The Canonical Wnt/β-catenin Signaling Pathway And Oxidative Stress In An Autistic Rat Model

Autism is a complex, pervasive developmental disorder that typically appears during the3years of life, which exerts a serious influence on children’s language formation, emotion cognition and social behaviors. Symptoms of autism include impaired linguistic communication, deficits in social interaction and aberrant ritualistic-repetitive behaviors. Such patients are unable to go to school, even unable to work in the future. Recently, statistical data demonstrate that there is an increasing incidence rate of autism. The etiology of autism remains unclear, but it very likely includes genetic and environmental factors that play a role during the critical period of brain development, for example, the prenatal exposure of valproic acid greatly increases the susceptibility to autism in the offspring. Non-genetic factors may interfere with the normal signaling pathways involved in prenatal brain development, whereas genetic agents may arise due to the mutation of certain genes participating in these signaling pathways.It is believed that the Wnt/β-catenin pathway plays a critical role in the proliferation, differentiation, apoptosis and process outgrowth of cells of the central nervous system during embryonic development. However, the Wnt/β-catenin pathway has two sides:one hand, it regulates the normal development and difference of the central nervous system, on the other hand, dysregulation of the Wnt signaling pathway could have any number of deleterious effects on neural development and thereby contribute to the pathogenesis of neurodevelopmental disorders, such as autism, in many different ways. The notion of Wnt2as an autism susceptibility gene was supported by screening Wnt2coding sequence for mutations in a large number of autistic probands. The dvl-1knockout mouse displayed social interaction and sensorimotor gating abnormalities, demonstrating that dvl-1may be involved in the social behavior of animals based on the findings that hippocampus dendritic growth was unusual in cultured hippocampal neurons from the dvl-1knockout mouse. The dvl-1and dvl-2deletion mouse performed neural tube closure defects. Transgenic mice expressing active stabilized forms of β-catenin in neuronal precursor cells develop grossly enlarged brains with an increased cerebral cortical volume, which may be relevant with the early excessive proliferation of the brain in autism. Therefore, these studies strongly suggest the Wnt pathway is closely related with the pathogenisis of autism.Recent research has focused in particular on the role of oxidative stress in autism patients, for example, increased biomarkers of oxidative stress, reduced resistance to endogenous oxidation, energy metabolism and mitochondria dysfunction. Dickkopfl (Dkk1) is a kind of secretory Wnt inhibitors. Existing research shows that, Dkkl transfection can significantly increase the levels of reactive oxygen species, the key source of oxidative stress. Nucleoredoxin (NRX), a kind of oxidizer reducer, negatively regulates the Wnt signaling pathway through Dvl. H2O2reduces the interaction between NRX and Dvl, and activates TCF temporarily. Contrary with these results, many studies show that H2O2induced ROS-dependent signal transduction inhibits the β-catenin/TCF transcriptional activity. ROS inhibits many phosphatases, however, how it works about phosphorylation on GSK3following H2O2treatment is not clear. Interestingly, studies have shown that dvl overexpression can improve inhibition of H2O2induced β-catenin/TCF transcriptional activity. Thus, these data suggest ROS has two roles in regulating the Wnt pathway, which is dependent on Dvl. This also has shown that there is a correlation between oxidative stress and the Wnt signaling pathway.However, whether oxidative stress occurs in rats exhibiting autism-like symptoms caused by prenatal VPA exposure? What is the relationship between oxidative stress and the Wnt/β-catenin signaling pathway? If oxidative stress does occur in these animals, may sulindac, a small molecule inhibitor of the canonical Wnt/β-catenin pathway ameliorate the autism-like behavioral abnormalities that develop in these rats? These problems still remain unknown. In this study, we focused on the relationship between the Wnt/p-catenin pathway related genes and oxidative stress in the autistic animal model and primary cultured neurons, exploring the possible pathomechanism of autism. The work had been done as follows:Part one:We established an autistic rat model successfully. This autistic animal model was obtained in the offspring of the female Wistar rat that received a single intraperitoneal injection of VPA on the12.5th pregnancy day, and then the behavioral and developent index tests were performed using the hot plate, inclined board, Morris Water Maze. The results demonstrated that, compared to the control rats, the autistic ones had abnormal developmental brain, delayed timing of eye opening, lower geotaxis function, attenuated pain threshold, lower swimming performance, enhanced ritualistic-repetitive behaviors, poor learn and memory abilities and lower spatial exploratory ability, which were similar to the symptoms in autistic patients.Part two:We did research on the Wnt/β-catenin pathway and oxidative stress in VPA autism models. Real-time PCR and Western blot were used to investigate the levels of GSK-3β, Phosphorylated GSK-3β (P-GSK-3β), β-catenin, Phosphorylated β-catenin (P-β-catenin),4-hydroxynonenal (4-HNE) and thioredoxin (Trx) in the prefrontal cortexes (PFC) and hippocampi (HC) of autistic rats. Then, we detected the effects of sulindac, a specific Wnt/β-catenin pathway inhibitor, on oxidative stress. The results of Western Blot showed that P-GSK-3β was upregulated, and P-β-catenin was downregulated in PFC and HC of VPA-exposed rats compared with the control group (P<0.001). Concomitantly,4-HNE was enhanced (P<0.01), while Trx was attenuated (P<0.05). Real-time PCR showed that the mRNA of GSK-3β, trx1and trx2were lower, while β-catenin was higher in the PFC and HC of VPA autism models. VPA and Sulindac treatment decreased P-GSK-3β and4-HNE, whereas increased P-β-catenin (P<0.05). A battery of behavioral tests showed that Sulindac treatment ameliorated the pain threshold, repetitive/stereotypic activity, learning and memory abilities of rats in our autism model. From the above results, it is demonstrated that Sulindac downregulates the Wnt/β-catenin pathway, thus decreases oxidative stress, which finally ameliorates the autism-like behavioral abnormalities.Part three:We further revealed the roles of the Wnt/β-catenin pathway and oxidative stress in autism using cell culture technology. Real-time RT-PCR, western blot and flow analytical cytometry methods were used to further investigate the relationship between the Wnt/β-catenin pathway and oxidative stress in primary cultured neurons. The results from Real-time RT-PCR and western blot showed that P-β-catenin and the mRNA and protein expressions of GSK-3β were significantly decreased in the VPA-exposed cultured neurons, while P-GSK-3β,4-HNE and the mRNA and protein expressions of β-catenin were increased compared to the controls. Furthermore, FACS showed that ROS was enhanced in the VPA-exposed cultured neurons. Compared with the VPA treatment alone, the protein expression levels of both GSK-3β and P-β-catenin were increased in the VPA and sulindac-exposed neurons, whereas P-GSK-3β, β-catenin and4-HNE were decreased. The results from Real-time RT-PCR showed that both NAC and VPA did not change the expression levels of GSK-3β mRNAå'ŒÎ²-catenin mRNA compared with VPA-exposed group. These results demonstrated that VPA induced the upregulation of the Wnt/β-catenin signaling pathway by increasing oxidative stress.In conclusion, our results demonstrated that enviromental factors such as VPA exposure induced the increased activity of the Wnt/β-catenin pathway, thus resulting in the upregulation of Wnt/β-catenin pathway. The upregulation of Wnt/β-catenin pathway produces an imbalance of oxidative homeostasis, which affects the neuronal development and synaptic junctions, resulting in behavioral, emotional and linguistical abnormality, and finally leading to autism.