The Role And Mechanism Of Up-regulation Of Cdc20 In The Nucleus Accumbens Neurons Intervening Cocaine Addictive Behavior In Rats Through Promoting Ubiquitination And Degradation Of FMRP

BackgroundsDrug addiction is a chronic and recurrent encephalopathy that seriously endangers the physical and mental health of human beings.It is characterized by the fact that the addicts are aware of the possible serious adverse consequences of drug use,but they still repeatedly and compulsively abuse different psychoactive substances,and leads to public health and security issues.The nucleus accumbens（NAc）is an important target for neuromodulation in the treatment of drug addiction,and it has always been a hot brain region for us to explore the mechanism underlying the drug addiction.We performed a clinical trial of NAc deep brain stimulation（DBS）in the treatment of opioid addiction（relapse prevention）,and the optimization and promotion of DBS urgently depended on the solution of some key problems,which were due to our lack of sufficient in-depth understanding of the structure and function of NAc.In recent years,single-cell RNA sequencing（sc RNA-seq）has been applied for functional analysis on the subpopulation of cells in a given brain nucleus.In this study,SD rats were treated as model animals,and the cocaine addiction model was established using the classical self-administration method,and single-cell and spatial transcriptomics（ST）sequencing were combined to help to uncover the NAc more comprehensively in terms of the molecular,cellular,anatomical and functional heterogeneity,and reveal its changes after animal addiction.Moreover,we will conduct further research based on a subgroup of cells and a set of marker genes or molecules to try to find a novel molecular,cellular,or anatomical target for intervention in drug addiction.ObjectivesThis research is an exploratory study.Through the analysis of sc RNA-seq data,the molecular,cellular,anatomical and functional heterogeneity of NAc cells in rats before and after cocaine addiction were initially revealed,and the structure and function of NAc will be further explored.After confirming the existence of a group of neurons with Cdc20as a main marker gene in the rat NAc,the expression of Cdc20 gene and its products in the NAc and the role of Cdc20 on the growth and development of neurons will be studied.Additionally,the novel phenotypes resulting from knockdown or overexpression of Cdc20in the adult rat NAc will be revealed.According to the characteristics of Cdc20 as an anaphase promoting complex/cyclosome（APC/C）co-activator for ubiquitination and degradation of substrate proteins,a new substrate of Cdc20 in NAc will be discovered,and the role and mechanism of its regulation for ubiquitination will be preliminarily elucidated.Finally,we plan to find whether this regulation is involed in drug addiction by using animal models and try to explore new targets for prevent drug addiction.Methods and Results1.Single-cell and spatial transcriptomic analysis of the NAc in cocaine-addicted rats.Adult male SD rats were treated with jugular vein catheterization to establish a cocaine intravenous self-administration（SA）model.The NAc tissue in the rats from saline control group and cocaine-addicted group was isolated,and sc RNA-seq and ST sequencing were performed using 10×Genomics technology.The work flows and analysis methods of these data were omitted.56,959 cells in the NAc tissues of 20 model rats from all 4 samples were classified into 12 main cell lineages:astrocytes,microglia,oligodendrocytes,neurons,endothelial cells,mural cells,ependymocytes,monocytes,macrophages,oligodendrocyte precursor cells（OPCs）,T lymphocytes,and granulocytes.The m RNA expression analysis of the four types of cell populations before and after addiction was performed respectively.The astrocytes were classified into 6 subgroups according to UMAP,and their representative marker genes were Agt,Luzp2,Grin2c,Tf,Vim and Mpc2 respectively.The relative abundance comparison revealed that the expression of Luzp2+and Mpc2+subgroup increased,and the expression of Agt+,Grin2c+,and Tf+subgroup decreased in the cocaine addiction group,while no obvious changes were found in the Vim+subgroup.However,the combined analysis of sc RNA and ST revealed that Vim+subgroup was obviously activated specifically in the NAc,which were then verified by fluorescence in situ hybridization.Gene set variation analysis（GSVA）showed that Luzp2+and Mpc2+subgroup with increased expression after cocaine addiction were mainly enriched in pathways such as OXIDATIVE PHOSPHORYLATION,constituting a major component of oxidative phosphorylation which was enhanced in astrocytes after addiction.Based on UMAP,the microglia were classified into 7 subgroups,whose representative marker genes were:Aif1,Dusp1,Itm2b,Ccl2,Il1b,Gja1 