孤独症模型大鼠经典Wnt信号通路与氧化应激变化的研究
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摘要
孤独症(autism)又称自闭症,是一种广泛的神经发育障碍性疾病,一般于3岁前发病,严重影响儿童的语言形成、情绪认知与社会行为。多数患儿有明显的语言交流艰难、社交行为障碍、重复刻板行为三大核心临床表现。这些患儿不能正常入托、上学,甚至将来也无法工作。近年来的统计数据资料表明,autism的发病率逐年增多。Autism的病因不明,目前普遍认为是遗传和环境因素在脑发育关键时期共同作用的结果。如,妊娠早期有丙戊酸盐(Valproic acid, VPA)用药史的产妇,其子代发生autism风险大大增加。非遗传因素可能与干扰胚胎脑发育的正常的信号通路传导有关,而遗传因素可能是信号通路传导异常导致的基因突变所致。
Wnt/β-catenin信号通路对个体发育,尤其是神经系统发育有重要的调控作用。在个体发育早期,它调控神经细胞的增殖、分化、凋亡与迁移以及突触的形成与联系,调控细胞、组织及器官正常有序的分化和生长发育。然而Wnt通路犹如双刃剑,既是调控神经细胞正常分化发育的重要通路,又是神经发育疾病发生的成因之一,通路中任何蛋白分子的异常,都将引起信号传递错误,导致细胞命运的改变,进而引发神经发育障碍疾病的发生,如autism。许多autism患者存在Wnt2编码序列基因突变,故Wnt2基因是autism易感基因。敲除通路中散乱蛋白(Dishevelled, Dvl)-1基因的小鼠,表现出社交行为和感觉运动障碍,说明Dvl-1基因可能参与动物社会行为的形成,这可能与Dvl-1调节神经发育有关,因为从Dvl-1敲除鼠组织培养的海马神经元树突发育异常。Dvl-1和Dvl-2缺失的小鼠,神经管闭合不全。β-catenin转基因小鼠,则能使神经前体细胞增殖,导致巨大脑、脑皮质容量增加,这可能与autism脑早期的过度增生有关。这些研究强烈提示Wnt通路与autism的发病密切相关。
近年来,大量临床资料报道表明autism患者存在氧化应激的失调,如氧化应激生物标记物分泌增加,体内内源性抗氧化能力减少,能量代谢失调,线粒体功能障碍。Dickkopfl (Dkkl)是一种分泌性的Wnt抑制剂。研究显示,Dkk1转染能显著增加氧化应激产生的活性氧(Reactive oxygen species, ROS)水平。Nucleoredoxin (NRX)作为一种氧化还原剂,通过与Dvl作用,对Wnt信号通路进行负调节。H202作用于细胞减少了NRX和Dvl的相互作用,导致T细胞因子(T cell factor, TCF)暂时激活。而与这些结果截然相反的许多研究显示,H202诱导ROS依赖性的信号转导抑制了β-catenin/TCF转录活性。ROS能抑制许多磷酸酶,而H202处理后GSK3怎么去磷酸化并不清楚。有趣的是,有研究显示,Dv1高表达能改善H202诱导的β-catenin/TCF转录活性抑制。因此,这些研究提示ROS依赖于Dvl在调节Wnt信号通路中起双重作用。这也说明氧化应激与Wnt信号通路之间存在关联。
那么,autism动物模型是否存在氧化应激异常?在autism产生过程中氧化应激与Wnt信号通路是否存在关联?用Wnt信号通路特异性抑制剂是否能改善autism行为学异常?目前尚不清楚。针对这些问题,本课题集中研究autism动物模型及培养神经元中氧化应激与Wnt/β-catenin通路相关分子的关系,以探讨autism发病的相关分子机制。我们主要进行了以下三方面的工作：
一、建立并检测了autism动物模型。采用Wistar大鼠,在妊娠第12.5天时,腹腔注射(Intraperitoneal injecting, ip) VPA制作子代autism大鼠模型,并运用热板致痛法、倾斜板实验、Morris水迷宫等方法对模型大鼠进行发育与行为学检测。结果显示,与正常对照组比较,autism模型组大鼠脑发育异常、睁眼时间推迟、方向趋向性机能及伤害性知觉下降、游泳能力差、重复刻板动作增加、学习记忆和空间探索能力差。这些表现与人类autism患者的某些特征极为相似,表明用VPA注射法较成功地制作出了autism大鼠模型。
