果蝇突触后谷氨酸受体不同亚型的稳态调控机制

The majority of excitatory synapses in the mammalian brain are glutamatergic. For the normal functioning of excitatory synapses, different types and subtypes of glutamate receptors (GluRs) have to be accurately controlled in their location and number at postsynaptic side. Previous studies show that overexpressing either GluRIIA or IIB subtype at Drosophila neuromuscular junction (NMJ) will lead to a reduction of the other. To further confirm this, we manipulated GluRIIA or GluRIIB expression at different levels and found that the other would always change in the opposite direction, i.e., when IIA is upregulated, IIB will be downregulated, and vice versa, indicating an antagonistic effect between the two subtypes. Additionally, we observed the subsynaptic architecture of Drosophila NMJ using super-resolution microscopy based on structured illumination technique for the first time. The result revealed that GluRIIA located in the center with GluRIIB encircled it outside, and we also found that loss of either GluRIIA or GluRIIB led to disruption of the integrity of the other. Taken all the pieces of evidence together, we hypothesize that the accurate localization and antagonistic relation between GluRIIA and GluRIIB are controlled through a homeostatic regulation. The goal of this proposal is to elucidate the molecular mechanisms underlying the antagonistic regulation between the GluRIIA and GluRIIB using multidisciplinary approaches including super-resolution microscopy. This study will shed novel light on the GluR-related physiology and pathology of excitatory synapses.

人类大脑中兴奋性突触的受体大都是谷氨酸受体。维持突触后不同类型或不同亚型谷氨酸受体的正确定位和比例对于突触的正常功能至关重要。先前的研究表明，果蝇神经肌肉突触谷氨酸受体IIA/IIB亚型之一过表达会造成另一亚型在突触后表达减少。我们在进一步确证了这一现象的基础上，利用超分辨显微技术（super-resolution microscopy）首次对突触特异蛋白包括突触后受体进行观察，结果显示IIA/IIB在突触后呈不同的分布特征：IIA在中心，IIB呈环状包绕IIA；并且任一受体亚型的缺失都会造成另一亚型结构的完整性被破坏。这些结果提示，IIA/IIB受体亚型在果蝇神经肌肉突触后的不同定位以及拮抗关系受到严格的稳态调控。本项目将利用多学科交叉技术手段，包括超分辨显微观测这一新兴技术，解析IIA/IIB受体亚型在突触后相互拮抗的分子细胞机制，从而加深对突触功能以及相关疾病病理发生机制的理解。

哺乳动物中枢神经系统绝大多数的兴奋性神经传导都是由谷氨酸能突触介导的。果蝇神经肌肉突触与哺乳动物的中枢神经系统兴奋性突触有诸多相同点，其谷氨酸受体与哺乳动物的非NMDA受体也有很强的保守性，因而是一个很好的研究谷氨酸受体调控的模式系统。突触可塑性是学习和记忆的机制所在。目前对于谷氨酸受体亚型的调控及其对突触可塑性的作用所知有限，因而利用这一系统我们可以更为便利地对受体亚型的调控进行研究。果蝇神经肌肉突触存在两种受体亚型：A型和B型。通过遗传学分析，我们发现不同受体亚型之间存在稳态调控，即一种受体亚型升高或降低后，另一种受体亚型的突触表达会降低或升高，而受体的突触表达总量不变。进一步的mRNA和蛋白总量检测发现，这种受体亚型的稳态调控发生在转录后水平，且在不同类型运动神经元突触中普遍存在。突触效能是由突触前神经活性和突触后受体活性来决定的。通过突变体分析，我们发现不论是增强还是抑制神经活性，均会诱导突触表达的A型受体升高，而B型受体不变，即突触受体总量升高。而通过突变体及药物分析，我们发现当抑制A型受体活性时，会诱发突触表达的A型受体升高，B型受体下降的稳态调控。为了进一步解析诱导受体稳态调控的机制，我们进行了候选基因遗传筛选，发现突触后特异性下调dunce可以诱导A型受体升高，B型受体降低的稳态调控。dunce下调cAMP水平。突变体表型分析表明突触后上调cAMP水平也可以诱导同样的受体亚型稳态调控。因此，我们以果蝇神经肌肉突触为模式发现了受体亚型的稳态调控，并发现这种稳态调控因突触效能的改变而被诱导，并且受到cAMP水平的调控。上述结果为我们理解突触可塑性提供了新见解。