背景:突触是高度特化的位点,其特征是复杂的蛋白质-蛋白质相互作用(PPI)网络对于维持健康的突触至关重要。因此,绘制这些网络可以解决关于人类认知的未解决的问题,突触可塑性,学习,在生理和病理条件下的记忆。分析活人中突触相互作用的局限性导致了从冷冻保存的人脑中分离突触末端(突触体)的方法的发展。
方法:这里,我们通过对从冷冻的人额叶皮质(FC)和海马(HP)分离的突触体(Syn-FlowPLA)应用流式细胞术邻近连接测定(FlowPLA),建立了一种检测突触PPI的方法。
结果:将此方法应用于突触体,我们能够检测到N-甲基-D-天冬氨酸谷氨酸受体(NMDARs)不同亚型与其锚定突触后密度95蛋白(PSD95)之间已知的突触后相互作用.此外,我们检测到已知的突触前相互作用之间的SNARE复合蛋白突触体相关蛋白25kDa(SNAP25),突触短蛋白(VAMP2),和语法1a(STX1A)。作为阴性对照,我们分析了线粒体超氧化物歧化酶2(SOD2)和PSD95之间的相互作用,这些相互作用预计不会在物理上相关。
方法:PPI主要通过免疫共沉淀进行体外研究,亲和层析,蛋白质片段互补测定(PCA),和流式细胞术。所有这些都是有效的方法;然而,他们需要更多的步骤或与其他技术的组合。PLA技术以高特异性和灵敏度识别PPIs。
结论:此处描述的Syn-FlowPLA允许对PPI进行快速分析,特别是在从冷冻尸检标本中分离出来的突触室中,实现更大的目标灵敏度。Syn-FlowPLA,正如这里所介绍的,因此是在生理和病理条件下研究人类突触PPI的有用方法。
Synapses are highly specialized sites characterized by intricate networks of protein-protein interactions (PPIs) important to maintain healthy synapses. Therefore, mapping these networks could address unsolved questions about human cognition, synaptic plasticity, learning, and memory in physiological and pathological conditions. The limitation of analyzing synaptic interactions in living humans has led to the development of methods to isolate synaptic terminals (synaptosomes) from cryopreserved human brains.
Here, we established a method to detect synaptic PPIs by applying flow cytometric proximity ligation assay (FlowPLA) to synaptosomes isolated from frozen human frontal cortex (FC) and hippocampus (HP) (Syn-FlowPLA).
Applying this method in synaptosomes, we were able to detect the known post-synaptic interactions between distinct subtypes of N-methyl-D-aspartate glutamate receptors (NMDARs) and their anchoring postsynaptic density 95 protein (PSD95). Moreover, we detected the known pre-synaptic interactions between the SNARE complex proteins synaptosomal-associated protein of 25 kDa (SNAP25), synaptobrevin (VAMP2), and syntaxin 1a (STX1A). As a negative control, we analyzed the interaction between mitochondrial superoxide dismutase 2 (SOD2) and PSD95, which are not expected to be physically associated.
PPIs have been studied in vitro primarily by co-immunoprecipitation, affinity chromatography, protein-fragment complementation assays (PCAs), and flow cytometry. All these are valid approaches; however, they require more steps or combination with other techniques. PLA technology identifies PPIs with high specificity and sensitivity.
The Syn-FlowPLA described here allows rapid analyses of PPIs, specifically within the synaptic compartment isolated from frozen autopsy specimens, achieving greater target sensitivity. Syn-FlowPLA, as presented here, is therefore a useful method to
study human synaptic PPI in physiological and pathological conditions.