二、研究了Wnt/β-catenin信号通路及氧化应激水平。运用Real-time PCR和Western blot技术检测autism模型大鼠前额叶皮层(Prefrontal cortex, PFC)和海马(Hippocampus, HC)两脑区Wnt通路信号蛋白GSK-3p,磷酸化GSK-3β(Phosphorylated GSK-3β, P-GSK-3β), β-catenin,磷酸化β-catenin (Phosphorylated β-catenin, P-β-catenin)及氧化应激标记物4-羟壬烯醛(4-Hydroxynonenal,4-HNE),硫氧还原蛋白(Thioredoxin, Trx)等相关分子的表达。进而我们检测了Wnt信号通路的特异性抑制剂Sulindac对氧化应激的影响。Western Blot结果显示,在autism模型大鼠PFC和HC脑区,Wnt通路关键信号蛋白失活的P-GSK-3β上调,抑制性的P-β-catenin下调,差异显著高于对照组(P<0.001)。同时,氧化应激标记物4-HNE表达增加(P<0.01),Trx表达减少(P<0.05)。Real-time PCR结果显示,autism模型组大鼠PFC和HC脑组织GSK-3βmRNA水平显著低于对照组(P<0.05),而β-catenin mRNA水平显著高于对照组(P<0.01)。同时,抗氧化基因trx1,trx2mRNA表达下调(P<0.05)。Wnt信号通路的特异性抑制剂Sulindac作用以后,与autism模型大鼠相比,VPA+Sulindac处理组失活的P-GSK-3p下调(P<0.05),抑制性的P-β-catenin上调(P<0.05),而氧化应激标记物4-HNE减少(P<0.05)。行为学研究结果显示,与autism模型大鼠相比,Sulindac预处理,大鼠的痛闽相对正常；重复刻板动作减少；学习和记忆能力有所提高。以上结果表明,Wnt信号通路抑制剂Sulindac预处理,能削弱Wnt信号通路的亢进机能,从而减少氧化应激的发生,最终使autism模型鼠的异常行为获得不同程度的改善。
三、采用细胞培养技术探讨了Wnt/β-catenin通路与氧化应激在autism发生过程中的作用。应用Real-time PCR, Western blot及流式细胞分析(Flow analytical cytometry system, FACS)技术深入研究VPA处理的原代培养神经元Wnt/β-catenin与氧化应激的关系。Real-time PCR及Western blot结果显示,在VPA处理培养神经元中,GSK-3βmRNA及蛋白表达显著低于对照组,失活的P-GSK-3p显著高于对照组；而P-catenin mRNA及蛋白表达显著高于对照组,抑制性的P-β-catenin显著低于对照组。同时,在VPA处理培养神经元中,4-HNE表达显著高于对照组。FACS研究结果显示,VPA处理组ROS表达水平显著高于对照组。Sulindac处理以后,与VPA处理组相比,β-catenin蛋白表达下调,P-β-catenin表达上调,GSK-3p表达上调,P-GSK-3p表达下调,4-HNE下调。Real-time PCR结果显示,抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)处理虽然减少了ROS的产生,但是并没有改变GSK-3β mRNA和P-catenin mRNA的表达。以上结果揭示,Wnt/β-catenin通路活性增强导致氧化应激的产生,VPA促进神经元氧化应激的产生由于该通路上调所致。
综上所述,我们的工作提示：环境因素(如VPA暴露)通过诱导脑组织Wnt/β-catenin通路活性改变,使脑内经典Wnt信号通路活性增强,进而增加氧化应激的产生、氧化稳态的异常,有可能进一步影响神经元的正常发育和联系,阻碍了正常的神经网络形成,从而引起社会交往、情绪和语言等行为方面的异常,最终导致autism的发病。
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 mRNA和β-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.
