3.1 Whether miR-181a could regulate the mTOR activity after CFC training.Previous results have shown that PRKAA1 and REDD1, which are parallel but are not upstream and downstream relation pathways, were shown to regulate mTOR activity in cells. Both PRKAA1 and REDD1 could inhibit mTOR activity. Therefore,we then aim to examine mTOR activity after CFC training. We used antagomirs blocked miR-181a and gave mice CFC training. Mice were randomly divided into 4 groups: HC+veh, HC+anta, CFC+veh, and CFC+anta. Four hours after CFC training,DG of hippocampus was taken for indicating whether miR-181a could regulate the mTOR activity after CFC training.3.2 The effect of miR-181a on contextual fear memory consolidation depends on mTOR pathway.3.2.1 Whether PRKAA1 and REDD1 participation in fear memory consolidation depended on mTOR.Previous studies have reported that both PRKAA1 and REDD1 could inhibit mTOR activity in cells. Since mTOR has been shown to participate in contextual fear memory consolidation, we then ask whether miR-181a could take part in contextual fear memory consolidation by mTOR pathway regulation. To this end, We divided mice into four groups, Scr+veh, siREDDl+C9, Scr+Rapamycin, and siREDDl+C9+Rapamycin and gave these four groups mice CFC training to find out whether PRKAA1 and REDD1 participation in fear memory consolidation depended on mTOR.3.2.2 Whether miR-181a could enhance mTOR activity to participate in fear memory consolidation.Since we have found PRKAA1 and REDD1 participated in contextual fear memory consolidation by inhibiting mTOR activity, we then ask whether miR-181a could enhance mTOR activity to participate in fear memory consolidation. To this end,we divided mice into four groups, Scr+veh, miR-181a-OE, Scr+Rapamycin, and miR-181a-OE +Rapamycin and gave these four groups mice CFC training to find out whether miR-181a participation in fear memory consolidation depended on mTOR.Results:1. miR-181a is upregulated in the hippocampus 1 h after CFC.We initially performed qPCR to quantify miR-181a levels 1 and 6 h after CFC training and showed that hippocampal miR-181 a levels increased significantly 1 h after training. Our results revealed that neither context nor shock alone could upregulate miR-181a levels.2. Manipulating of miR-181a in hippocampus affects the CFC consolidation.We stereotactically injected miR-181a antagomirs and then tested CFC in two mice groups, scramble and miR-181a antagomirs. Both groups exhibited intact freezing response during the training process, suggesting that miR-181a abolition had no effect on contextual fear memory acquisition. We then examined short-term memory (STM) and long-term memory (LTM) 1 h and 24 h after training. Both groups did not exhibit significantly different freezing times during the STM test.However, in the LTM test, freezing time was significantly shorter in the antagomir-injected group than in the scramble group. Our results suggested that miR-181a blocking could not affect spontaneous exploratory activity and anxiety-like behavior.3. PRKAA1 and REDD1 are targets of miR-181aWe aimed to determine miR-181a-regulated targets participating in hippocampal memory by scanning the 3' UTR of mRNAs for potential miR-181 a binding sites using different commonly mined algorithms (Targetscan, miR-22,miRDB,microcosm). These algorithms generate many putative targets. To limit the scope,KEGG analysis was performed on the predicted targets to examine miR-181a-regulated molecular pathways. We noticed that the mTOR pathway was predicted by all algorithms used, suggesting that miR-181a could play important roles in mTOR pathway regulation. PRKAA1 and REDD1 were two proteins in the mTOR pathway signaling predicted to be regulated by miR-181a. Previous studies have shown that the mTOR pathway was essential for protein synthesis, synaptic plasticity,and memory formation. Therefore, we focused on the mTOR pathway moving forward. We then performed luciferase reporter assay to determine whether PRKAA1 and REDD1 are indeed the molecular targets of miR-181a. These results suggest that miR-181a targeted PRKAA1 and REDD1.4. PRKAA1 and REDD1 is induced by miR-181a after CFC training.The above results show that PRKAA1 and REDD1 were the targets of miR-181a.We then investigated whether these targets were regulated by miR-181a after CFC training. We first examined PRKAA1 and REDD1 protein levels after CFC training and found that both proteins were decreased 4 h after CFC training. Previous results have shown that PRKAA1 and REDD1 could inhibit mTOR activity. Therefore, we examined mTOR activity after CFC training and found that p-mTOR levels of were increased 0 h and 4 h after CFC training. When we block miR-181a, CFC training could not change the PRKAA1, REDD1 and mTOR levels. Taken together, these results suggest that miR-181a could reduce PRKAA1 and REDD1 levels to enhance mTOR activity after CFC training. Previous studies have reported that some proteins such as HOXA11, Bcl2 and so on are the targets of miR-181a in cancer cells. To found out whether these targets were regulated by miR-181a after CFC training in vivo, we examined the protein levels of HOXA11, Bcl2 and Msx2, which were all the validated targets of miR-181a, at the different