HDAC7 Ubiquitination By Cbx4 Is Involved In Contextual Fear Conditioning Memory Formation

ObjectiveLearning and memory is one of the advanced functions of the brain,which plays the important roles in our survival and lives.Identification of the essence of learning and memory could not only help us to know the world better,but also provide the therapeutic method for the neuropsychiatric disorders with the main characteristics of defect in learning and memory,such as Alzheimer disease,posttraumatic stress disorder,habituation etc.Specific genes that affect neuronal plasticity are crucial for long-term memory.However,the molecules translated by these genes last only from several hours to several days,which may not maintain sufficient protein levels for long-term memory.In recent studies,epigenetics has been demonstrated to be the basis for the sustained regulation of gene transcription.Specifically,histone acetylation has been thoroughly investigated in many mental diseases in recent years.Acetylation may occur in specific sites in histones and is regulated by two classes of enzymes,including histone acetyltransferase(HAT)and histone deacetylase(HDAC).The de-acetylation of histones is induced by HDACs to ensure DNA is tightly associated with histones and chromatin is "closed" for transcription.HDAC has been reported to play important roles in learning and memory.There are 4 groups and 18 types of HDAC(HDAC 1-11 and SIRT 1-7)in the body,which plays different roles.For example,knock-out of HDAC3 in CA1 promoted object location but not spatial memory.Moreover,knock-out of HDAC5 damaged the spatial memory but not the formation of fear memory in mice.The overexpression of HDAC1 promoted the extinction but not the formation of fear memory.It has been reported that the non-specific inhibitors of HDACs may significantly improve the performance of animals in contextual fear memory,object location,object recognition and spatial memory tests,which is promising for the development of effective drugs to rescue memory deficits in multiple neurological disorders.However,the member diversity and the efficacy complexity of HDACs make this development not as easy as it appears.Therefore,additional research is necessary to clarify the roles of individual HDACs in learning and memory.The role of HDAC7 in learning and memory is not clear by now,so we focus on this part.Contextual fear conditioning is a traditional model in research of learning and memory,which could test the acquisition of learning,short term and long term of memory.We found that many HDACs,including HDAC7,changed in CA1 region after CFC training in gene microarray screen,which indicated that HDACs functioned in learning and memory.HDAC7 is a member of the class II HDACs and has aroused substantial interest.The functions of HDAC7 have predominately been reported in the non-nervous system,such as the cardiovascular and immune systems.HDAC7 expression in the hippocampus changed after CFC training;however,there has been no direct evidence to date to indicate the functional roles of HDAC7 in CFC memory.We performed a series of experiments to investigate the role and mechanism of HDAC7 in CFC memory.We determined that the HDAC7 protein level is selectively decreased in the DH after CFC training.Overexpression or knock-down of HDAC7 in the DH effectively regulated the formation of CFC memory.The decrease in HDAC7 is caused by ubiquitin-related degradation,and CBX4 and Nur77 are the E3 ligase and the target gene of HD AC 7 separately during CFC formation.Our investigation discovered the new function of HDAC7 in learning and memory,which provided the therapeutic methods for clinical diseases.Methods1.The physiological change of HDAC7 during CFC training1.1 Establishment of CFC memory model and detection of HDAC7.Animal:12 weeks old wild type C57BL/6J mice.Mice were separately housed and the cages were placed in the CFC training room 2h before starting experiments.The mice were placed in a standard fear conditioning chamber(25×25×25 cm)and allowed to habituate for 2 min without stimulation(habituation).Each mouse received 3 consecutive footshocks(Two second duration each;weak,0.4 mA or normal,0.7 mA)through a stainless steel grid floor.Each footshock was separated by a 58 s time period.After an additional 58 s following the last shock,the mice were returned to the home cage.The footshock US was generated by a programmable animal shocker,and the CS comprised the experimental context.It takes about 5 min for the training.Mice were executed at different time course as planned,the head was cut off and brain was taken out.We use western blot,real-time PCR and immunohistochemistry to test the change of HDAC7 after CFC training.1.2 Change of HDAC7 in different encephalic regions.Mice were executed 6h after CFC training and brains were taken out.Then the brains were put on the brain mould.The dorsal hippocampus,the ventral hippocampus and the basolateral amygdale regions were separated.Then we use WB to test the change of HDAC7 in different encephalic regions,including the change of different HDACs in dorsal hippocampus.1.3 Change of HDAC7 due to the coupling of environment and fear memory.To separate the impact of the "context" and the "shock" on the HDAC 7 expression in the DH,two additional experiments were performed.To determine the effect of the context