Wnt/β-catenin信号通路对个体发育,尤其是神经系统发育有重要的调控作用。在个体发育早期,它调控神经细胞的增殖、分化、凋亡与迁移以及突触的形成与联系,调控细胞、组织及器官正常有序的分化和生长发育。然而Wnt通路犹如双刃剑,既是调控神经细胞正常分化发育的重要通路,又是神经发育疾病发生的成因之一,通路中任何蛋白分子的异常,都将引起信号传递错误,导致细胞命运的改变,进而引发神经发育障碍疾病的发生,如autism。许多autism患者存在Wnt2编码序列基因突变,故Wnt2基因是autism易感基因。敲除通路中散乱蛋白(Dishevelled, Dvl)-1基因的小鼠,表现出社交行为和感觉运动障碍,说明Dvl-1基因可能参与动物社会行为的形成,这可能与Dvl-1调节神经发育有关,因为从Dvl-1敲除鼠组织培养的海马神经元树突发育异常。Dvl-1和Dvl-2缺失的小鼠,神经管闭合不全。β-catenin转基因小鼠,则能使神经前体细胞增殖,导致巨大脑、脑皮质容量增加,这可能与autism脑早期的过度增生有关。这些研究强烈提示Wnt通路与autism的发病密切相关。
近年来,大量临床资料报道表明autism患者存在氧化应激的失调,如氧化应激生物标记物分泌增加,体内内源性抗氧化能力减少,能量代谢失调,线粒体功能障碍。Dickkopfl (Dkkl)是一种分泌性的Wnt抑制剂。研究显示,Dkk1转染能显著增加氧化应激产生的活性氧(Reactive oxygen species, ROS)水平。Nucleoredoxin (NRX)作为一种氧化还原剂,通过与Dvl作用,对Wnt信号通路进行负调节。H202作用于细胞减少了NRX和Dvl的相互作用,导致T细胞因子(T cell factor, TCF)暂时激活。而与这些结果截然相反的许多研究显示,H202诱导ROS依赖性的信号转导抑制了β-catenin/TCF转录活性。ROS能抑制许多磷酸酶,而H202处理后GSK3怎么去磷酸化并不清楚。有趣的是,有研究显示,Dv1高表达能改善H202诱导的β-catenin/TCF转录活性抑制。因此,这些研究提示ROS依赖于Dvl在调节Wnt信号通路中起双重作用。这也说明氧化应激与Wnt信号通路之间存在关联。
那么,autism动物模型是否存在氧化应激异常?在autism产生过程中氧化应激与Wnt信号通路是否存在关联?用Wnt信号通路特异性抑制剂是否能改善autism行为学异常?目前尚不清楚。针对这些问题,本课题集中研究autism动物模型及培养神经元中氧化应激与Wnt/β-catenin通路相关分子的关系,以探讨autism发病的相关分子机制。我们主要进行了以下三方面的工作：
一、建立并检测了autism动物模型。采用Wistar大鼠,在妊娠第12.5天时,腹腔注射(Intraperitoneal injecting, ip) VPA制作子代autism大鼠模型,并运用热板致痛法、倾斜板实验、Morris水迷宫等方法对模型大鼠进行发育与行为学检测。结果显示,与正常对照组比较,autism模型组大鼠脑发育异常、睁眼时间推迟、方向趋向性机能及伤害性知觉下降、游泳能力差、重复刻板动作增加、学习记忆和空间探索能力差。这些表现与人类autism患者的某些特征极为相似,表明用VPA注射法较成功地制作出了autism大鼠模型。
