The Mechanism Study Of ErbB4 Promoting Inhibitory Synapse And Perineuronal Nets Formation

Neurons are one of the basic structural and functional unit of the central nervous system.The communications between neurons are the basis for the brain functions.Conforming to the role of neurons,it can be divided into excitatory neurons and inhibitory neurons.In the brain,pyramidal neurons and interneurons are the two main types of neurons,and the coordination of their neuronal activities and firing is pivotal for the animal behaviors.Among them,the neurotransmitter of pyramidal neurons is glutamate,which belongs to excitatory neurons;and interneurons that use ?-aminobutyric acid(GABA)as the neurotransmitter are the main source of inhibitory signals.Interneurons play a vital role in maintaining the excitation and inhibition balance(E/I balance)of the brain.Its imbalance is believed to cause some neurological and neuropsychiatric disorders like epilepsy,schizophrenia,and autism.The genes encoding Neuregulin1(NRG1)and its receptor Erb B4 have been identified as susceptibility genes for mental diseases such as schizophrenia,bipolar disorder,and major depressive disorder.Erb B4 belongs to the family of singletransmembrane receptor tyrosine kinases.Upon binding to the ligand NRG1 through its extracellular domain(ECD),Erb B4 dimerizes for autophosphorylation of tyrosine kinase within its intracellular domain(ICD),thus be activated to trigger downstream signaling cascades.In the post-natal or adult brain,Erb B4 is specifically expressed in GABAergic interneurons,and the NRG1/Erb B4 signaling pathway regulates GABAergic synaptic transmission in the adult brain.Inhibitory synapses are the structural basis for the connection between interneurons and other neurons,and are critical in the assembly of inhibitory neural circuits.The cellular and molecular mechanisms of synapse formation have been extensively studied,and many cell adhesion molecules that regulate excitatory synapse formation have also been discovered and confirmed.However,little is known about the molecular mechanisms underlying inhibitory synapse formation.Studies have demonstrated that Erb B4 in interneurons promotes the formation and maintenance of the GABAergic synapse onto pyramidal neurons,but it is still unclear whether the tyrosine kinase activity of Erb B4 is required.To this end,we first adopted an artificial synapse formation assay in vitro,in which HEK293T(Human Embryonic Kidney 293T)cells were co-cultured with primary hippocampal neurons,and used immunocytochemical staining with anti-gephyrin antibody to observe the induction of gephyrin puncta(an inhibitory postsynaptic marker)at the dendrites of neurons,thereby reflecting the formation of GABAergic synapses onto pyramidal neurons.We found that HEK293 T cells expressing Erb B4 can induce gephyrin puncta.But Erb B4-K751 M,a kinase-dead mutant of Erb B4 in which the lysine 751 was mutated to methionine,can also induce gephyrin puncta,suggesting Erb B4 can induce the formation of inhibitory synapse onto pyramidal neurons in a kinase activity-independent manner.To validate this phenotype in vivo,we generated Erb B4-K751 M knockin mice.Compared with WT(wildtype)mice,the numbers of interneurons and inhibitory synapses onto pyramidal neurons did not change in the brains of K751 M mice,but were decreased in Erb B4-deficient(Null)mice.Next,to explore how Erb B4 regulates the formation of inhibitory synapses onto pyramidal neurons,we utilized cell aggregation assay and coimmunoprecipitation experiments in HEK293 T cells,and found that Erb B4 can in trans interact with Slitrk3,an inhibitory postsynaptic transmembrane cell adhesion molecule,through the extracellular RLD domain of Erb B4.In the artificial synapse formation assay,HEK293 T cells expressing RLD domain deletion mutant of Erb B4(Erb B4-?RLD)cannot induce gephyrin puncta;further overexpression of the secretable RLD domain in primary neurons to disrupt Erb B4-Slitrk3 interaction decreased gephyrin puncta.Finally,we expressed and purified the RLD domain in vitro,then injected it into the hippocampal CA1 area of Erb B4-K751 M mice by stereotactic injection.Using electrophysiological methods,we found that inhibiting Erb B4-Slitrk3 interaction impaired GABAergic neurotransmission.Our results suggest that Erb B4 acts as a cell adhesion molecule to promote the formation of inhibitory synapse onto pyramidal neurons by in trans interacting with Slitrk3 in a kinase activity-independent manner.It also provides a novel mechanism for Erb B4 in inhibitory synapse formation.As the specialized extracellular matrix of the central nervous system,perineuronal nets(PNNs)mainly covers the cell body,proximal dendrites,and axon initial segment of GABAergic interneurons.PNNs are highly distributed in the cortex and hippocampus,and are involved in different brain functions.The impaired PNNs can lead to a variety of behavioral changes like cognition,learning and memory.Clinically,the expression of PNNs is significantly decreased in many psychiatry disorders.However,the mechanism of PNNs formation and how PNNs regulate neuron functions are less understood at present.To explore the effect of neuronal activity on PNNs,we first set up an environmental enrichment model for mice to enhance the neuronal activity.Through behavioral tests such as Y-maze and novel object recognition test,we found that the spatial working memory and cognition of mice were enhanced under this model,and the expression of PNNs in the medial prefrontal cortex(m PFC)was increased.On the other hand,the spatial working memory and cognition of mice were impaired after ch ABC digesting PNNs in m PFC.Interestingly,we also found the increased expression of NRG1 and Erb B4,as well as elevated Erb B4 kinase activity in mouse m PFC under the environmental enrichment model,indicating that the enriched environment may promote the NRG1/Erb B4 signaling pathway.Next,to explore the effect of the NRG1-Erb B4 signaling pathway on PNNs' formation,we added recombinant NRG1 to primary cultured neurons and found that NRG1 can induce the expression of PNNs on Erb B4+ interneurons,and this induction can be blocked by tyrosine kinase inhibitor of Erb B4,indicating that the NRG1/Erb B4 signaling pathway promotes the formation of PNNs.Finally,the inducible Erb B4 knockout mice(Erb B4-i KO)and Erb B4-T796G(the threonine 796 of Erb B4 was mutated to glycine,which did not alter its kinase activity,but can be specifically bound by the chemical inhibitor 1NMPP1 to inhibit its tyrosine kinase activity)knockin mice were used to evaluate the effects of NRG1-Erb B4 on the formation of PNNs in vivo.We found that knockout of Erb B4 or chronically inhibition of Erb B4 kinase activity in adulthood reduced the PNNs' formation and impaired spatial working memory and cognition in mice.Taken together,we found for the first time that the NRG1/Erb B4 signaling pathway promotes the formation of PNNs,which may affect the behaviors of mice.In summary,Erb B4,as a single transmembrane receptor tyrosine kinase specifically expressed in GABAergic interneurons,can not only induce inhibitory synapses formation through cell adhesion function,but also promote the formation of PNNs through canonical NRG1/Erb B4 signaling pathway.These roles of Erb B4 regulate the function of interneurons,and affect the inhibitory neural circuits,thereby regulating animal behaviors.