and Agpat4.The relative abundance comparison revealed that the expression of Ccl2+and Il1b+subgroup increased,and the expression of Aif1+,Dusp1+,Itm2b+,Gja1+,and Agpat4+subgroup decreased in the cocaine addiction group.GSVA showed that the Il1b+subgroup（M1type）with increased expression after addiction was mainly enriched in TNFA SIGNALING VIA NFKB,MITOTIC SPINDLE,PI3K AKT MTOR SIGNALING,IL6JAK STAT3 SIGNALING,INTERFERON ALPHA RESPONSE,TGF BETA SIGNALING,ANDROGEN RESPONSE,PROTEIN SECRETION and other pathways,constituting a major component of these pathways which were enhanced in microglia after addiction.The oligodendrocytes were classified into 6 subgroups according to UMAP,and their representative marker genes were:Qdpr,Pex5l,Klk6,Tnfaip6,Plpp3 and Sparc.The relative abundance comparison revealed that the expression of Pex5l+,Klk6+,and Plpp3+subgroup increased,and the expression of Qdpr+,Tnfaip6+,and Sparc+subgroup cells decreased in the cocaine addiction group.GSVA showed that the Pex5l+and Klk6+subgroups with increased expression after addiction were mainly enriched in PROTEIN SECRETION and MITOTIC SPINDLE,constituting a major component of these pathways which were enhanced in oligodendrocytes after addiction.The neurons was classified into 5 subgroups according to UMAP,and their representative marker genes were:Stmn2,Dut,Gpr37,Slc1a3 and Ctss.The relative abundance comparison revealed that the expression of Dut+（neural precursor cells,NPCs）and Slc1a3+subgroup increased,and the expression of Stmn2+,Gpr37+,and Ctss+subgroup decreased in the cocaine addiction group.GSVA analysis showed that the Dut+subgroup with increased expression after addiction was mainly enriched in G2M CHECKPOINT,E2F TARGETS,MITOTIC SPINDLE,MYC TARGETS V1,MYC TARGETS V2 pathways,etc.,constituting a major component of these pathways which were enhanced in neurons after addiction.Analysis of m RNA expression differences between the core and shell region of NAc was also performed.Calb1 and Crym were mainly distributed in the core,while Nts and Nmb were predominantly distributed in the shell.Regardless of the brain slices from the control group or the addiction group,GO enrichment analysis showed that the differential genes between the core and shell of NAc were enriched in the biological processes including learning or memory,cognition,learning,and associative learning,the cell components including neuron to neuron synapse,transport vesicle,axon terminus,neuron projection terminus,distal axon,exocytic vesicle,and perikaryon,and the molecular functions including neuropeptide receptor binding.GSVA showed that the differential genes between the core and shell of NAc were enriched in signaling pathways such as OXIDATIVE PHOSPHORYLATION,APICAL SURFACE,HEDGEHOG SIGNALING,PROTEIN SECRETION,and CHOLESTEROL HOMEOSTASIS.2.There are GABAergic neuron subgroup with Cdc20 as its marker gene in rat NAc.By sc RNA-seq and analysis of NAc tissue of adult rats in physiological state,28,935 cells were also classified into 12 main cell lineages.Clustering using UMAP revealed that4,894 neurons could be classified into two main subgroups:mature neurons and NPCs.The NPCs were further divided into three subpopulations:Ube2c+,Ran+and Tuba1b+neurons,and Cdc20 and Fmr1 were specifically enriched in the Ube2c+subpopulation.The RNAscope for FISH was used to verify the specific expression of these two markers Ube2c and Cdc20 in some GABAergic neurons,where the Ube2c and Gad1 m RNA,and the Cdc20 and Gad1 m RNA were co-labeled respectively.3.Spatial-temporal expression patterns of Cdc20 molecules in the rat NAc and its effect on primary neuron development.By using the male SD rats at different postnatal stages,the western blotting（WB）results showed that Cdc20 proteins in NAc were decreased significantly 3 weeks after birth（P<0.05）and was consistently maintained at the adult stage,indicating that Cdc20 proteins decreased with the growth and development of the rats.Moreover,the decrease was found to be more substantial in the NAc shell as compared with the NAc core（P<0.0001）using the immunohistochemistry（IHC）,indicating that Cdc20 expression was conserved to a greater extent in the NAc core.The immunofluorescence（IF）results showed that Cdc20 could be both expressed in neurons containing dopamine D1 and D2 receptors.This protein was found to be distributed in the nucleus and cytoplasm after the nucleocytoplasmic separation of NAc cells was completed.Furthermore,the IF images of NAc brain slices and primary striatal neurons showed that Cdc20 was not only expressed in the nucleus and cytoplasm of neurons,but also in the cytomembrane and dendrites,indicating that Cdc20 might be related to the regulation of the structure and function of neuronal dendrites.The co-labeling of Cdc20 and Cdc27 in a structure（APC）in the NAc