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Augmenting AMPA receptor signaling after spinal cord injury increases ependymal-derived neural stem/progenitor cell migration and promotes functional recovery

Nature Neuroscience volume 28, pages 2054–2066 (2025) Cite this article

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Abstract

Ependymal cells in the adult spinal cord become activated after spinal cord injury (SCI), gaining stem/progenitor cell properties. Although growing evidence has implicated these cells as potential players in the endogenous repair process after injury, their activation to a stem-cell-like state is transient and insufficient for adequate regeneration. Moreover, the drivers of their activation state remain largely unknown. Previous work suggested that AMPA receptors (AMPARs) regulate cultured ependymal-derived neural stem/progenitor cells (epNSPCs). In this study, we identified an AMPAR-dependent mechanism of epNSPC regulation after SCI. Using lineage tracing in adult mice, we demonstrate that conditional knockout of GluA1–GluA3 AMPAR subunits in epNSPCs abolishes glutamate-induced AMPA currents and impairs the acute activation of these cells after SCI. Augmenting AMPAR signaling with the ampakine CX546 alters the transcriptional profile of epNSPCs, maintaining their acute maturation reversal after SCI into the chronic injury period, increasing connexin-43 signaling, promoting their migratory capacity and enhancing ependymal–glial cell contacts, which may contribute to the spatial distribution and migratory pattern of ependymal cells after injury. CX546 treatment ameliorates the subacute decrease in corticospinal tract excitability after SCI and leads to long-term functional improvements. Together, this work identifies a neurotransmitter receptor-dependent mechanism of epNSPC activation after injury, which may be targeted to harness the regenerative potential of the spinal cord.

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Fig. 1: Pharmacological blockade of AMPARs inhibits the acute activation of epNSPCs after SCI.
The alternative text for this image may have been generated using AI.
Fig. 2: Electrophysiological characterization of the Foxj1-CreER-tdT-Gria1–3fl/fl mouse line.
The alternative text for this image may have been generated using AI.
Fig. 3: Impaired epNSPC response to SCI in GluA1–GluA3 cKO mice.
The alternative text for this image may have been generated using AI.
Fig. 4: snRNA-seq reveals transcriptomic changes in epNSPCs with CX546 treatment.
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Fig. 5: CX546 treatment increases epNSPC Cx43 signaling and migration after SCI, which is abolished with GluA1–GluA3 cKO.
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Fig. 6: Decreased corticospinal excitability is partially reversed by the activation of AMPARs.
The alternative text for this image may have been generated using AI.
Fig. 7: CX546 promotes long-term behavioral recovery after SCI.
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Data availability

RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE295949. Source data are provided with this paper.

Code availability

All analyses were performed with freely available software as described in the Methods. All codes used in this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank G. Collingridge and P. Dirks for their insight, guidance and scientific discussions; J. Frisen and E. Llorens for providing the Foxj1-CreER-tdTomato mouse line; D. Dietrich and A. Levine for scientific discussions. We thank J. Hong for scientific discussions and assistance with animal perfusions. We thank R. van Bendegem, L. Lee, S. Kouhzaei, J. Wang, M. Movahed and Y. Dechi Castro for animal care, behavioral assessments and technical support. We thank the following platforms: University Health Network Advanced Optical Microscopy Facility (T. X. Yu and W. Walker), Princess Margaret Genomics Center (T. Ketala), University Health Network Animal Resource Centre and Canadian Centre for Computational Genomics-McGill University. Schematic illustrations were created with BioRender.com. This research was funded by the Canadian Institutes of Health Research (project 487036; M.G.F., C.H.T. and L.D.H.), the Neurosurgery Research and Education Foundation/Academy of Neurological Surgeons (L.D.H.), the Physician Services Incorporated Foundation (L.D.H.), the Vanier Canada Graduate Scholarship (L.D.H.), the Surgeon Scientist Training Program, University of Toronto (L.D.H.), the Campeau/Tator Chair in Brain and Spinal Cord Research (M.G.F.), the University Health Network Foundation (C.H.T.) and the Krembil Foundation (M.G.F., C.H.T. and T.A.V.). The funders had no role in study design, data collection and analysis, preparation of the manuscript or decision to publish.

