利用NucleuSynapse-Tag对细胞核和突触进行平行分析，揭示表观遗传驱动的突触重塑

IF 2.3 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-08-18 DOI:10.1016/j.jneumeth.2025.110550

Yunuen Moreno-López, Citlali A. Suárez-Rangel, José A. Bonilla, G. Aleph Prieto

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引用次数: 0

摘要

神经元的功能依赖于细胞间协调的分子机制，以及细胞核和突触之间的通信。然而，核和突触内容的多重定量分析仍然是一个重大的技术挑战。新方法将AAV-EGFP病毒颗粒立体定向注入小鼠背齿状回（DG），用EGFP标记神经元。随后进行海马背侧解剖、均质和差速离心，获得细胞核（P1）和突触体粗组分（P2）；P1和P2均来自同一海马匀浆。过滤后进行免疫标记，流式细胞术分析。结果在EGFP+事件中，NeuN和PROX1鉴定了DG神经元的细胞核，突触体通过大小、FM4-64和VGluT1鉴定。共聚焦显微镜和电子显微镜证实了从EGFP标记的DG神经元中分离的EGFP+核和EGFP+突触体的身份和完整性。通过使用AAV-EGFP-shRNA-SUV39H1颗粒对EGFP标记的突触体和细胞核进行平行分析，NucleuSynapse-Tag提供了第一个直接证据，支持表观遗传机制调节突触中BDNF蛋白水平的观点。与现有的聚焦于细胞核或突触的方法相比，NucleuSynapse-Tag方案提供了一种定量方法，通过跟踪基因荧光标记的特定神经元群体，从单个大脑样本中并行研究突触和核分子标记。结合AAV、Cre小鼠和示踪剂等工具，我们的方法能够全面分析控制神经元功能和可塑性的两个相互依赖的亚细胞区室的分子重塑。

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Unveiling epigenetic-driven synaptic remodeling by parallel analysis of nuclei and synapses using NucleuSynapse-Tag

Background

Neuronal functions rely on coordinated molecular mechanisms across cell compartments, with communication between the nucleus and synapses. However, a multiplex and quantitative analysis of nuclear and synaptic content in parallel remains a significant technical challenge.

New method

Neurons were tagged with EGFP by stereotaxic injection of AAV-EGFP viral particles into the dorsal dentate gyrus (DG) of mice. This was followed by dorsal hippocampal dissection, homogenization, and differential centrifugation to obtain the nuclear (P1) and synaptosomal crude fractions (P2); both P1 and P2 were obtained from the same hippocampal homogenate. After filtration, fractions were immunolabeled and analyzed by flow cytometry.

Results

In EGFP⁺ events, NeuN and PROX1 identified nuclei from DG neurons, while synaptosomes were identified by size, FM4–64, and VGluT1. Bright-field, confocal, and electron microscopy confirmed the identity and integrity of EGFP⁺ nuclei and EGFP⁺ synaptosomes isolated from DG neurons tagged with EGFP. Through parallel analyses of synaptosomes and nuclei tagged with EGFP using AAV-EGFP-shRNA-SUV39H1 particles, NucleuSynapse-Tag provided the first direct evidence supporting the idea that epigenetic mechanisms modulate BDNF protein levels at the synapse.

Comparison with existing methods

Unlike current methods that focus either on nuclei or on synapses, the NucleuSynapse-Tag protocol offers a quantitative approach for studying synaptic and nuclear molecular markers in parallel from a single brain sample, by tracking genetically fluorescent-tagged specific populations of neurons.

Conclusions

Combined with tools like AAV, Cre mice, and tracers, our approach enables a comprehensive analysis of molecular remodeling in two interdependent subcellular compartments that control neuronal function and plasticity.

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.