Expression changes of CX3CL1 and CX3CR1 proteins in the hippocampal CA1 field of the gerbil following transient global cerebral ischemia

Chen

et al. 2020

Preprint

Background: Postsynaptic density 93 (PSD-93) mediates glutamate excitotoxicity induced by ischemic brain injury, which then induces microglial inflammatory response. However, the underlying mechanisms of how PSD-93 mediates the crosstalk between neurons and microglia in the postsynaptic dense region remain elusive. CX3 chemokine ligand 1 (CX3CL1) is a chemokine specifically expressed in neurons while its receptor CX3CR1 is highly expressed in microglia. In this study, we aimed to investigate the role of PSD-93 and CX3CL1 interaction in the crosstalk between neuron and microglia in acute ischemic stroke.Methods: Male C57BL/6 mice were used to establish middle cerebral artery occlusion model and co-immunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points following cerebral ischemic/reperfusion (I/R). ELISA was used to detect soluble CX3CL1. Yeast two-hybrid and co-immunoprecipitation were used to identify special amino acid sequences responsible for the interaction between PSD-93 and CX3CL1. Finally, a fusion small peptide Tat-CX3CL1 was designed to inhibit PSD-93 and CX3CL1 interaction.Results: The binding of PSD-93 and CX3CL1 peaked at 6 h after I/R. The binding sites were located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. Tat-CX3CL1 (357-395aa) could inhibit the interaction of PSD-93 and CX3CL1 and inhibited the pro-inflammatory cytokine IL-1β and TNF-α expression and provided neuroprotection following reperfusion.Conclusions: PSD-93 binds CX3CL1 to activate microglia and initiate neuroinflammation. Specific blockade of PSD-93-CX3CL1 interaction reduces I/R induced neuronal cell death, and provides a new therapeutic target for ischemic stroke.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

PSD-93 mediates the crosstalk between neuron and microglia and facilitates acute ischemic stroke injury by binding to CX3CL1

Chen

et al. 2020

Preprint

“…In addition, PSD-93 can interact with other proteins with its function structure domain and change the structure and function of other proteins [25][26] . As a transmembrane protein speci cally expressed in neurons, CX3CL1 can be cut into soluble CX3CL1 and reach the surface of the microglia to bind CX3CR1 and activate microglia when neurons are damaged or exposed to glutamate [19][20] . In this study, we found that PSD-93 could bind NR2B and CX3CL1 to form a complex and promote the release of soluble CX3CL1.…”

Section: Discussionmentioning

confidence: 99%

“…CX3 chemokine ligand 1 (CX3CL1) is a chemokine speci cally expressed in neurons, while its receptor CX3CR1 is highly expressed in microglia. When neurons are damaged or exposed to glutamate, soluble CX3CL1 disintegrates from its structural domain and binds to CX3CR1 on microglia to activate microglia [19][20] . Therefore, neurons and microglia can communicate through CX3CL1/CX3CR1 signals.…”

Section: Introductionmentioning

confidence: 99%

PSD-93 Mediates the Crosstalk between Neuron and Microglia and Facilitates Acute Ischemic Stroke Injury by Binding to CX3CL1

Li²,

Chen³

et al. 2020

SSRN Journal

Background: Postsynaptic density 93 (PSD-93) mediates glutamate excitotoxicity induced by ischemic brain injury, which then induces microglial in ammatory response. However, the underlying mechanisms of how PSD-93 mediates the crosstalk between neurons and microglia in the postsynaptic dense region remain elusive. CX3 chemokine ligand 1 (CX3CL1) is a chemokine speci cally expressed in neurons while its receptor CX3CR1 is highly expressed in microglia. In this study, we aimed to investigate the role of PSD-93 and CX3CL1 interaction in the crosstalk between neuron and microglia in acute ischemic stroke. Methods: Male C57BL/6 mice were used to establish middle cerebral artery occlusion model and coimmunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points following cerebral ischemic/reperfusion (I/R). ELISA was used to detect soluble CX3CL1. Yeast two-hybrid and co-immunoprecipitation were used to identify special amino acid sequences responsible for the interaction between PSD-93 and CX3CL1. Finally, a fusion small peptide Tat-CX3CL1 was designed to inhibit PSD-93 and CX3CL1 interaction. Results: The binding of PSD-93 and CX3CL1 peaked at 6 h after I/R. The binding sites were located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. Tat-CX3CL1 (357-395aa) could inhibit the interaction of PSD-93 and CX3CL1 and inhibited the pro-in ammatory cytokine IL-1β and TNF-α expression and provided neuroprotection following reperfusion. Conclusions: PSD-93 binds CX3CL1 to activate microglia and initiate neuroin ammation. Speci c blockade of PSD-93-CX3CL1 interaction reduces I/R induced neuronal cell death, and provides a new therapeutic target for ischemic stroke.