exposure("context"),the mice were allowed to freely explore for 5 min in the training chamber without receiving any footshock.To determine the effect of the shock and minimize the context exposure("immediate shock"),the mice were given a 2 s footshock(0.7 mA)immediately after being placed in the training chamber and were quickly removed and returned to their home cage.Mice in the naive group were only housed in the standard home cage during CFC training.We used WB to test the change of HDAC7 in different stimulations.2.The function of HDAC7 during the formation of CFC memory2.1 Construction of lentivirus and the infection efficiency test of the lentivirus.We use the 3rd generation method to generate lentivirus expressing small-hairpin RNA targeting HDAC7(sh-HDAC7)and GFP by transfecting plasmids into HEK293T cells.Cell cultural medium were collected and virus were concentrated 72 h after the transfection.Then we injected the virus into DH region and used IF to test the infection efficiency,and used WB method to test the knock down efficiency and overexpression levels of HDAC7.2.2 The influence on CFC memory when knock down or overexpress HDAC7 in DH.We knock down or overexpressed HDAC7 in DH and performed the CFC training.Short-term memory(STM)was tested at 1 h and long-term memory(LTM)was tested at 24 h after training.Two separate groups of mice were used for the STM and LTM tests.The mice were returned to the previous chamber in which the training occurred and were tested for 5 min without footshock;memory was assessed by the duration of freezing behavior.2.3 The specific brain region of HDAC7 in CFC.We knock down or overexpressed HDAC7 in AMY region and performed CFC training as described above.Also tested STM and LTM as described above.3.The function of HDAC7 in different hippocampal-dependent learning and memory process.We knock down or overexpressed HDAC7 in DH and performed Morris water maze test and object location test as described below to test the learning memory on space and object location.Morris water mazeThe apparatus consisted of a circular water tank(120 cm diameter,40 cm height)filled with water(22?)to a depth of 25 cm.A circular escape platform(6 cm in diameter)was placed 1 cm below the water surface.A complete experiment consisted of a learning period(platform in place)with four trials per day for 5 consecutive days and a probe test(platform moved)on the sixth day.During the period of learning,the platform was always placed in the center of the same quadrant(target quadrant).Each trial consisted of a maximum of 60 s starting from one of the four quadrants with the animal facing the wall.In the learning process,the escaped latencies for a single day were averaged to produce a daily mean.On the 6th day,the platform was removed,and the mice were allowed to swim for 60 s.The number of platform crossings and the time spent in the four quadrants for each mouse were recorded with a video tracking system(Smart).Object location testThe testing apparatus comprised an open field arena(40 x 40 cm)with 35 cm high walls.The floor was white with wall-mounted visual cues(located on the north and east sides),which were visible in the arena.The behavior recording camera and the lamp were installed above.Two identical objects were placed in the NW and NE corners of the arena,and the mice were allowed to explore these objects for 5 min.Memory was subsequently assessed during a single,5 min test trial after a 24 h delay(for LTM).During the test trial,replicas of the training objects were placed in a familiar[NW]corner and a novel[SE]corner.The times that the mice explored the object in the new and old positions were independently recorded.4.The mechanism for the change of HDAC74.1 Detection of HDAC7 synthesis and degradation.CFC training were performed 2 weeks after pipe laying in mice.Three groups were established:vehicle,protein synthesis inhibitor(CHX)and protein degradation inhibitor(?LAC).All 3 drugs were injected in DH.Then CFC training was performed and the brains were taken out.We used WB to test the change of HDAC7 protein level.4.2 The screen of E3 ligase of HDAC7.We used protein mass spectrum to screen the E3 ligase.We used the exogenous and endogenous Co-IP assay,protein purification and in vitro ubiquitin assays to verify the E3 ligase of HDAC7.4.3 The influence of E3 ligase on CFC memory.We generated lentivirus expressing small-hairpin RNA targeting CBX4.Then we injected the virus in DH and test the freezing time as described above.5.The mechanism of HDAC7 impacting on CFC5.1 Screen of the histone acetylation sites regulated by HDAC7.We used lentivirus to knock down HDAC7 in DH and performed CFC training.Then we tested the change of different histone acetylation sites by WB.5.2 The downstream target genes regulated by HDAC7 and their mechanism.Mice were performed CFC training and we tested Nur77 protein level by using real-time PCR.We also knock down or overexpressed HDAC7 in cultured neurons and in DH and then tested Nur77 protein levels.We tested the influence of HDAC7 on Nur77 promoter by CHIP assay.5.3 The influence on CFC from downstream target genes.We generated lentivirus expressing Nur77 knock down small hairpin RNA.Then we knock down Nur77 in DH and performed CFC training to test the freezing time.Results1.The change of HDAC7 during CFC memory1.1 HDAC7 decreased significantly during