Nascent Proteome Remodeling following Homeostatic Scaling at Hippocampal Synapses.

Neuronal networks are subject to fluctuations in both the magnitude and frequency of inputs, requiring plasticity mechanisms to stabilize network activity. Homeostatic synaptic scaling is a form of synaptic plasticity that adjusts the strength of neuronal connections up or down in response to large changes in input. Although homeostatic plasticity requires changes in gene expression, there is only limited data describing the molecular changes associated with homeostatic scaling, focusing mostly on the expression mechanisms involving glutamate receptors. The fact that neuronal networks can be scaled up (in response to reduced activity) or down (in response to enhanced activity) provides a unique opportunity to examine the molecular and proteomic response to opposite ends of the phenotypic spectrum of synaptic plasticity. Here we first demonstrate that homeostatic scaling required protein synthesis. We then examined the plasticity-induced changes in the newly-synthesized neuronal proteome of neurons to identify the landscape of proteomic changes that contribute to opposing forms of homeostatic plasticity. Cultured rat hippocampal neurons (21 DIV) underwent homeostatic upscaling or downscaling (treatments with TTX and Bicucculine, respectively). We used BONCAT (BioOrthogonal Non-Canonical Amino acid Tagging) to metabolically label, capture and identify newly-synthesized proteins, detecting and analysing 5940 newly-synthesized proteins using liquid chromatography-coupled tandem mass spectrometry and label-free quantitation. Neither up- or down-scaling produced changes in the number of different proteins translated. Rather, our findings indicate that synaptic up- and down-scaling elicit opposing translational regulation of several molecular pathways, producing targeted adjustments in the neuronal proteome. We detected ~ 300 differentially regulated proteins involved in neurite outgrowth, reorganization of nerve terminals, axon guidance and targeting, neurotransmitter transport, filopodia assembly, excitatory synapses and glutamate receptor complexes. These proteins include well-characterized mediators of synaptic plasticity, e.g. the ionotropic glutamate receptor complex that is down-regulated during down-scaling and coordinately upregulated during upscaling. We also identified differentially regulated proteins that in addition to their regulation in homeostatic plasticity, are also associated with multiple diseases and disorders, including intellectual disability, schizophrenia, epilepsy, and Parkinson’s disease.

神经元网络会受到输入信号幅度与频率波动的影响，需借助可塑性机制维持网络活动的稳态。稳态突触缩放（homeostatic synaptic scaling）是一类突触可塑性形式，可根据输入信号的大幅变化双向调控神经元连接的突触强度。尽管稳态可塑性需要基因表达的改变，但目前描述与稳态突触缩放相关的分子变化的研究数据仍较为有限，且大多聚焦于谷氨酸受体相关的表达调控机制。神经元网络可被上调（对应活动减弱时）或下调（对应活动增强时），这为探究突触可塑性表型谱两端对应的分子与蛋白质组学响应提供了独特契机。
本研究首先证实稳态突触缩放依赖于蛋白质合成。随后我们分析了可塑性诱导的神经元新合成蛋白质组的变化，以明确参与两种对立稳态突触缩放形式的蛋白质组调控全貌。我们对体外培养21天（DIV）的大鼠海马神经元分别进行稳态突触上调与下调处理（分别采用河豚毒素[tetrodotoxin, TTX]与荷包牡丹碱[bicuculline]处理）。采用生物正交非经典氨基酸标记（BioOrthogonal Non-Canonical Amino acid Tagging, BONCAT）技术对新合成蛋白质进行代谢标记、捕获与鉴定，并通过液相色谱-串联质谱联用技术与无标记定量方法，检测并分析了5940种新合成蛋白质。
无论是突触上调还是下调处理，均未改变翻译产生的蛋白质种类数量。相反，本研究结果显示，突触上调与下调会对多条分子通路产生双向相反的翻译调控，从而实现神经元蛋白质组的靶向性调节。我们共检测到约300种差异调控蛋白质，这些蛋白质参与神经突生长、神经末梢重构、轴突导向与靶向定位、神经递质转运、丝状伪足组装、兴奋性突触及谷氨酸受体复合物的形成等过程。其中包括已被充分研究的突触可塑性介质，例如离子型谷氨酸受体复合物：其在突触下调过程中表达下调，而在突触上调过程中则协同上调。我们还鉴定出一些差异调控蛋白质，这些蛋白质除参与稳态可塑性调控外，还与多种疾病及病症相关，包括智力障碍、精神分裂症、癫痫以及帕金森病。