The Study Of EphB6 In Modulation Of Autism-like Behaviors And The Underlying Mechanisms

BACKGROUND&OBJECTIVEAutism is a set of heterogeneous neurodevelopmental conditions,first described in the early 1940s by psychiatrists Kanner.It’s core characterised are difficulties in social interaction and communication,and unusually restricted,repetitive behaviour and interests.The behavioral symptomatology of Autism spectrum disorders(ASD)encompasses a spectrum of wide-ranging phenotypes which are differences between the patients.Autism affects four times more male than female individuals.However,the worldwide population prevalence is about 1%until 2014,the incidence rate has been increased year by year.Currently,there is no large scale epidemiological statistical data of autism in China,but according to the other countries,the population of autism will be 1 million in China.With growing number of children being diagnosed with autism,it has become an urgent issue to clarify the pathogenesis of autism.ASD is often accompanied by other mental disease.Epilepsy is observed in up to 25%of people with an ASD,a high rate when compared with a population prevalence of less than 1%.Epilepsy in ASD is correlated with having a low IQ,and with more severe symptoms,and is also more common in females.About 42-56%children with anxiety.There are also reported in patients with Rett syndrome,fragile X chromosome syndrome and other diseases.About 70%individuals with an ASD may also have an intellectual disability.However,at least 25%of cases show normal or superior intellectual function at least in some elements such as in a variety of cognitive or artistic domains,but declarative memory is most consistently accentuated.Understanding of ASD has evolved with a growth in research since the mid-1990s.ASD is a complex genetic disorder,and more than 1000 genes are implicated.Synapses mediate communication between neurons and enable the brain to change in response to experience,which is essential for electrophysiology.Neuronal excitability is affected by the number of synapses,and imbalance between excitation and inhibition.Abnormalities in synapse and ratio of excitatory and inhibitory synaptic transmission are associated with a broad range of brain disorders,including ASD.The number of microglia and astrocyte are increased in autism postmortal samples.Interesting,not only the immunity factors are changed but also digestive tract inflammation are more sever in autism.These suggest that inflammation may play an important role ASD.Eph receptors are the largest subfamily of receptor tyrosine kinase family.The mammalian Eph receptors family is divided into 2 subgroups:the EphA receptors composed of 9 members(EphA1-8 and EphA10)and the 5 EphB receptors(EphB1-4 and EphB6).EphA receptors typically bind most or all types of ephrinA ligands(Ephrin-A 1-5),and EphB receptors typically bind most or all ephrinB ligands(Ephrin-B1-3).One exception is the EphA4 that can bind to both ephrinA and most ephrinB ligands.EphrinA is tethered to the membrane by a glycosyl phosphatidylinositol anchor,and ephrinB associated with other molecules through a transmembrane domain that is followed by a short cytoplasmic region.Ephrins and Ephs are expressed pre-and/or postsynaptically at the developing excitatory synapse and act to regulate central neuronal processes such as changes in synaptic transmission and morphology and are intimately involved in synaptic plasticity.The roles of Eph/ephrin signaling during development were studied extensively especially in the nervous system.For example,directing of axons to their distant target tissues is mediated in several instances by the Eph/ephrin reverse or forward signaling that can lead to repulsion or attraction of the axons.Eph/ephrin signals can also regulate the formation and morphology of dendrites,and dendritic spines.It is reported that Ephs and ephrins are also involved in cancer.Ephs and ephrins have been shown to affect the growth,migration,and invasion of cancer cells in culture as well as tumor growth,invasiveness,angiogenesis,and metastasis in vivo.In addition,Ephs and ephrins are involved in brain disorders and diseases with memory impairment symptoms,including Alzheimer’s disease and anxiety.EphB6 is one of the Receptor tyrosine kinases,high level expression in brain,thymus and pancreas.Sequence analysis of EphB6 revealed 6 unusual amino acid alterations in its kinase domain,which cause EphB6 kinase-dead.EphB6 exerts its effects by forming heterodimers with other Eph receptor kinases,such as EphB1,which has been reported to interact with EphB6,and/or other signalling components.EphB6 appears to represent an unusual situation,as it is abundantly expressed in normal,nonmalignant tissues,unlike another kinase-defective RTK,ErbB3,which is often expressed at lower amounts in normal tissues than in cancer.It is also reported that EphB6 have related to immune systerm.Mice lacking EphB6 develop normally but have defects in T cell function.Currently,in addition to function of EphB6 has been reported in cancer and the immune system.Unexpectedly,EphB6 has involved with autism.A recent study published in Nature pointed out that there are two loci mutation in EphB6 gene in the genome test from ASD,suggesting that EphB6 may associate with ASD.However whether EphB6 gene mutation can cause the symptoms of ASD has not been reported,so I proposed EphB6 whole cell knockout(EphB6-/-)mice to simulate human ASD process.With this mice model to investigate the role of EphB6 in ASD and its pathogenesis,in order to provide a new way for the