二、研究了Wnt/β-catenin信号通路及氧化应激水平。运用Real-time PCR和Western blot技术检测autism模型大鼠前额叶皮层(Prefrontal cortex, PFC)和海马(Hippocampus, HC)两脑区Wnt通路信号蛋白GSK-3p,磷酸化GSK-3β(Phosphorylated GSK-3β, P-GSK-3β), β-catenin,磷酸化β-catenin (Phosphorylated β-catenin, P-β-catenin)及氧化应激标记物4-羟壬烯醛(4-Hydroxynonenal,4-HNE),硫氧还原蛋白(Thioredoxin, Trx)等相关分子的表达。进而我们检测了Wnt信号通路的特异性抑制剂Sulindac对氧化应激的影响。Western Blot结果显示,在autism模型大鼠PFC和HC脑区,Wnt通路关键信号蛋白失活的P-GSK-3β上调,抑制性的P-β-catenin下调,差异显著高于对照组(P<0.001)。同时,氧化应激标记物4-HNE表达增加(P<0.01),Trx表达减少(P<0.05)。Real-time PCR结果显示,autism模型组大鼠PFC和HC脑组织GSK-3βmRNA水平显著低于对照组(P<0.05),而β-catenin mRNA水平显著高于对照组(P<0.01)。同时,抗氧化基因trx1,trx2mRNA表达下调(P<0.05)。Wnt信号通路的特异性抑制剂Sulindac作用以后,与autism模型大鼠相比,VPA+Sulindac处理组失活的P-GSK-3p下调(P<0.05),抑制性的P-β-catenin上调(P<0.05),而氧化应激标记物4-HNE减少(P<0.05)。行为学研究结果显示,与autism模型大鼠相比,Sulindac预处理,大鼠的痛闽相对正常；重复刻板动作减少；学习和记忆能力有所提高。以上结果表明,Wnt信号通路抑制剂Sulindac预处理,能削弱Wnt信号通路的亢进机能,从而减少氧化应激的发生,最终使autism模型鼠的异常行为获得不同程度的改善。
三、采用细胞培养技术探讨了Wnt/β-catenin通路与氧化应激在autism发生过程中的作用。应用Real-time PCR, Western blot及流式细胞分析(Flow analytical cytometry system, FACS)技术深入研究VPA处理的原代培养神经元Wnt/β-catenin与氧化应激的关系。Real-time PCR及Western blot结果显示,在VPA处理培养神经元中,GSK-3βmRNA及蛋白表达显著低于对照组,失活的P-GSK-3p显著高于对照组；而P-catenin mRNA及蛋白表达显著高于对照组,抑制性的P-β-catenin显著低于对照组。同时,在VPA处理培养神经元中,4-HNE表达显著高于对照组。FACS研究结果显示,VPA处理组ROS表达水平显著高于对照组。Sulindac处理以后,与VPA处理组相比,β-catenin蛋白表达下调,P-β-catenin表达上调,GSK-3p表达上调,P-GSK-3p表达下调,4-HNE下调。Real-time PCR结果显示,抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine, NAC)处理虽然减少了ROS的产生,但是并没有改变GSK-3β mRNA和P-catenin mRNA的表达。以上结果揭示,Wnt/β-catenin通路活性增强导致氧化应激的产生,VPA促进神经元氧化应激的产生由于该通路上调所致。
综上所述,我们的工作提示：环境因素(如VPA暴露)通过诱导脑组织Wnt/β-catenin通路活性改变,使脑内经典Wnt信号通路活性增强,进而增加氧化应激的产生、氧化稳态的异常,有可能进一步影响神经元的正常发育和联系,阻碍了正常的神经网络形成,从而引起社会交往、情绪和语言等行为方面的异常,最终导致autism的发病。
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 mRNA和β-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.
引文
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