time after CFC training. Our results revealed that HOXA11 and Bcl2 protein levels were increased 4h after CFC training and Msx2 protein levels were not changed, which suggested that these validated targets were not regulated by miR-181a after CFC training.5. Effect of miR-181a on CFC consolidation depends on PRKAA1/ REDD1.Mice were given CFC training and testing in a lower electric. We first examined the synergetic effect of PRKAA1 and REDD1 in CFC memory. Mice injected with REDD1-OE and AICAR in the DG were given CFC training and exhibited similar freezing times when compared with GFP+veh-injected mice during training and 1h after training, which suggested that enhancing PRKAA1 and REDD1 had no effect on fear memory acquisition and short-term memory. We then examined contextual fear memory consolidation 24 h after training and found that mice injected with REDD1-OE, AICAR and REDD 1-OE+AICAR showed significantly lower freezing times than vehicle injected mice, suggesting that elevating PRKAA1 and REDD1 impaired contextual fear memory consolidation. Next, we injected siREDD1 and C9 into the DG of adult mice to block PRKAA1 and REDD1 functions at a lower current intensity. Results revealed that blocking PRKAA1 and REDD1 did not affect contextual fear memory acquisition and STM. However, when given the LTM test,mice injected with siREDD1, C9, and siREDD1+C9 exhibited significantly elevated freezing times compared with vehicle injected mice. suggesting that blocking PRKAA1 and REDD1 could enhance contextual fear memory consolidation.We then asked whether miR-181a's effect on contextual fear memory consolidation depends on PRKAA1 and REDD1. Results showed that miR-181a overexpression enhanced contextual fear memory consolidation. which is consistent with our above data. Moreover, mice in REDD1-OE+AICAR group exhibited lower freezing times than those in the GFP+veh group, suggesting that increased PRKAA1 and REDD1 functions suppressed contextual fear memory consolidation. However,freezing time was significantly lower in the 181a-OE+REDD1-OE+AICAR group than in GFP + veh group, while no significant difference was found compared with the REDD1-OE+AICAR group, which suggested that increased PRKAA1 and REDD1 functions could block miR-181a-OE-induced contextual fear memory consolidation enhancement. These results indicate that miR-181a's effect on the hippocampal contextual fear memory consolidation depends on PRKAA1 and REDD1.6. Effect of miR-181a on CFC consolidation depends on the mTOR pathwayPrevious studies have reported that both PRKAA1 and REDD1 could inhibit mTOR activity in cells. Since mTOR has been shown to participate in contextual fear memory consolidation, we then ask whether miR-181a could take part in contextual fear memory consolidation by mTOR pathway regulation. To this end, we first examined whether PRKAA1 and REDD1 participation in fear memory consolidation depended on mTOR, which was still unclear from previous research. Our results revealed that the siREDDl+C9 group exhibited significantly higher freezing times than the Scr+veh group, suggesting that abolishing PRKAA1 and REDD1 function could enhance contextual fear memory consolidation. Moreover, the Scr+Rapamycin group exhibited lower freezing times than the Scr+veh group. However,the siREDD1+C9+Rapamycin group still showed lower freezing times when compared to the siREDDl+C9 group but not when compared with the Scr+Rapamycin group,which suggested that inhibiting mTOR activity could abolish the enhanced memory consolidation induced by the loss of PRKAA1 and REDD1 functions. These results suggest that PRKAA1 and REDD1 were involved in contextual fear memory consolidation by inhibiting mTOR activity.Since we have found PRKAA1 and REDD1 participated in contextual fear memory consolidation by inhibiting mTOR activity, we then ask whether miR-181a could enhance mTOR activity to participate in fear memory consolidation. When given Rapamycin,the 181 a-OE+Rapamycin group exhibited significantly lower freezing times than the GFP+veh group but had similar freezing responses when compared to the GFP+Rapamycin group, which suggested that mTOR activity inhibition could abolish the enhanced memory consolidation induced by increased miR-181a levels in the hippocampus. These results indicated that the effect of miR-181a on contextual fear memory consolidation depends on the activity of mTOR pathway.Conclusions:1. In this study, we observed that miR-181a was responsible for hippocampal contextual fear memory consolidation.2. Using different commonly mined algorithms and luciferase reporter assay, we found that miR-181a targeted PRKAA1 and REDD1, two mTOR upstream inhibitor proteins.3. We indicated that upregulated miR-181a could reduce PRKAA1 and REDD1 protein levels and then promote mTOR activity to facilitate hippocampal fear memory consolidation.4. PRKKA1 and REDD1 were responsible for hippocampal contextual fear memory consolidation.5. Our study enhanced our understanding of the functions of miRN A in learning and memory. Considering that miRNA are essential for many brain disorders, we recommend further studies on miR-181a as a potentially important therapeutic target. |
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