Morphologic Changes In Neurons And Expressing Alterations In β-catenin And GSK-3β In Brain Regions Of Rat Autism Model

Autism is one of the most serious of the pervasive developmental disorders and is characterized by deficits in social relatedness,communication,and cognitive flexibility.Onset of the unusual behavior typically occurs in infancy and is fully present by the child's 3 years.In 2 to 3-year-old,a significant developmental characteristic to the majority of autistic patients is the greater head circumference than the normal children with same age,while these wloud be followed by the less head circumference in adolescent than the normal.The incidence is approximately 4 in 1000 live births with a 4:1 predominance of males to females.The cause of autism is still unclear,and may be genetic or environmental.Nongenetic causes are speculated to involve prenatal interruptions in the normal signaling pathway of brain development.Genetic causes may arise from mutations in genes along this developmental pathway.Wnts are a large class of signaling molecules implicated in a variety of processes throughout development such as stem cell self renewal,cell-fate determination,axon guidance,and synaptogenesis.There are over 16 Wnt proteins in vertebrates,which all have a conserved cysteine residue that is palmitoylated before the proteins are secreted.In the canonical pathway,Wnt binds to the Frizzled coreceptor,which signals downstream to Disheveled(Dvl).Activated Dvl then inhibits a trio of proteins, GSK3β,Axin and APC.Inhibition leads to dephosphorylation ofβ-catenin,which triggers its activation and allows its translocation to the nucleus,where it binds to TCF/LEF transcription factors.It has been suggested that abnormalities of this signaling transduction may be present in schizophrenia,unipolar or bipolar depression and autism.It also has been shown in animal experiments that mice completely deficient of Dvl-1 display abnormalities in social behavior and sensorimotor gating characteristic to several psychiatric disorders including autism.Sinceβ-catenin represents the key protein that transduces activation of the Wnt pathway into the nucleus while GSK-3βis the central negative regulator of cytoplasmicβ-catenin levels,the present study was designed to examine these two signaling proteins,β-catenin and GSK-3β,in an animal model of autism as following:Ⅰ:An animal model of autism was obtained in offspring of the Wistar rat by a single intraperitoneal injection of sodium valproate to the rat at the pregnancy day of 12.5.The present study examined the expression of two signaling proteins,β-catenin and GSK-3β,which take the key position in Wnt pathway,in prefrontal cortex, hippocampus and cerebellum of the autism model of rat with different ages by means of the Western blot technique.The results showed that,the level of expression ofβ-catenin was higher in animal model tissue than in control with the exception of the hippocampus and cerebellum at the age of postnatal(P) 7,and there was a trend to a greater difference of expression in these regions during the course of brain development.GSK-3βprotein levels tended to be less in the animal models when compared to controls.The only exception to this was in cerebellum in which no difference was observed between models and controls.Consistent withβ-catenin,the difference was greater with the development of the rats.These results indicated that the Wnt signaling pathway was over activated in the autism models,and this activation might be the one of the causes of abnormal development of neurons in the brain of autism.Therefore,the present results suggested that Wnt signaling pathway plays an important role in the etiology of the autism.Ⅱ:For evidence from the morphology to our results,the present study selectively observed the GSK-3βimmunoreactive neurons of cerebellum by immunohistochemical staining.The same animal model of autism was obtained by VPA treated pregnant Wistar rats.We examined the expression of GSK-3βin the cerebellum at the age of P7,P14,P21,P28 and P90.The results showed that the immunoreactivity of the GSK-3βwas contained in the body of the cerebellar Purkinje cells.At P7,the GSK-3βimmunoreactive neurons exhibited similar distribution patterns and morphology in both the model and contral animals.Howere,differences with statistics significance in number of neurons between the model and contral rats were boserved in P14,P21,P28 and P90,and the patterns of distribution of the GSK-3βimmunoreactive neurons was observed in a diffusing mannar in animal models compared to controls.These results indicated that the GSK-3βexpression in the cerebellum significantly reduced,which is consistent with the western blot results in partⅠof the present study.Accordingly,the reduction of the GSK-3βis bound to cause the reduction of theβ-catenin degradation in the Wnt signaling pathway,and therefore leading to Wnt signaling enhanced.Additionally,Golgi staining was carried out to show the morphology and number of the pyramidal neurons in prefrontal cortex at the age of 90d.The results showed that the density of pyramidal neurons increased in the animal model.The top dendrites of the neurons in the model animals were shortened,and the dendrites were excessively branched in disorders.This morphology of the pyramidal neurons might increase the volume of the cortex,and cause abnormal neuronal networks both in local neurons and the long-distance between brain regions, and further cause the imbalance of excitory and inhibitory neuronal activity.To sum up,the present study suggested that the Wnt signaling pathway in the brain of autistic patients might be over-activated,and this,in turn,might cause the number and morphological changes of the neurons,for example,the excessive proliferation of the neurons,and then lead to the increased volume of the brain.Further more,the over-growed neurons might affect the role of the environment to the brain development,which lead to incorrect neural connections,and impede the formation of normal local and long-distance neuronal networks between brain regions,and eventually the autistic symptom,impairment in social behavior,emotion and language, occur.