ASD research,and provide a new therapeutic target for ASD.STRATEGY1.Identification genotyping of EphB6 mice.In order to identify the expression of EphB6 DNA in EphB6 knockout mice,we applied the technique of PCR for the recombinant allele in EphB6-/-mice,and western blotting for the expression levels of EphB6 protein.As result showed the genotyping of EphB6-/-mice had only a recombinant allele,without wild type allele on agarose gels.Western blotting analysis showed that expression of EphB6 protein level was significantly diminished in EphB6-/-mice,compared to the control mice.Due to the Eph family have important functions in neuron migration,we used immunofluorescence to detect neurons distribution in autistic-related cortex in EphB6-/-mice.Experimental results showed that the number of neurons in the medial prefrontal cortex and hippocampus in EphB6-/-mice was same to the controls,and the distribution of neurons was normal.We applied western blotting to explore the expression of c-Fos protein in autism-related regions.Result showed the expression of c-Fos protein in the medial prefrontal cortex of EphB6-/-mice was significantly increased while hippocampus and cerebellum were normal,compared to the control mice2.Autism-like behavior tests for EphB6-/-mice.To confirm that EphB6-/-mice have autism-like behavior,a series of behavior experiments were performed.Firstly,motor ability and anxiety-like behavior were detected by Open field test.Results showed that there was no significant difference between control mice and EphB6-/-mice in the total distance,indicating that EphB6-/-mice has normal locomotor activity.Habituation olfactory/dishabituation test was used to detect olfactory ability.Results show that EphB6-/-mice had comparable exposure to non-social odors,but spent less time in sniffing social odors suggesting that EphB6-/-mice has normal olfactory function,but impaired the sensory abilities to discriminate social/non-social odours,compared to wildtype mice.Partition test and Three-chamber test to detect social ability and social noverity.As results show that EphB6-/-mice cannot identifed social and non-social object,and lose interest in mice whether it is familiar or strange.It is suggest that EphB6-/-mice have defect in social ability and social novelty.Self-grooming test and Marble burying test were applied to detect repetitive stereotyped behavior in mice.Results showed that high levels of repetitive self-directed grooming were evident in EphB6-/-mice,compared to controls,but there had no significant difference between control mice and EphB6-/-mice in marbles buried,indicated EphB6-/-mice has repetitive behaviors.Morris water maze applied to detect repetitive stereotyped and reversal behavior.Experiment results showed that EphB6-/-mice can find platform quickly and form habits in acquisition and reversal test,but mice showed before repeating behavior once removed platform.These results illustrated that EphB6-/-mice characterized by repetitive stereotyped behavior.Elevated plus maze was used to detect anxiety-like behavior in mice.Results showed that the counts of explored into the open arm,time spent in the open arm were significantly reduced while more in the closed arm in EphB6-/-mice compared with the control.These indicated that EphB6-/-mice has anxiety-like behavior.3.Electrophysiology in EphB6-/-mice.In order to investigate whether EphB6 knockout infuence the electrophysiology in mPFC of mice,we first recorded miniEPSCs in mPFC of EphB6-/-mice and control mice to test the basic synaptic transmission,and the results showed that the miniEPSCs of EphB6-/-mice were similar to control mice,not only of frequency but also the amplitude.Secondary we recorded sEPSCs to detect action potential-dependent synaptic transmission,the results showed that EphB6 knockout increased the frequency and amplitude of sEPSCs in mPFC pyramidal neurons.Finally we recorded the action potentials of EphB6-/-mice and control mice to detect the neuronal excitability.We found that the Rheobase of action potential in knockout mice were deceased compared to control mice.The number of APs showed the excitability of EphB6-/-mice was enhanced compared to control mice.Above all,these results suggest that EphB6 knockout increased the excitability of mPFC pyramidal neurons,and also increased in action potential-dependent synapse transmission.4.The number of microglia and autism-related inflammatory factors in mPFC of EphB6-/-mice.It was reported that EphB6 knockout mice had normal growth and development,but resulted in compromised T cell function.Here we want to know whether a mouse model exhibiting many features of autism also displays altered immune function.We used immunofluorescence and Q-PCR technique to detect microglia and mRNA of autism-related inflammatory factors in mPFC.Results showed that the number of active microglia,non-activate microglia and total microglia was increased in EphB6-/-mice.Q-PCR results showed that the expression of IL-1,IL-4,IFNγ and TNF-α were enhanced,and IL6 and IL-12 were decreased in EphB6-/-mice,compared to control mice,but there was no significant.These suggested EphB6 knockout could affect the function of immune in central nervous system,promote microglia proliferation and activation,may affect the inflammatory factors release in mPFC of mice.CONCLUSION1.EphB6-/-mice showed autism-like behavior.2.The excitability of pyramidal neurons was increased and the action potential dependent synaptic transmission was enhanced in mPFC of EphB6-/-mice.3.The number of microglia was increased in mPFC of EphB6-/-mice.