Enhancement of AMPA Receptor Signaling After Spinal Cord Injury Promotes Ependymal-Derived Neural Stem/Progenitor Cell Migration and Functional Recovery —— Synopsis of the Latest Research in Nature Neuroscience

1. Academic Background: Challenges in Spinal Cord Injury Repair, Potential of Ependymal Cells, and Exploration of AMPA Receptor Mechanisms

Spinal cord injury (SCI) represents a severe form of central nervous system trauma, typically leading to irreversible neurological deficits and paralysis. With the limited regenerative capacity of the mammalian spinal cord, effective neural regeneration and functional recovery post-SCI remain a long-standing challenge in neuroscience and clinical rehabilitation. In recent years, studies have found that ependymal cells around the central canal of the spinal cord can be activated following injury, acquiring neural stem/progenitor cell (NSPC) characteristics, showing transient proliferation and migration abilities. These ependymal-derived neural stem/progenitor cells (epNSPCs) play a key reparative role in lower vertebrates (e.g., amphibians and fish), but in mammals, their activated state is short-lived and their reparative power far inferior. Therefore, how to sustain and enhance the activation and stemness of ependymal cells, thereby unlocking their regenerative potential, has become a research hotspot in SCI repair mechanisms.

Previous explorations of epNSPCs activation have mostly focused on pathways like Wnt, Oncostatin, and Purinergic, but no decisive regulatory factor has been identified. During the early stages of injury, local glutamate levels rise dramatically, leading to excitotoxicity, and glutamate signaling strongly affects neural stem cell development, differentiation, and migration. Recent in vitro studies indicated that the AMPA receptor (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR) regulates epNSPCs proliferation and differentiation. Hence, it is hypothesized that the glutamate signaling mediated by AMPAR after injury could be a critical driver of ependymal cell activation and migration and is a potential breakthrough target for SCI repair.

2. Source of the Paper and Author Information

This study was carried out by Laureen D. Hachem, Homeira Moradi Chameh, Gustavo Balbinot, Andrea J. Mothe, Alain Pacis, Rui Tong Geng Li, Taufik A. Valiante, Wei Lu, Charles H. Tator, and Michael G. Fehlings, mainly affiliated with the University of Toronto’s Division of Neurosurgery, Krembil Brain Institute, and University Health Network, with collaborators from Simon Fraser University, McGill University, and NIH. The paper, titled “augmenting ampa receptor signaling after spinal cord injury increases ependymal-derived neural stem/progenitor cell migration and promotes functional recovery,” was published in October 2025 in the top neuroscience journal Nature Neuroscience (DOI: https://doi.org/10.1038/s41593-025-02044-8).

3. Detailed Research Workflow

1. Research Model and Overall Design

A series of innovative experiments were designed to explore the mechanisms by which AMPA receptors regulate the activation of spinal cord ependymal cells and functional recovery after SCI. Adult female C57BL/6J mice were used for a standardized bilateral compression/contusion injury at the C6/7 spinal segment. The main experimental processes were as follows:

1.1 Pharmacological Inhibition Experiments

To verify AMPAR’s role in epNSPC activation, the investigators administered NBQX and GYKI-53655 (the former also blocks kainate receptors, the latter is more specific) intrathecally before injury and used immunohistochemistry to detect the proportion of Ki67+ proliferative epNSPCs in the central canal area of the injury side. Foxj1-CreER-tdTomato reporter mice were used to track the migration patterns of epNSPCs.

1.2 Genetic Knockout Model Construction and Verification

To exclude off-target drug effects, Foxj1-CreER-tdTomato; Gria1–3flox/flox transgenic mice were generated, where Gria1/2/3 genes (AMPA receptor subunits 1-3) in epNSPCs are precisely knocked out via tamoxifen induction. Patch clamp recording was used both in vivo and in vitro to verify the abolition of AMPA receptor activity in epNSPCs.

1.3 Analysis of Injury-Induced Activation and Migration Assessment

With the above knockout models, the team assessed proliferation and migration distribution (migration distance and proportion from the central canal to the injury site) of epNSPCs at 3 and 7 days after injury, dissecting the impact of AMPAR knockout on cell activation.

