INTRODUCTION:Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder of early onset, characterized by impaired sociability, cognitive function and stereotypies. The etiology of ASD involves a multidimensional link between genetic, neurobiological and environmental factors. Since existing, comprehensive animal models for ASD are time consuming and laborious, the need for simple, quick approaches to study subsets of ASD-associated characteristics has always been in demand for better understanding of disease. The aim of the present study was to develop a cost and time effective zebrafish model with quantifiable parameters to facilitate mechanistic studies as well as high-throughput screening of new molecules for autism. METHODS:Zebrafish embryos were treated with valproic acid (75 μM) beginning at 4-h post fertilization to 5-days post fertilization. A series of behavioral tests (anxiety, inattentive behavior and circling behavior) and molecular studies were performed as surrogate parameters of ASD-like characteristic on the larvae at 7-dayspost fertilization for a quick screen. The study was followed by validation of model by screening positive control and negative control drugs. The social interaction test was performed on 21-days post fertilization to confirm that the surrogate phenotypes were indicative of social deficit (a core symptom of ASD). RESULTS:The model showed a significant behavioral impairment (2-4fold difference) in valproic acid treated larvae compared to control larvae, which was further supported by alterations in select high-risk genes and proteins, implicated in human ASD. Reversal of behavioral impairments using standard drugs marketed for symptomatic treatment in ASD and no effect on behaviors when treated with paracetamol (negative control) signifies the role of model in preliminary drug screening. CONCLUSION:The model shows robust parameters to study behavior, molecular mechanism and drug screening approach in a single frame. Thus, we postulate that our 7-day larval model could be a useful preliminary screening tool to identify novel targets as well as potential drugs for autism and also can be applied to develop a high-throughput screening approach.

译文

简介:自闭症谱系障碍(ASD)是一种较早发作的复杂神经发育障碍,其特征是社交能力,认知功能和定型观念受损。 ASD的病因学涉及遗传,神经生物学和环境因素之间的多维联系。由于现有的全面的ASD动物模型既费时又费力,因此一直需要简单,快速的方法来研究与ASD相关的特征的子集,以便更好地了解疾病。本研究的目的是开发一种具有可量化参数的成本和时间有效的斑马鱼模型,以促进机理研究以及针对自闭症的新分子的高通量筛选。
方法:从受精后4小时至受精后5天,用丙戊酸(75μM)处理斑马鱼的胚胎。受精后7天,对幼虫进行一系列行为测试(焦虑,注意力不集中和盘旋行为)和分子研究,作为ASD样特征的替代参数,以便进行快速筛查。在研究之后,通过筛选阳性对照和阴性对照药物来验证模型。受精后21天进行社交互动测试,以确认替代表型指示社交缺陷(ASD的核心症状)。
结果:该模型显示丙戊酸处理的幼虫与对照组幼虫相比,行为上有明显的损伤(差异是2倍),这进一步得到与人类ASD有关的精选高风险基因和蛋白质的改变的支持。使用市售用于对症治疗的对症治疗标准药物来逆转行为障碍,用扑热息痛(阴性对照)治疗对行为无影响,表明该模型在初步药物筛选中的作用。
结论:该模型显示了可靠的参数,可在单一框架内研究行为,分子机理和药物筛选方法。因此,我们假设我们的7天幼虫模型可能是有用的初步筛选工具,用于识别新的靶标以及自闭症的潜在药物,也可以用于开发高通量筛选方法。

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