Mechanisms Of AtLAS-Syn2b In The Regulation Of Social Hierarchy

Background: Social hierarchy is a fundamental phenomenon in grouped animals and human beings which serves as a guiding principle for the population structure maintenance,resources distribution and thriving population.The formation and maintenance of social hierarchy is related to survival rate,resource acquisition and stress,but the role of epigenetic regulation in it remain elusive.In previous reports,we found a Lnc RNA,AK013786 was dramatically altered among mice with different social stage using Arraystar lnc RNA microarray and up or down regulating AK013786 can bidirectional control social hierarchy,besides we found its downstream target protein Syn2.Syn2 have two different isoforms,Syn2 a and Syn2 b which are generated by alternative polyadenylation(APA),compared with subordinate mice,the expression of Syn2 b in dominant mice is decreased while the level of Syn2 a is increased,but the mechanism of how AK013786 regulates the ratio of Syn2a/2b in is not clear.Objective: To investigate the mechanism of how AK013786 regulates the ratio of Syn2a/2b in different social ranks.Methods: RACE was used to get the full sequence information of AK013786,and the cell and tissue expression specificity of AK013786 was determined by in situ hybridization.The subcellular localization of AK013786 was determined by separating the cytoplasmic and nuclear components.By up-regulation or down-regulation of AK013786,the ratio of Syn2a/2b was detected by western blotting and RT-PCR.The RNA binding proteins regulating the alternative splicing of Syn2 were predicted by RBPmap,and RIP and RNA Pull down were used to confirm that Celf4 regulates the generation of Syn2 a and 2b.The binding sequence of Celf4 to Syn2 pre-m RNA was detected by cell transfection and PCR.EMSA experiments were used to detect the effect of At LAS for the binding of Celf4 with pre-m RNA.Results: We performed 3? RACE and 5? RACE to get the full sequence of AK013786 which is identical to that from the UCSC genome database.AK013786 was expressed mainly in Cam KII-positive neurons,and AK013786 can be detected in m PFC,striatum,hippocampus,olfactory bulb,but the reduction of AK013786 in the dominant mice can only be detected in the m PFC rather than other brain regions.By isolating the nuclear and cytoplasmic fractions,we found that in the dominant mice the loss of AK013786 largely happened in the nuclear region rather than in the cytoplasmic compartment,supporting the hypothesis that At LAS is involved in the events taking place in nuclei such as gene regulation.We named AK013786 At LAS according to its genomic location.By up-regulation or down-regulation of AK013786 can bidirectional control the ratio of Syn2a/2b.By using RBPmap and the i CLIP data from mouse database of CLIPbp2,we found Celf4 mediated the expression of Syn2 isoforms by regulating the alternative polyadenylation(APA)of Syn2 and promote the syn 2a isoform expression.We get the sequence which is important for the binding of Celf4 with pre-syn2,and by using the AMO targeting the region can disrupt the binding of Celf4 with pre-syn2 and promote the syn 2b isoform expression.At LAS binds with the pre-m RNA of Syn2 and regulates the Syn2a/b ratio by inhibiting Celf4-mediated alternative polyadenylation,which leads to the increased expression of Syn2 b.Conclusions: At LAS is significantly reduced in the dominant mice,and plays an important role in the nuclei,and At LAS bidirectional controls the ratio of Syn2a/2b by regulating the alternative polyadenylation(APA)of Syn2.Background: Social hierarchy has a fundamental impact in maintaining population stability and distribution of resources.Compared with subordinate mice,activities of the m PFC neurons are up-regulated in the dominant mice.We have found a Lnc RNA,At LAS which was dramatically up-regulated in the subordinate mice using Arraystar lnc RNA microarray,and we found its downstream target protein synapsin II(Syn2).Syn2 has two isoforms,and it's Syn2 b not Syn2 a participates in the social dominance.We generated Syn2b-knockdown mice(Syn2b-KD)with CRISPR/Cas9 strategy,the mice displayed higher hierarchy and larger amplitudes in m EPSCs relative to the WT littermates.Syn2 belongs to a class of neuron-specific phosphoproteins which is abundant in nervous system and participates in modulation of neurotransmitter release and synaptogenesis.However,the mechanism of how At LAS/Syn2 b regulates social hierarchy via alternation in synaptic efficacy remains elusive.Objective: To investigate the mechanism of how At LAS/Syn2 b regulates social hierarchy.Methods: The localization of Syn2 in different components was detected by purifying different fractions of synaptic components and western blotting,and the PFC primary neurons were cultured to DIV12 and immunolabeled with syn2 and presynaptic or postsynaptic marker.The potential differences in the synaptic strength of neurons infected with sh-At LAS virus were measured.The physically association of Syn2 with subunits of AMPA receptors was detected by immuneprecititation,and we generated mutations to reveal the details of how Syn2 b interacts with AMPARs.We designed a membrane-permeable peptide(P-2B)which contains the C-terminus of syn2 b,and treated the primary neurons with P-2B,then the level of AMPAR at the plasma membrane and the synaptic strength of neurons were measured.The social rank of mice after intraperitoneally injecting with P-2B for consecutive 10 days were identified by tube test.Results: We found that Syn2 a and Syn2 b were localized at both the presynaptic and postsynaptic compartments by purifying different fractions of synaptic components for western blotting,and Syn2 co-localizes with both presynaptic and postsynaptic protein marker.We found that compared to the control mice,the sh-At LAS mice displayed larger amplitudes in miniature excitatory postsynaptic currents(m EPSCs),indicating the involvement of postsynaptic AMPARs in At LAS/Syn2b-dependent regulation of synaptic efficacy.Indeed,only Syn2 b was found to have the physical association with Glu A1 and Glu A2 subunits of AMPARs,and Syn2 b physically associates with AMPARs via its C-terminus.We designed a membrane-permeable peptide containing the C-terminus of Syn2 b,and confirmed that P-2B was able to disrupt the binding of Syn2 b to AMPARs.Incubation of cultured prefrontal cortex neurons with P-2B resulted in the increase of AMPAR expression at the plasma membrane and the larger amplitude of m EPSCs.And mice intraperitoneally injected with P-2B for consecutive 10 days displayed higher ranks than animals receiving scrambled control peptide.Conclusions: Syn2 b binds with AMPAR via its unique C-terminus to restrain the membrane insertion of AMPAR,the weakened synaptic strength in the m PFC renders social submissive roles in these animals.At LAS-Syn2 b constrains the postsynaptic AMPAR membrane expression.P-2B peptide disrupts Syn2b-AMPAR binding and leads to the enhanced synaptic efficacy and elevated social hierarchy.