1.4 Pharmacological Augmentation (Ampakine CX546) Experiments

To “positively regulate” AMPAR, the ampakine CX546 (a positive allosteric modulator of AMPAR) was administered by daily intraperitoneal injection starting at 7 days post-injury for five weeks. Single-nucleus RNA sequencing (snRNA-seq), immunohistochemistry, and behavioral assessments were used to evaluate CX546’s regulatory effects on epNSPC transcriptional profiles, migratory activity, and spinal cord functional recovery during the subacute and chronic injury stages.

1.5 Specific Verification and Intercellular Signaling Analysis

Using Gria1–3 knockout mice again, the study examined whether CX546’s effects depend on AMPAR expression. Cell–cell communication inference was used to analyze signaling changes (e.g., Connexin-43, Cadherin, FGF2) between ependymal cells, astrocytes, and neurons, exploring microenvironmental influences during CX546 intervention.

1.6 Behavioral and Electrophysiological Functional Assessments

Included Basso Mouse Scale (BMS) open field test, Forelimb Locomotor Assessment Scale (FLAS), Catwalk gait analysis, Von Frey sensory threshold measurement, and grip strength testing. Motor evoked potentials (MEP) were recorded at 1 week after injury and other time points (parameters: maximum amplitude, latency, recruitment curve), to correlate corticospinal tract excitability with residual spinal neurons.

2. Innovative Methods and Technologies

• Foxj1-CreER-tdTomato mice: Employed for in situ immunofluorescent tracking of epNSPCs and derivatives.
• Single-nucleus RNA sequencing (snRNA-seq): Used to resolve spinal cell subtypes; fine analyzes of ependymal cell transcriptional responses to drugs.
• Augur algorithm: Used to assess response strength of various cell types to injury or drug treatment.
• Cell communication inference & GSEA enrichment: Deciphers changes in intercellular pathways such as Connexin-43 and FGF2, and key processes like cell adhesion.
• Automated behavioral and multiparametric electrophysiology analysis: Objectively reflects mouse motor, sensory, strength, and neural conduction changes.

4. Detailed Key Experimental Results

1. Pharmacological Inhibition Demonstrates AMPAR’s Role in Genetic Regulation of epNSPC Activation

In the early injury phase, glutamate excitotoxicity is prominent. Experiments in Foxj1-CreER-tdTomato mice show that intrathecal injection of NBQX and GYKI-53655 significantly reduces the proportion of Ki67+ epNSPCs and the number of migratory cells in the central canal, but has little effect on the total number of epNSPCs. This suggests that AMPAR mainly regulates activation and migration, not overall cell survival.

2. Gria1–3 Gene Knockout Accurately Blocks AMPAR Current and Injury-Induced Activation

Patch-clamp results indicate that while wild-type epNSPCs elicit strong AMPAR currents and multi-unit activity upon glutamate stimulation, Gria1–3 knockout cells show a dramatic decrease in AMPA current and virtually no glutamate response, with much reduced unit activity, confirming that AMPAR electrophysiological function is essentially abolished.

3. AMPAR Knockout Restricts Ependymal Cell Activation and Migration

At 3 days post-injury, Gria1–3 knockout mice show significantly reduced proportions of Ki67+ epNSPCs; at 7 days, the proportion and migration distance of migratory cells are both markedly lower than controls, confirming AMPAR’s central driving force for ependymal cell activation and migration in early injury.

4. Ampakine CX546 Enhances AMPAR Signaling and Sustains epNSPC Migration Activation

snRNA-seq shows that epNSPCs are most strongly responsive to CX546. Related proliferation/migration regulatory genes, such as Erbb4, Magi2, and Rnf220 (AMPAR modulators), are upregulated; several negative migration regulators (Magi2, Csmd1, Ptprd, Rora, etc.) are downregulated. This suggests that CX546 maintains ependymal cells in an immature, migration-prone stem-like state.