Author information

Author notes

  1. These authors contributed equally: Homeira Moradi Chameh, Gustavo Balbinot.

Authors and Affiliations

  1. Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada

    Laureen D. Hachem, Taufik A. Valiante, Charles H. Tator & Michael G. Fehlings

  2. Krembil Brain Institute, University Health Network, Toronto, ON, Canada

    Laureen D. Hachem, Homeira Moradi Chameh, Gustavo Balbinot, Andrea J. Mothe, Rui Tong Geng Li, Taufik A. Valiante, Charles H. Tator & Michael G. Fehlings

  3. Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada

    Gustavo Balbinot

  4. Canadian Centre for Computational Genomics, McGill University, Montreal, QC, Canada

    Alain Pacis

  5. Synapse and Neural Circuit Research Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

    Wei Lu

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Contributions

L.D.H., C.H.T. and M.G.F. designed and conceived all parts of the study. L.D.H., H.M.C. and T.A.V. performed and analyzed the in vitro electrophysiological data. L.D.H. and G.B. performed and analyzed the in vivo electrophysiological data. L.D.H., A.J.M. and R.G.L. performed image processing and analysis. L.D.H. and A.P. performed the snRNA-seq analysis. L.D.H., A.J.M. and W.L. performed management of lines. L.D.H. wrote the original and revised drafts. All authors read and approved the manuscript. M.G.F. and C.H.T. supervised all aspects of the study.

Corresponding authors

Correspondence to Laureen D. Hachem, Charles H. Tator or Michael G. Fehlings.

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Nature Neuroscience thanks Victoria Moreno and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of proliferative astrocytes with AMPAR inhibition.

a, Quantification of the fraction of proliferative astrocytes in vehicle or NBQX (6nmol) treated animals 3dpi. Data are mean ± s.e.m., n = 5 mice/group. Two way ANOVA (P < 0.0001 between treatments) followed by Sidak’s post hoc multiple comparisons test (−0.336 mm P = 0.0219; −0.224 mm P = 0.0003; −0.112 mm P = 0.0020; 0 mm P = 0.0003). b, Quantification of the fraction of proliferative astrocytes in vehicle or GYKI-53655 (3nmol) treated animals 3dpi. Data are mean ± s.e.m., n = 3 mice/group. Two way ANOVA followed by Sidak’s post hoc multiple comparisons test. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Source data

Extended Data Fig. 2 Supplemental cell quantification for treatment paradigms.

a, Treatment with NBQX or GYKI-53655 did not significantly alter the total number of tdT+ epNSPCs 7dpi. N = 6 mice in vehicle, n = 3 mice in NBQX, n = 3 mice in GYKI-53655. Two way ANOVA. b, Total number of tdT+ epNSPCs was not significantly different between control and cKO groups at 7dpi. N = 4 mice in Foxj1-CreER-tdT, n = 5 mice in Foxj1-CreER-tdT-Gria1-3fl/fl. Two way ANOVA. c, Average distance of epNSPCs from origin in vehicle-treated and CX546-treated groups 6 wks after SCI. N = 15 mice in vehicle, N = 16 mice in CX546. Two way ANOVA. d, Total tdT+ cell counts in vehicle-treated and CX546-treated groups was not significantly different (6 wks after SCI). N = 15 mice in vehicle, N = 16 mice in CX546. Two way ANOVA. e, f, Average distance of epNSPCs from origin and total tdT+ cell counts in CX546-treated and vehicle- treated groups with and without cKO of Gria1-3 genes. N = 15 mice in vehicle, N = 16 mice in CX546, N = 7 mice in cKO+Veh, N = 9 mice in cKO+CX546. Two way ANOVA followed by Tukey’s post hoc multiple comparisons test. Asterisk in e indicates significant difference on pairwise comparisons at 0 mm (P = 0.0384). All data are mean ± s.e.m. *P < 0.05.