CFC memory formation.We found that HDAC7 decreased significantly compared to naive group after CFC training,which reached the lowest point at 6 h and lasted to 24 h.This result was also proved by IHC assay.1.2 HDAC7 decreased specially in DH.We tested HDAC7 protein level in different encephalic regions and found that,compared to control group,only HDAC7 in DH decreased while HDAC7 in VH and AMY stayed constant.Also,in DH,only HDAC7 decreased significantly after CFC training while HDAC1 and HDAC6 did not change.1.3 The coupling of environment and fear memory facilitated the change of HDAC7.We tested the different stimuli on HDAC7 protein level and found that compared to naive,one stimuli only(contest or shock)did not influence HDAC7 while the coupling CFC decreased HDAC7.2.HDAC7 damaged CFC memory formation2.1 HDAC7 in DH regulated the formation of CFC memory.We overexpressed or knock down HDAC7 using virus in DH and performed CFC training and test.We found that compared to control group,HDAC7-overexpressed group did not perform significant change in STM,while in LTM,the freezing time declined markedly.On the other hand,HDAC7-knock down group also did not perform significant change in STM,while in LTM,the freezing time increased markedly.2.2 Knock down or overexpress HDAC7 in AMY did not influence CFC formation.We knock down or overexpressed HDAC7 using virus in AMY and performed CFC training and test.We found that compared to control group,there is no significant change in STM or LTM in HDAC7 knock down or overexpression group.3.HDAC7 is involved in different hippocampal-dependent learning and memory processes.3.1 Knock down HDAC7 facilitated the spatial memory and object location memory.We knock down HDAC7 in DH and performed water maze test and objection location test.We found that compared to control group,the time spent on finding platform significantly decreased in HDAC7 knock down group.We also found that compared to control group,the time spent on new objects significantly increased in HDAC7 knock down group.3.2 Overexpressed HDAC7 damaged the spatial memory and object location memory.We overexpressed HDAC7 in DH and performed water maze test and objection location test.We found that compared to control group,the time spent on finding platform significantly increased in HDAC7 overexpressed group.We also found that compared to control group,the time spent on new objects significantly decreased in HDAC7 overexpressed group.4.HDAC7 degraded via ubiquitination during CFC memory4.1 HDAC7 ubiquitination level increased during CFC.We tested the change of HDAC7 mRNA in DH during CFC by using real-time PCR assay and found that,compared to naive group,there is no significant change of HDAC7 mRNA level at different time course after CFC training.Then we detected HDAC7 ubiquitination level by Co-IP assay and found that it increased markedly compared to control group after CFC.Then we microinjected CHX and(3LAC in DH and found that compared control group,HDAC7 in ?LAC group did not change significantly while HDAC7 in CHX decreased observably.4.2 CBX4 as HDAC7 E3 ligase induced its ubiquitination degradation during CFC.We screened CBX4 as E3 ligase of HDAC7 and verified this result through endogenous Co-IP,exogenous Co-IP and in vitro purified protein Co-IP and found that HDAC7 interacted with CBX4 markedly.Then we found that CBX4 could ubiquitin HDAC7 by using endogenous Co-IP and in vitro ubiquitin assay.4.3 CBX4 expression increased during CFC.We found that compared to naive group,CBX4 increased significantly in CFC.Also,we found that when knock down CBX4 using virus in DH,HDAC7 increased significantly compared to control group.4.4 Knock down CBX4 damaged the formation of CFC memory.We knock down CBX4 by using lentivirus in DH.Then we performed CFC training and test and found that compared to control group,freezing time of mice decreased markedly in CBX4 knock down group.5.HDAC7 modulated CFC memory via Nur775.1 Nur77 increased during CFC formation.We found that compared to naive group,Nur77 increased significantly in DH but not VH in CFC by using RT-PCR and WB assay.5.2 HDAC7 regulated Nur77 expression via inhibiting acetylation of H4K12 site.We tested the mechanism of HDAC7 regulating Nur77 by WB and CHIP assay and found that compared to control group,H4K12 site acetylation level increased significantly in HDAC7 knock down group,also,Nur77 promoter and protein level increased markedly.5.3 Knock down of Nur77 damaged the formation of CFC memory and resisted HDAC7 to a certain degree.We knock down Nur77 and/or HDAC7 in DH as indicated in figures and performed CFC training and test.We found that compared to control group,knock down Nur77 only could significantly decrease the freezing time,while knock down both Nur77 and HDAC7 also significantly decreased the freezing time.ConclusionThe current findings demonstrated the effects of HDAC7 on hippocampal-dependent memories.Moreover,we determined the mechanism of decreased HDAC7 in CFC through ubiquitin-dependent protein degradation.We also verified CBX4 was one of the HDAC7 E3 ligases.Finally,we demonstrated that Nur77,as one of the important targets for HDAC7,was involved in CFC memory formation.All these proteins,including HDAC7,CBX4 and Nur77,could be the potential therapeutic targets for preventing the memory deficits in aging and neurological diseases.