5. Connexin-43 (CX43) Signaling Enhancement and Cell Adhesion Activation

Immunohistochemistry confirms that CX546 upregulates CX43 protein in epNSPCs. Cell communication inference finds significant upregulation of CX43 pathways between epNSPCs and astrocytes; CX546 promotes their interaction and FGF2 signaling, enhancing cell–cell adhesion and cooperative migration. Behavioral metrics, migration distance, and proportion of migratory cells all increase significantly.

6. Gria1–3 Knockout Confirms CX546’s Dependence on AMPAR

CX546 treatment in Gria1–3 knockout mice results in a marked reduction in CX43 expression and epNSPC migration, confirming that CX546’s effects are highly dependent on AMPAR expression—AMPAR regulation serves as the direct molecular basis for its action.

7. Electrophysiological Enhancement and Significant Functional Recovery

CX546 not only promotes epNSPC activation and migration at the cellular level but also reverses decreased corticospinal excitability seen after injury. MEPs—maximum amplitude, latency, and recruitment curves—all improve significantly. Grip strength, BMS, FLAS, and Catwalk gait analysis persistently show improvement, indicating that AMPAR modulation promotes both stem/progenitor cell migration and overall spinal network and motor function recovery.

8. Other Findings: Neuroprotection and Roles of Base Adhesion Molecules

CX546 directly promotes neuronal protection in the injury area (NeuN+ cell increase) and positively regulates the function and migration of several neuronal subtypes (e.g., PDYN, RORB, SOX5, MAF); by facilitating cadherin signaling between ependymal cells and various neurons, it may support axon regeneration and network remodeling.

5. Main Conclusions and Scientific Significance

This study systematically details how the AMPA receptor regulates ependymal-derived neural stem/progenitor cell (epNSPC) rapid activation and migration after SCI. Pharmacological enhancement via CX546 effectively prolongs and enhances the migratory and de-maturation state of epNSPCs, maintaining a long-lasting stem/migratory phenotype that ultimately translates to improved spinal electrophysiology and systemic motor function recovery. This mechanism has great clinical translational potential.

Key scientific significances include: - First detailed molecular mechanism proof of AMPAR-mediated ependymal stem cell activation and migration. - Development of a new SCI repair intervention around CX546 pharmacology. - Discovery of pivotal roles for CX43, cadherin, and related signaling in microenvironmental migration and intercellular adhesion. - Presentation of a novel theoretical model integrating AMPAR activation and cell–cell communication for SCI repair. - Proposes that activating ependymal stemness and migratory potential is the crucial step to promote spinal neural regeneration and network reconstruction.

6. Research Highlights and Innovations

• Dual-pathology validation: Both pharmacological inhibition and genetic knockout confirm AMPAR’s core function, with logical rigor.
• Stem cell activation and migration control: Innovatively proposes AMPAR-mediated regulation of ependymal cell stemness and migratory ability, breaking the mold of single-molecule targeting.
• Cell communication mechanism: First multi-level integration of single-cell sequencing and cell communication analysis, revealing synergy among CX43, FGF2, cadherin, and the interaction between stem cells, neurons, and glia.
• Direct functional recovery proof: Multiple behavioral and electrophysiological indicators show robust improvement, establishing a clear mechanism from molecule/cell to systems.
• High translational value: The safety of ampakines provides a strong theoretical base for future SCI drug development and clinical trials.

7. Additional Information and Prospects

The study also discusses the differences in ependymal cell maturation and regenerative ability between humans and animals, inspiring further research into human-origin stem cells and activation mechanisms. The paper cites more than 60 references, systematically reviewing SCI and ependymal cell literature from development, differentiation, to regeneration.

If validated in human patients, this mechanism could allow for low-injury, highly efficient activation and migration of ependymal stem cells after SCI, heralding a new era of spinal cord repair.

8. Summary

From theoretical basis to experimental validation, this research rigorously and innovatively proposes a strategy of controlling ependymal stem cell activation and migration via AMPAR signal enhancement, achieving a new breakthrough in SCI repair mechanism. It has significant implications for both basic neuroscience and clinical rehabilitation. In the future, it may provide new adjunctive routes for SCI-targeting drugs and stem cell therapies, greatly improving the quality of life for SCI patients.