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Updated: Aug 25, 2025
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
Blocking postsynaptic density-93 binding to C-X3-C motif chemokine ligand 1 promotes microglial phenotypic
Xiao-Wei Cao1, Hui Yang2, Xiao-Mei Liu3
1Department of Neurology of Drum Tower Hospital, Medical School and the State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University; Nanjing Drum Tower Clinical College of Xuzhou Medical University, Nanjing, Jiangsu Province, China; Institute of Brain Sciences, Nanjing University; Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University; Jiangsu Province Stroke Center for Diagnosis and Therapy; Nanjing Neurology Clinic Medical Center, Nanjing; Department of Neurology, Lianyungang Municipal Hospital, Affiliated Hospital of Xuzhou Medical University, Lianyungang, Jiangsu Province, China.
Abstract:
We previously reported that postsynaptic density-93 mediates neuron-microglia crosstalk by interacting with amino acids 357-395 of C X3 C motif chemokine ligand 1 (CX3CL1) to induce microglia polarization. More importantly, the peptide Tat-CX3CL1 (comprising amino acids 357-395 of CX3CL1) disrupts the interaction between postsynaptic density-93 and CX3CL1, reducing neurological impairment and exerting a protective effect in the context of acute ischemic stroke. However, the mechanism underlying these effects remains unclear. In the current study, we found that the pro-inflammatory M1 phenotype increased and the anti-inflammatory M2 phenotype decreased at different time points. The M1 phenotype increased at 6 hours after stroke and peaked at 24 hours after perfusion, whereas the M2 phenotype decreased at 6 and 24 hours following reperfusion. We found that the peptide Tat-CX3CL1 (357-395aa) facilitates microglial polarization from M1 to M2 by reducing the production of soluble CX3CL1. Furthermore, the a disintegrin and metalloprotease domain 17 (ADAM17) inhibitor GW280264x, which inhibits metalloprotease activity and prevents CX3CL1 from being sheared into its soluble form, facilitated microglial polarization from M1 to M2 by inhibiting soluble CX3CL1 formation. Additionally, Tat-CX3CL1 (357-395aa) attenuated long-term cognitive deficits and improved white matter integrity as determined by the Morris water maze test at 31-34 days following surgery and immunofluorescence staining at 35 days after stroke, respectively. In conclusion, Tat-CX3CL1 (357-395aa) facilitates functional recovery after ischemic stroke by promoting microglial polarization from M1 to M2. Therefore, the Tat-CX3CL1 (357-395aa) is a potential therapeutic agent for ischemic stroke.
Insights
The peptide Tat-CX3CL1 (357-395aa) promotes functional recovery after ischemic stroke by shifting microglia from a pro-inflammatory M1 state to an anti-inflammatory M2 state, reducing neurological impairment.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Postsynaptic density-93 (PSD-93) mediates neuron-microglia communication via C-X3-C motif chemokine ligand 1 (CX3CL1).
- The peptide Tat-CX3CL1 (amino acids 357-395) disrupts PSD-93/CX3CL1 interaction, offering neuroprotection in ischemic stroke.
- The precise mechanism of Tat-CX3CL1's therapeutic effect in stroke remains to be elucidated.
Purpose of the Study:
- To investigate the mechanism by which Tat-CX3CL1 (357-395aa) confers neuroprotection in acute ischemic stroke.
- To determine the effect of Tat-CX3CL1 on microglial polarization phenotypes (M1 and M2) post-stroke.
- To evaluate the therapeutic potential of Tat-CX3CL1 in attenuating long-term cognitive deficits and improving white matter integrity.
Main Methods:
- Assessed microglial M1 and M2 phenotypes at 6 and 24 hours post-ischemic stroke.
- Administered Tat-CX3CL1 (357-395aa) and an ADAM17 inhibitor (GW280264x) to investigate their effects on microglial polarization and soluble CX3CL1 production.
- Evaluated long-term cognitive function using the Morris water maze test and white matter integrity via immunofluorescence staining.
Main Results:
- Pro-inflammatory M1 microglia increased, while anti-inflammatory M2 microglia decreased post-stroke.
- Tat-CX3CL1 (357-395aa) promoted M1 to M2 microglial polarization by reducing soluble CX3CL1.
- ADAM17 inhibition also facilitated M1 to M2 polarization by decreasing soluble CX3CL1.
- Tat-CX3CL1 treatment attenuated long-term cognitive deficits and improved white matter integrity.
Conclusions:
- Tat-CX3CL1 (357-395aa) promotes functional recovery after ischemic stroke by facilitating microglial polarization from M1 to M2.
- Reducing soluble CX3CL1 is a key mechanism underlying Tat-CX3CL1's therapeutic effects.
- Tat-CX3CL1 (357-395aa) represents a promising therapeutic candidate for ischemic stroke treatment.
