Shank3基因缺失致自闭症的纹状体直接、间接通路平衡失调研究

Autism spectrum disorders(ASDs) are developmental disorders typically defined by three core symptoms:impaired social relationships, impaired communication and language, repetitive behaviors and restricted interests. Currently, there are no effect pharmacotherapies for managing the core symptoms of the disorder. So developing efficacious treatments for these symptoms is considered to be the primary unmet need in ASDs research. For this to occur, basic research is required to help elucidate the aetiology of ASDs. Recently, Variants of Shank3 have been linked to autism in patients. The Shank family (Shank1-3) gives rise to postsynaptic scaffold proteins implicated in the dendritic spine development and glutamatergic synapse formation. Our previous works found Shank3 knock out (KO) mice exhibit autistic like behaviors. Shank3 is highly expressed in the striata of humans and mice. Our electrophysiological and morphological studies reveal obvious reduction in function and structures of cortical-striatal glutamate synapses. The project neurons of the striatum are medium spiny neurons (MSNs), which can be divided into two major subpopulations: Direct pathway MSNs, which predominantly express D1 dopamine receptors (D1 MSNs), send inhibitory synapses to internal globus pallidus(GPi) and substantia nigra pars reticulata(SNr), whereas indirect pathway MSNs, which predominantly express D2 receptors(D2 MSNs), project mostly to external globus pallidus(GPe) then indirectly excitating GPi/SNr.The balance of two pathways play remarkable role in normal basal ganglion function by dopamine modulation to glutamate synapses. So the imbalance of direct and indirect pathways found be highly related with abnormal behaviors of basal ganglion diseases, such as Parkinson's disease, drugs addiction and Obsessive-Compulsive disorder. Previous works in autism research did not seperate D1 and D2 MSNs pathways, which may be mislead the exact function of direct and indirect pathways balance in autistic like behaviors. Our preliminary electrophysiological data showed reduction of excitatory synapses numbers in D2 MSNs but not D1 MSNs, which provide the evidence of imbalance between direct and indirect pathways causing autisms like behavior in Shank3 KO mice. We are planning do further works to research excitatory synaptic function and dopamine modulation of cortical-striatal pathways by seperating D1 and D2 MSNs in Shank3 KO mice with bacterial artificial chromosome trangenic technology. Then we can find a way to rescue autistic like behavior by rebalancing D1 and D2 MSNs activities in vivo. From that, we can demonstrate clear role of direct and indirect pathways balance in ASDs and help to find the target for pharmacotherapies of the core symptoms of ASDs.

自闭症导致儿童社会交往和语言障碍、强迫行为，缺乏有效治疗手段。Shank3在兴奋性突触形成和功能中具有重要作用，该基因缺失是导致自闭症的重要因素。我们前期发现，敲除Shank3基因小鼠出现自闭症样行为，皮质-纹状体谷氨酸能突触功能和结构异常。纹状体由表达多巴胺D1受体的直接通路和D2受体的间接通路组成。两条通路在基底神经节输出控制中相互拮抗，受D1、D2受体调制，其平衡确保基底神经节正确输出，而平衡失调则导致帕金森、成瘾等。自闭症是否与纹状体直接、间接通路失衡有关，尚不清楚。我们发现Shank3敲除小鼠出现间接通路兴奋性突触数目降低，结合环路特征推测自闭症可能由纹状体直接通路强于间接通路失衡导致。本研究将系统研究Shank3敲除小鼠纹状体直接和间接通路兴奋性突触受体功能和多巴胺调控的失衡表现，以期阐明自闭症中纹状体直接、间接通路平衡失调的机制，寻找可能的作用靶点，为自闭症药物治疗提供依据。

自闭症是严重影响儿童身心健康的神经发育障碍性疾病。患儿存在社会交往障碍和刻板行为或兴趣狭窄典型表现，无法适应社会，极大增加了家庭和社会的负担。现已明确遗传异常是自闭症发病的重要因素。新近发现，Shank3 单基因突变即可导致自闭症的发生。我们利用前期建立的Shank3敲除小鼠自闭症模型，对自闭症重要的脑区——纹状体进行了系统的研究。利用多巴胺受体转基因荧光小鼠，我们可辨别纹状体表达多巴胺D1受体的直接通路投射神经元（D1 MSNs）和表达多巴胺D2受体的间接通路投射神经元（D2 MSNs），并对纹状体D1 MSNs和D2 MSNs空间分布特征进行了研究，发现纹状体内D1 MSNs密度略高于D2 MSNs。在此基础上，我们发现Shank3敲除小鼠纹状体D2 MSNs出现突触传递功能下降、长时程突触可塑性丧失和树突棘密度下降，而D1 MSNs突触功能和结构相对正常。采用化学遗传学特异性激活间接通路D2 MSNs的电活动，可以显著改善Shank3敲除小鼠的重复刻板行为，而激活直接通路D1 MSNs则对刻板行为无明显作用。此外，多巴胺是纹状体通路平衡重要的调节递质。我们发现多巴胺对D1 MSNs和D2 MSNs上的内向整理钾通道（Kir）具有截然相反的调控作用。多巴胺激活D1受体，增加D1 MSNs上Kir电流的幅度，其内在的信号通路机制是激活了AC-cAMP-PKA通路。而多巴胺激活D2受体，则抑制D2 MSNs上Kir电流的幅度，通过PLC-PKC通路进行调控。D1受体Kir电流的增加可以导致D1 MSNs树突整合能力下降，从而负反馈抑制D1受体对D1 MSNs突触传入和内在兴奋性的增加作用，使得D1 MSNs在D1受体作用下增加的输出信息控制在一定的范围内。在D2 MSNs多巴胺也有类似的作用，机制是降低Kir电流而增强D2 MSNs的树突整合能力，整体上也是对D2 MSNs信息输出的内在负反馈。上述结果提出了自闭症刻板行为调控的纹状体通路平衡新机制，展现了多巴胺对纹状体通路平衡的精密调控，为理解自闭症刻板行为产生的环路机制和潜在的调控靶点提供了理论基础和思路。