nNOS表达上调通过AMPA受体GluA4转运上膜改善孤独症表型的机制研究

Autism spectrum disorder (ASD) is an immensely challenging neurodevelopmental disorder that can cause significant social, communication and behavioral challenges. Excitation-inhibition imbalance has been postulated to underlie phenotypic deficits in ASD. Neuronal activity- regulated pentraxin (Narp) was shown to bind and cluster GluA4-containing AMPA receptors onto the interneuronal membranes and result in enhanced GABAergic synaptic transmission. We have previously shown that expression of neuronal nitric oxide synthase (nNOS) was significantly decreased in mice treated prenatally with valproic acid (VPA) in the basolateral amygdala. Consistently, nNOS overexpression increases surface expression of Narp and GluA4 on nNOS-containing interneurons in response to enhanced nNOS interneuronal activity in VPA-treated mice, and alleviates ASD-like phenotypes. Based on above observations, we presume that up-regulation of nNOS mitigates autism symptoms through promoting the clustering of GluA4 onto nNOS-expressing interneurons mediated by Narp. In this project, using patch clamp, immunofluorescence, co-immunoprecipitation and small-molecule peptide interference, we first examine synaptic transmission in nNOS-positive interneurons and pyramidal cells in mice treated with VPA. Then, in order to increase nNOS protein expression, we will inject lentiviral vector mediated overexpression of nNOS into the basolateral amygdala and modulate PI3K/Akt/mTOR pathway in VPA-exposed mice. Furthermore, we investigate the effect of up-regulated expression of nNOS on synaptic transmission by Narp-induced GluA4 clustering and the subsequent amelioration of ASD-associated behavioral defects in VPA-treated mice. Accordingly, these findings will offer a rational basis for targeted pharmacological treatment of ASD.

孤独症是一种影响患者社会功能的神经发育障碍性疾病，主要发病机制是兴奋性和抑制性突触传递失衡。神经元活性调节蛋白（Narp）介导中间神经元上AMPA受体GluA4转运上膜，增强抑制性突触传递。前期我们发现，孤独症小鼠基底外侧杏仁核神经元型一氧化氮合酶（nNOS）表达降低；nNOS过表达使孤独症nNOS中间神经元活性增强、Narp和GluA4膜表面表达增加，并改善孤独症症状。据此推测，nNOS表达上调通过Narp介导GluA4转运上膜改善孤独症表型。本项目拟采用膜片钳、免疫荧光、免疫共沉淀和小分子肽干扰等技术，研究孤独症小鼠nNOS中间神经元和锥体细胞的突触传递特征。进一步通过nNOS基因过表达和调控PI3K/Akt/mTOR信号通路，研究nNOS上调对孤独症nNOS中间神经元上Narp介导GluA4突触传递的影响，探讨其改善孤独症症状的作用机制。这将为治疗孤独症提供新的理论基础和药物靶标。

孤独症是一种影响患者社会功能的神经发育障碍性疾病，主要发病机制是兴奋性和抑制性突触传递失衡。神经元活性调节蛋白（Narp）介导中间神经元上AMPA受体GluA4转运上膜，增强抑制性突触传递。我们前期研究表明，孤独症小鼠基底外侧杏仁核神经元型一氧化氮合酶（nNOS）表达降低。据此，本项目拟采用膜片钳、免疫共沉淀、小分子肽干扰和行为学等技术，研究nNOS中间神经元在孤独症样行为中的突触传递机制。结果表明：（1）基底外侧杏仁核nNOS基因敲减的小鼠表现为孤独症样核心症状，包括社交障碍、增强重复刻板和焦虑样行为，伴有兴奋性突触传递效能减弱（自发兴奋性突触后电流频率和幅度降低，微小兴奋性突触后电流频率和幅度降低），膜蛋白NMDA受体亚基（GLuN2B）和突触后致密蛋白95（PSD95）表达降低；（2）运用蛋白质组学发现孤独症小鼠基底外侧杏仁核nNOS阳性中间神经元上膜蛋白Narp和AMPA受体亚型GluA4亚单位表达下调；（3）nNOS 基因过表达和L-精氨酸调控 PI3K/Akt/mTOR信号通路引起孤独症小鼠基底外侧杏仁核nNOS 蛋白表达上调，通过增加孤独症小鼠基底外侧杏仁核膜蛋白Narp和GluA4表达，导致孤独症小鼠 nNOS 中间神经元微小和诱发兴奋性突触后电流幅度均显著增加，进而减轻孤独症小鼠社交障碍、重复刻板和焦虑样行为；本研究为治疗孤独症提供新的理论基础和药物靶标。三年内已发表SCI论文5篇，另外1篇正在投稿中，获得河南省科技进步二等奖1项。总之，根据计划书的要求，我们基本完成了各项指标。