neurons was verified by IF.The Cdc20^flox/flox homozygous primary striatal neurons were transfected with Cre adenoviruses at a multiplicity of infection（MOI）of 200 on day 3 of in vitro culture.After 21 days of culture,all the cells were harvested for the subsequent experiments.The conditional knockout（CKO）of Cdc20 had been verified by IF.Compared with wild type,the total length and number of dendrite branches,the length and number of branches at different levels were significantly reduced（P<0.005）.4.The effects of overexpression of Cdc20 in the NAc of adult rats on synaptic plasticity and behavior.Stereotactic injection of Cdc20 overexpressing（OE）adeno-associated virus（AAV）and a dual-AAV expression system that enabled sparse labelling into bilateral NAc.3 weeks later,the dendritic spine density of neurons transfected with Cdc20 OE AAV was markedly less than that of neurons transfected with the control AAV.Cdc20-overexpressing neurons exhibited significant decreases in the densities of not only total spines（P<0.0001）but also the types of spines（thin,filopodia,mushroom,and stubby,P<0.05）in both the secondary and tertiary dendrites.The open field tests were performed 3 weeks after microinjection of Cdc20 OE AAV.The center distance and number of central zone entries were significantly decreased in the Cdc20 OE group compared with the group injected with the control AAV（P<0.05）,and the total distance and mean velocity were not decreased significantly in the Cdc20 OE group compared with the control AAV group（P>0.05）.In the elevated plus maze test,the time spent in an open arm,time spent in a closed arm,numbers of entries into an open arm were significantly different in the Cdc20 OE male group compared with the male group injected with the control AAV（P<0.05）.The results of female rats in the test were similar to those of male rats.The upregulation of Cdc20 in bilateral NAc neurons increased the anxiety-like behaviour reduced exploratory activity in the central zone and open arm for the adult rats in the behavioral tests.Correlation analysis between dendritic spine density and behavioural test results showed the total density of dendritic spines negatively correlated with anxiety-like behaviours.5.The effects of knockdown of Cdc20 in the NAc of adult rats on synaptic plasticity and behavior.Stereotactic injection of Cdc20 sh RNA AAV and the dual-AAV expression system into bilateral NAc.3 weeks later,the total density of dendritic spines in neurons transfected with Cdc20 sh RNA AAV did not change significantly compared with neurons transfected with the positive control AAV（P>0.05）.The open field tests were performed3 weeks after microinjection of Cdc20 sh RNA AAV.Compared with the positive control group,the center distance and number of central zone entries were significantly increased in the Cdc20 sh RNA group（P<0.05）,while the total distance and mean velocity had no significant changes（P>0.05）.It indicated that down-regulation of Cdc20 expression in the bilateral NAc led to increased exploratory activity in the central zone for the adult rats.6.Study on the mechanism of Cdc20 regulating ubiquitination degradation of FMRP.Apcin（100 and 200μM）treatment for 48 h significantly inhibited the division and proliferation of neuro-2a cells（P<0.0001）.After neuro-2a cells and rat primary striatal neurons were treated with apcin（100μM）for 48 h,the WB results showed that the expression level of FMRP protein in the cells was significantly increased（P<0.05）.After neuro-2a cells were transfected with the Cdc20 overexpressing plasmids,the WB results showed that the expression level of FMRP was decreased,and the co-immunoprecipitation（Co-IP）results confirmed that Cdc20 could bind to FMRP and Cdc27（APC3,a subunit of APC）.The neuro-2a cells transfected with Myc-FMRP and HA-Ub plasmids and pretreated with DMSO,MG-132 or apcin were harvested,lysed,and immunoprecipitated with a Myc antibody.Compared with DMSO,MG-132 and apcin led to more and less FMRP ubiquitination,respectively.The bands on PVDF membranes incubated with the total and Ub-k48 antibodies exhibited similar patterns.Since Cdc20 bound to FMRP and the apcin inhibited the FMRP ubiquitination incubated with Ub-k48 antibody,Cdc20 was thought to regulate the ubiquitination and degradation of FMRP through K48-linked polyubiquitination.We next obtained Cdc20^flox/flox primary striatal neurons from fetal mice that were generated by female Cdc20flox/flox mice crossed with male Cdc20^flox/flox mice.These neurons were transfected with Cre adenoviruses at MOI of 200 to knock out Cdc20,which was confirmed by IF.The WB results revealed that the expression level of Cdc20was substantially reduced（P<0.005）and that of FMRP was increased（P<0.05）in the Cdc20 CKO group compared to the control.The endogenous ubiquitinated FMRP was substantially