特异性敲除小脑浦肯野细胞Scn8a基因对小鼠孤独症相关行为学的改变及分子机制研究

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder, characterized by persistent impairment of social communication, restricted interests and repetitive behaviors. Recent studies have implicated hundreds of genetic variants in the cause of ASD. Despite these advances in the genetic delineation of syndromic forms of ASD, the underlying molecular mechanisms remain poorly understood. Recent evidence has highlighted a crucial role of the cerebellum in ASD pathology, with post-mortem studies showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. Recent studies have also established a strong link between abnormal PC function, caused by the mutation of Tsc1, Tsc2, PTEN, and behavioral deficits that are relevant to ASDs. ..Scn8a gene encodes the voltage-gated sodium channel α subunit Nav1.6, which is highly expressed in cerebellum, particularly in PCs. Our previous study showed that patients with Scn8a mutations exhibit motor impairment, epileptic encephalopathy, social impairment and repetitive behavior. Mice heterozygous for mutations in the Scn8a gene have also been shown to display motor impairments and neuropsychological abnormalities. Conditional PC-specific Scn8a knockout mice exhibit ataxia, impaired coordination, deficits in delay eye blink conditioning and Morris water maze tests, which are also consistent findings in other animal models with autistic features. Similar to findings in PC-specific Tsc1 and PTEN knockout mice, Scn8a knockout PCs show lower frequency of spontaneous firing, and are thus less excitable. ..In the proposed study, we will use the Cre-loxP recombination system to generated conditional knockout mice in which Scn8a inactivation restricted to PCs. We will use a battery of behavioral tests to investigate sociability, repetitive behavior, motor learning, and cognitive inflexibility. In addition, we will use immunohistochemical staining/Golgi staining to analyze the morphology of PCs and measure levels of oxidative stress, cell death of PCs in PC Scn8a-mutant mice. Our study will be the first to analyze the specific contributions of cerebellar Purkinje cell Scn8a loss to ASD. The proposed PC-specific knockout model of Scn8a will allow us to determine the contribution of cerebellar pathology to ASD-related neuronal networks. The proposed experiments will thus provide novel insights into mechanisms contributing to the pathogenesis o ASDs, and will lay the basis for further mechanism-based therapies for syndromic and nonsyndromic ASD treatment.

孤独症是一种神经系统发育障碍，发病机制至今未明。近期研究发现小脑在孤独症发生中起关键作用。孤独症患者脑标本一致发现小脑浦肯野细胞丢失，小脑损伤也增加孤独症的发病风险。多种基因敲除鼠模型发现小脑浦肯野细胞异常与孤独症行为有关。Scn8a基因编码电压门控钠离子通道Nav1.6，在小脑浦肯野细胞中广泛表达。我们临床发现Scn8a突变的患者运动协调障碍、癫痫、孤独症行为和小脑萎缩，既往研究也发现Scn8a突变小鼠出现运动受损、精神障碍等，Scn8a突变的浦肯野细胞功能与其他孤独症模型中类似，因此我们认为小脑中Scn8a突变为孤独症发病的重要原因。我们使用Cre-loxP系统获得小脑浦肯野细胞特异性敲除Scn8a基因的小鼠，通过行为学评估小鼠孤独症行为；组织染色观察浦肯野细胞和突触形态及氧化应激指标。本课题首次利用浦肯野细胞Scn8a基因突变的小鼠开发孤独症模型，为孤独症的发病机制和防治提供新思路。

孤独症谱系障碍（Autism Spectrum Disorder, ASD）是一种广泛的神经系统发育疾病，特点是社会交往障碍，狭窄的兴趣和刻板的动作。最近越来越多的证据表明小脑在孤独症的发病中起到重要作用。在ASD患者脑标本中最一致的病理发现即小脑浦肯野细胞的丢失，此外生后早期小脑损伤也会增加ASD的发病风险。近年来的研究也应用一些合并有孤独症的单基因病模型，发现Tsc1、Tsc2、PTEN、Shank2等基因突变引起的小脑浦肯野细胞功能异常与小鼠的孤独症样行为有关。Scn8a基因编码电压门控钠离子通道Nav1.6，在小脑浦肯野细胞中广泛表达。Scn8a突变的患者存在运动协调障碍、癫痫、孤独症行为和小脑萎缩，且常存在精神行为异常。既往研究也发现Scn8a杂合突变小鼠表现出睡眠障碍、运动受损、精神障碍等，小脑特异性Scn8a基因敲除小鼠存在学习功能异常和延迟眨眼反射受损。电生理研究也发现Scn8a突变的浦肯野细胞功能与其他孤独症模型中类似，因此我们认为小脑中Scn8a突变可能会引起孤独症样的临床表现。在本研究中我们使用Cre-loxP系统获得小脑浦肯野细胞特异性敲除Scn8a基因的小鼠，通过行为学评估小鼠孤独症行为，组织染色观察浦肯野细胞和突触形态。我们应用加速转棒实验及步态分析发现小脑浦肯野细胞特异性敲除Scn8a基因的小鼠存在运动协调性及运动学习能力受损，共济失调步态。应用三箱实验、社会嗅觉实验发现突变小鼠表现出明显的孤独症样行为，对陌生小鼠兴趣较对照组明显减小（P<0.01）。突变小鼠比对照组小鼠在10分钟内表现出更多的刻板动作（P<0.05）。通过旷场实验、明暗箱实验及高架臂实验，我们发现突变小鼠的自发活动水平和对照组小鼠相比没有明显差别，但突变小鼠表现出明显的焦虑倾向。应用逆转T型水迷宫实验，我们发现突变小鼠逆转学习能力受损。通过对小脑标本大体分析及小鼠头颅MRI成像分析，发现突变小鼠小脑体积明显缩小。对小脑切片进行免疫荧光染色我们发现小鼠小脑浦肯野细胞进行性丢失。首次在国内构建了小鼠小脑浦肯野细胞特异性基因敲除鼠的孤独症模型，验证了特异性敲除小脑浦肯野细胞Scn8a基因通过加速浦肯野细胞死亡、引起浦肯野细胞及突触形态发生改变，促进小脑进行性萎缩，从而产生孤独症、焦虑等行为学改变。为今后更好的理解孤独症的发病机制及精准治疗提供思路。