Shank3基因缺失导致皮层兴奋-抑制失衡在孤独谱系障碍感觉异常中的机制研究

Autism spectrum disorders (ASDs) are developmental disorders typically defined by core symptoms: impaired social relationships, repetitive behaviors and restricted interests. Recent studies showed that 90% of ASD patients have some degree of sensory abnormalities. Atypical sensory responses are now part of the ASD diagnostic criteria in the most recent edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). SHANK3 is a critical postsynaptic scaffolding protein for glutamatergic synaptic development and function. Deletions as well as mutations of the SHANK3 gene cause neurodevelopmental disorder (Phelan-McDermid syndrome) with high prevalence of ASD. Genetic deletions/mutations of the Shank3 gene in mice lead to glutamatergic synaptic dysfunction and ASD-relevant behavioral abnormalities. In previous works, we examined the neural representations of sensory perception in the neocortex of Shank3 knockout mouse (Shank3-/-) model of ASD. Shank3-/- mice were more sensitive to relatively weak tactile stimulation in a vibrissa motion detection task. In vivo population calcium imaging in primary somatosensory cortex (S1) revealed increased spontaneous and stimulus-evoked firing in pyramidal neurons but reduced activity in interneurons. The balance of excitation and inhibition plays important roles in the neural representations of sensory perception in the neocortex. Does the imbalance of excitation and inhibition directly relate to sensory abnormality in Shank3-/- mouse? And, what is the sensory changes of Shank3 deletion/mutation in either excitatory neurons or inhibitory neurons? These are two important questions are unclear in sensory abnormalities in Shank3-/- mouse. In this proposal, we plan to focus on answering these two questions. Especially, we will put more effort on dissecting the roles of Shank3 gene deletion in the interneurons of sensory processing in Shank3-/- mouse. Through this study we can find the mechanism of excitation and inhibition imbalance in sensory cortex of Shank3-/- mouse. And, we will find a way to rescue sensory abnormalities in Shank3-/- mouse by rebalancing the excitatory and inhibitory neuronal activities in vivo. This study will help to find the new drug target for pharmacotherapies of the sensory abnormalities of ASDs.

感觉异常在孤独谱系障碍（Autism spectrum disorder，ASD）患者中普遍存在，并且会加重ASD的社交障碍和强迫行为。我们前期证实Shank3基因敲除小鼠ASD模型触觉过敏，感觉皮层中兴奋性神经元活动增加而抑制性神经元活动降低。感觉皮层兴奋-抑制平衡对于正确感觉信息处理极为重要。Shank3敲除小鼠感觉皮层兴奋-抑制失衡和触觉过敏是否直接相关及可能的作用机制尚不清楚。本研究拟采用Shank3条件敲除小鼠结合多种Cre工具小鼠和病毒载体，在感觉皮层兴奋性和抑制性神经元中分别敲除Shank3基因，并综合运用行为学、活体双光子钙成像、电生理学等技术，以ASD模型触觉信息处理的中枢机制为切入点，探讨感觉皮层兴奋性神经元和抑制性神经元在维持兴奋-抑制平衡中的作用及参与调控ASD感觉异常的机制。该研究将有助于揭示ASD感觉异常发生的中枢神经机制，为寻找可能的治疗手段提供理论基础。

感觉异常在孤独症患者中普遍存在，已经被纳入孤独症新的诊断标准之一。目前孤独症感觉异常产生的中枢神经机制尚不清楚。我们前期工作发现孤独症致病基因- Shank3基因敲除小鼠除了有孤独症样行为，还存在对胡须触觉刺激过敏的表型，与孤独症患者感觉超敏的临床症状极为相似。感觉皮层是躯体感觉信号处理的关键脑区。我们通过活体动物双光子钙成像技术发现Shank3基因敲除小鼠感觉皮层兴奋性神经元活性较野生型小鼠显著增高，而抑制性中间神经元活性较野生型小鼠显著降低。利用Shank3基因条件性敲除小鼠，我们使用兴奋性神经元启动子和抑制性中间神经元启动子，分别在该小鼠感觉皮层兴奋性神经元和抑制性中间神经元敲除Shank3基因，发现只有在感觉皮层抑制性中间神经元敲除Shank3基因后可以导致感觉皮层兴奋性神经元活性显著增高。小鼠行为学测试结果也发现，仅仅在小鼠感觉皮层抑制性中间神经元敲除Shank3基因就可以导致小鼠出现对胡须触觉刺激过敏的表型。这些结果证明Shank3基因敲除小鼠感觉皮层抑制性中间神经元受损是导致感觉皮层兴奋性神经元活性增高和行为学上出现感觉过敏的主要原因。进一步，我们发现感觉皮层PV阳性抑制性中间神经元可能是主要功能受损的抑制性中间神经元亚类。以上研究系统揭示了孤独症感觉障碍的中枢神经机制，为孤独症感觉障碍诊疗提供了新的理论学依据。除此之外，我们建立了新型小鼠癫痫模型。利用该小鼠模型首次观测到Shank3基因敲除小鼠对癫痫诱发表现易感性的表型，为进一步研究Shank3基因缺失导致癫痫发生奠定了坚实的基础。为了更好地在小鼠大脑标记特定类型神经元突起及树突棘等微小结构。我们前期利用法尼基化（Farnesylated）可以促使蛋白更易于与细胞膜结合并锚定在神经元膜表面，通过将EGFP改造成法尼基化EGFP，用来标记神经元突起及树突棘微小结构。本项目中将其改造成Cre 重组酶依赖的版本，实现了对特定类型神经元的选择性稀疏标记。这些技术改进应用到Shank3基因敲除小鼠神经元结构改变的研究中，极大提高了工作效率。