reduced in the Cdc20 KO group,indicating that Cdc20 triggered the ubiquitination and degradation of FMRP in primary striatal neurons.To verify the interaction between Cdc20 and FMRP in vivo,apcin was microinjected into the bilateral NAc of adult rats for 24～48 hours.The FMRP expression was significantly increased compared to that in the negative and DMSO controls（P<0.005）.Additionally,after Cdc20 was overexpressed in the bilateral NAc by transfecting Cdc20 OE AAV,the FMRP expression was found to be reduced by WB,and immunoprecipitation of the NAc tissue confirmed that FMRP and Cdc27 could bind to Cdc20.After the bilateral NAc was transfected with Cdc20 sh RNA AAV,the WB results showed that the knockdown of Cdc20 was effective and the FMRP expression increased.All these data strongly supported the conclusion that FMRP was a novel substrate of Cdc20-APC in the NAc.7.The effect of cocaine on FMRP expression and behavioral sensitization in the primary striatal neurons and addicted rats.The WB experiments were performed on the primary striatal neurons which had been intermittently exposed to cocaine with different concentrations.It was found that the expression of FMRP was significantly increased at1.0μM（P<0.005）.The NAc tissue from cocaine self-administration rats at different stages was obtained for immunoblotting.It was found that the expression of FMRP was significantly increased 14 days after the training（P<0.01）.When the rats received intraperitoneal injection of cocaine for 7 days,quit cocaine treatment for 1 and 28 days,and relapsed 28 days after cocaine withdrawal,the expression levels of FMRP in NAc were significantly higher than those in the saline group at the corresponding stages（P<0.01）.The open field tests were used to test the locomotor activity（behavioral sensitization）of model rats in the different stages of addiction.When the rats received intraperitoneal injection of cocaine for 7 days,relapsed by cocaine 28 days after cocaine withdrawal,the total distances of the rats for 15 minutes were increased significantly in these groups than those in the control groups without cocaine injection（P<0.01）.8.The intervention of Cdc20 upregulation in the NAc on FMRP expression and behavioral sensitization of the cocaine-addicted rats.Three weeks after the stereotaxic injection of Cdc20 OE AAV into the bilateral NAc of the adult rats,intraperitoneal injection of cocaine was performed for 7 days,and it was found that the FMRP expression was significantly increased than that in the positive control AAV group（P<0.05）.After the above procedures were completed,the open field tests which was tested for behavioral sensitization was performed 30 minutes after the last cocaine injection,and it was found that the total distance of the rats within 15 minutes was significantly reduced than that in the positive control AAV group（P<0.05）,indicating that the exogenous overexpression of Cdc20 in the NAc could be effective to attenuate the behavioral sensitization induced by cocaine.However,when the rats only received saline injection,the total distances of the rats in which Cdc20 had been overexpressed were not significantly changed compared with those without overexpression of Cdc20（P>0.05）,indicating that the exogenous upregulation of Cdc20 in the NAc did not weaken the locomotor activity of the rats which had not been cocaine-dependent.Conclusions1.The single-cell and spatial transcriptomic sequencing and analysis of NAc in adult rats revealed the effects of cocaine addiction on the different cell populations,subpopulations and their marker genes,which led to altered signaling pathways and functions.When the rats became addicted to cocaine,the expression of Luzp2+,Mpc2+,and Vim+subcluster in astrocytes,Ccl2+and Il1b+subcluster in microglia,Pex5l+subcluster in oligodendrocytes,and Dut+subcluster in neurons increased significantly.2.The GABAergic neuron subcluster with Cdc20 as its marker gene was verified in the NAc of rats.The expression level of Cdc20 protein in the NAc decreased with the growth and development of the rats,and it was necessary for the development of dendrites in primary striatal neurons.3.Upregulation of Cdc20 in bilateral NAc of the adult rats reduced the density of dendritic spines and led to the anxiety-like behaviors for rats.This might be resulted from the degradation of FMRP regulated by the Cdc20-APC through enhanced K48-linked polyubiquitination.4.Cocaine increased the expression of FMRP in the primary striatal neurons and the NAc in cocaine-addicted rats.However,exogenous overexpression of Cdc20 in the NAc reduced the cocaine-induced increase in FMRP expression and attenuated the cocaine-induced behavioral sensitization of the rats.It suggests that Cdc20-FMRP signaling pathway is expected to be regarded as a new target for intervening drug addiction.