“…Its only receptor CX3CR1 is highly expressed in microglia and its main forms are membrane binding and soluble. When neurons are damaged or exposed to glutamate, soluble CX3CL1 disintegrates from its structural domain and binds to CX3CR1 on microglia to activate microglia [18][19] , directly inducing multiple functions of microglia. Therefore, neurons and microglia can communicate through CX3CL1/CX3CR1 signals.…”

Section: Introductionmentioning

confidence: 99%

PSD-93 mediate the dialogue between neuron and microglia and facilitate acute ischemic stroke by binding 357-395 amino acid sequence of CX3CL1

Chen

et al. 2020

Preprint

Background: Our previous experiments demonstrated that PSD-93 mediates glutamate excitotoxicity induced by ischemic brain injury, which promotes the release of inflammatory cytokines in early ischemic brain injury by activating the NMDA receptor. Glutamate activity is the key to neuronal excitatory toxicity and microglial cell inflammatory response in the joints. However, the underlying mechanisms of how does PSD-93 mediate the dialogue between neurons and microglia in the postsynaptic dense region remain elusive. And CX3 chemokine ligand 1 (CX3CL1) is a chemokine that is specifically expressed in neurons. Its only receptor CX3CR1 is highly expressed in microglia and its main forms are membrane binding and soluble. In this study, we aim to clarify the specific amino acid sequence of the binding of psd-93 and CX3CL1 and investigate role of PSD-93 on regulating the crosstalk between neuron and microglia in acute ischemic stroke. Methods: In this study, male C57BL/6 mice aged 8-12 weeks and weighted 22-26g were applied with Middle Cerebral Artery Occlusion (MCAO) model and randomly divided into different groups. Firstly, co-immunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points 3h, 6h, 12h 24h, 48h and 72h following cerebral ischemic/reperfusion. Meanwhile, ELISA was used to investigate the expression of soluble CX3CL1 at the same time points to confirm the relationship between of the expression of soluble CX3CL1 and the combination of PSD-93 and CX3CL1. Secondly, two bait plasmids pSos-PSD-93-full length, pSos-CX3CL1-full length and five mutant plasmids: pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4, and pMyr-CX3CL1-mut, were constructed and used a yeast two-hybrid system to screen and identify positive clones and to determine the sequence in which the two proteins bind to each other. Thirdly, the proteins corresponding to the three positive clones obtained in the yeast two-hybrid experiment were used to construct plasmids for transfection of eukaryotic cells and the protein expression binding was verified again by in vitro co-immunoprecipitation. Finally, a specific fusion small peptide Tat-CX3CL1 were designed according to above experiment to inhibit the integration of PSD-93 and CX3CL1 and to explore their role on neuron death following reperfusion. Results: We found that the binding capacity of PSD-93 and CX3CL1 proteins peaked at 6h after ischemia/reperfusion and then decreased gradually. The specific amino acid sequence of PSD-93 and CX3CL1 binding was obtained by yeast double hybridization and in vitro immunoprecipitation. We identified that their binding sites are located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. And a specific fusion small peptide Tat-CX3CL1 (357-395aa) were designed to inhibit the integration of PSD-93 and CX3CL1 and perform neuroprotection on neuron death following reperfusion. Conclusions: Our results suggest that PSD-93 promotes the formation of its soluble form by binding to CX3CL1, which is recruited to the surface of microglia to bind to CX3CR1, thereby activating microglia to initiate inflammation. Thus, specific blockade of PSD-93-CX3CL1 coupling can reduce ischemia-reperfusion induced neuronal cell death, which provide a new target to treat ischemic stroke.