The Chemokine CCL2 Promotes Excitatory Synaptic Transmission in Hippocampal Neurons via GluA1 Subunit Trafficking
In the latest research paper “Chemokine CCL2 Promotes Excitatory Synaptic Transmission in Hippocampal Neurons via GluA1 Subunit Trafficking” published in “Neurosci. Bull.”, researchers from multiple institutions, including the Shanghai Institute of Neuroscience, Chinese Academy of Sciences, and the School of Life Sciences, Peking University, have detailed how the chemokine CCL2 promotes excitatory synaptic transmission in hippocampal neurons by regulating the surface expression of the GluA1 subunit.
Research Background and Objectives
Cytokines are small secreted proteins known to play important roles in immune cell development, maturation, and disease processes. However, cytokines can also regulate synaptic transmission and neuronal excitability in the central nervous system (CNS). It has been reported that TNF-α plays an important role in maintaining excitatory synaptic strength, while IFN-γ can enhance GABAergic transmission and increase the excitability of CA3 pyramidal cells. Previous studies have shown that CCL2 (also known as monocyte chemoattractant protein-1, MCP-1) can enhance excitatory synaptic transmission in hippocampal CA1 and CA3 pyramidal cells, primary somatosensory cortex L2/3 pyramidal cells, and dentate gyrus granule cells. However, the molecular mechanisms by which CCL2 specifically regulates these processes remain unclear.
Research Methods
The research team employed a series of in vitro and in vivo experimental methods to explore how CCL2 regulates GluA1 surface expression through its receptor CCR2. The specific experimental procedures include:
1. Animal Experiments
All animal experiments followed the animal care standards of the National Institutes of Health and were approved by the relevant ethics committee. CCR2 knockout mice and primary hippocampal cell cultures from rats were used to study the effects of CCL2 on GluA1 surface expression.
2. Drug Treatment
Recombinant CCL2 protein was applied to cultured neurons in different experiments to examine the role of CCL2 in regulating GluA1 surface expression. To induce neuroinflammation, mice were intraperitoneally injected with lipopolysaccharide (LPS).
3. DNA Constructs and Real-time Quantitative PCR (RT-qPCR)
DNA constructs encoding vertebrate proteins were used for cell transfection, and RT-qPCR was used to analyze the expression levels of various inflammatory factors such as CCL2 and TNFα in hippocampal tissue.
4. Live Cell Imaging and Electrophysiological Recording
Through live cell imaging and electrophysiological recording methods, the research team investigated the real-time effects of CCL2 on GluA1 surface expression and synaptic transmission. GluA1 constructs containing pH-sensitive green fluorescent protein (super ecliptic pHluorin, SEP) were introduced to directly observe the expression and changes of GluA1 on the synaptic surface.
5. Western Blot and Immunocytochemistry
Western blot techniques were used to quantify related proteins and their phosphorylation levels in hippocampal tissue, and immunocytochemistry methods were used to study the expression regions and intensity of GluA1 on the neuronal surface.
Research Results
The research results showed that exogenous CCL2 application significantly increased the surface expression of GluA1 in cultured hippocampal neurons, and this effect was mediated through CCR2. Specifically:
1. SEP-GluA1 Live Cell Imaging
Five minutes after CCL2 treatment, the spot area and fluorescence intensity of SEP-GluA1 increased significantly. This effect was blocked by the addition of the CCR2 antagonist RS504393, indicating that CCL2-induced GluA1 surface expression depends on CCR2.
2. Immunofluorescence Staining of GluA1 Surface Expression
CCL2 treatment significantly increased the total area and total intensity of surface GluA1 in cultured hippocampal neurons, while this increase was completely blocked in CCR2 knockout mice. Additionally, in the hippocampal tissue of LPS-treated mice, the membrane-associated levels of GluA1 and phosphorylation levels at Ser831 and Ser845 sites were significantly increased, and these effects were blocked in CCR2 knockout mice.
3. Electrophysiology and Calcium Imaging
Through whole-cell patch-clamp recording, CCL2 significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons, but these effects were blocked in CCR2 knockout mice. Furthermore, calcium imaging results showed that CCL2 treatment greatly increased the frequency of calcium transients in hippocampal neurons, a process mediated by the Gαq signaling pathway and activated by calcium/calmodulin-dependent protein kinase II (CaMKII).
Research Conclusions and Significance
This study elucidates the mechanism by which CCL2 regulates GluA1 membrane expression and excitatory synaptic transmission through CCR2 and the downstream Gαq, Ca^2+, and CaMKII signaling pathways. This finding not only deepens our understanding of the molecular mechanisms by which chemokines regulate synaptic transmission but also provides new perspectives for exploring therapeutic strategies for neurological diseases associated with neuroinflammation in the future.
Research Highlights
The highlights of this research include: 1. Precise analysis of the molecular mechanism by which CCL2 regulates GluA1 surface expression through CCR2 and the CaMKII signaling pathway. 2. Provision of a systematic research method combining in vivo and in vitro experiments, which helps to comprehensively evaluate the role of CCL2 in different neuron types. 3. Proposal of a novel regulatory pathway for modulating synaptic plasticity under neuroinflammatory conditions.
The mechanism revealed in this study may be widely applicable to various neuron types and provides important references for in-depth research on the mechanisms of action of chemokines and their roles in regulating nervous system functions. Future research can further explore the specific roles of more chemokines and cytokines in regulating synaptic transmission and neuronal excitability, as well as their potential clinical application value.