Source data

Extended Data Fig. 3 Electrophysiological characterization of cultured epNSPCs from Foxj1-CreER-tdT-Gria1-3fl/fl mouse line.

a, Schematic of adult mouse epNSPC isolation, culture and FACS sorting for culture recordings. b, Images of cultured epNSPCs. Images were captured using a x40 objective under an IR microscope. epNSPC were labeled with tdT (right panel) and captured with TexRed using a x40 objective. Scale bar, 10µm. c, To record the current-voltage response of epNSPCs both at baseline and following the addition of glutamate, cells were initially held at –70 mV. Subsequently, they underwent a voltage sweep from –130 mV to 50 mV, with a step size of 20 mV. AMPAR currents were recorded in the presence of GABAergic blockers (Bicuculine 10 μM and CGP 10 μM), voltage-gated sodium channel blockers (TTX 2 μM), and NMDA receptor blocker (APV 20 μM). CTZ (10 μM) was added to decrease the quick desensitization of AMPA receptors. The amount of AMPAR current in both experimental groups (control and knockout) was recorded before and after adding 500 μM glutamate at a time of 0.5 s after each holding potential voltage (as indicated by the triangle in the left panel figure). The right panel shows the recorded AMPA currents following each holding potential in both control and knockout groups before and after adding 500 μM glutamate. The control group exhibits an increased AMPAR response in both baseline and after the addition of glutamate compared to the knockout group. Data are mean ± s.e.m., N = 5 cells in Control, N = 2 cells in Control-Glutamate, N = 3 cells in Knockout, N = 5 cells in Knockout-Glutamate. Schematic was created using Biorender; Hachem, L. (2025) https://BioRender.com/11mhl45

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Extended Data Fig. 4 snRNA-seq cluster markers and cell proportions.

a, Violin plots of nuclei cluster markers. b, Feature plot of key marker genes for the different cell types in the cervical spinal cord. c, Cell proportions in each treatment group: Group 1 uninjured, Group 2 SCI + vehicle, Group 3 SCI + CX546. There was no significant difference in cell proportions between SCI+veh and SCI + CX546 groups across any cell clusters. Black line in each group represents mean. Differential composition analysis was performed using the R package sccomp. Astrocyte: Group 1 vs 2 FDR = 0.0004; Group 1 vs 3 FDR < 0.0001. Ependymal: Group 1 vs 2 FDR = 0.018. Microglia/macrophages Group 1 vs 2 FDR < 0.0001; Group 1 vs 3 FDR < 0.0001. Neurons: Group 1 vs 2 FDR = 0.0038. Oligodendrocytes: Group 1 vs 2 FDR = 0.0015. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Extended Data Fig. 5 Cell-cell communication inference analysis.

a, Overlapping signaling pathways between groups were ranked based on their pairwise Euclidean distance where a larger distance indicates a greater difference.

Extended Data Fig. 6 Histological analysis of CX546 treatment.

a, Quantification of SOX9 (astroependymal marker) and SOX10 (oligodendroglial marker) staining in tdT+ epNSPCs 6wpi. Cells are largely SOX9+ with very low expression of SOX10. SOX9 analysis: N = 6 mice/group. SOX10 analysis: N = 6 mice in veh, n = 7 mice in CX546. Two-tailed t test. b, Representative confocal images of SOX9 (astroependymal marker) staining in tdT+ epNSPCs under vehicle and CX546 groups 6wpi. Scale bar, 10µm. c, Quantification of total cord expression of GFAP. N = 8 mice in veh, N = 10 mice in CX546. Two way ANOVA (P = 0.0861 between treatments). d, Quantification of spared myelin. N = 8 mice in veh, N = 10 mice in CX546. Two way ANOVA. e, NeuN quantification at 6 wpi in animals treated with vehicle or CX546. Analysis was conducted using HALO cytonuclear detection on full cross sectional images stained with NeuN and counterstained with DAPI. Neuronal counts are an average of 3 sections centered at each distance from the epicenter. N = 8 mice in veh, N = 10 mice in CX546. Two way ANOVA (P = 0.0471 between treatments). Bottom panel shows representative images of NeuN immunostaining in the ventral horn. Scale bar, 50µm. All data are mean ± s.e.m. *P < 0.05.

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Extended Data Fig. 7 Ependymal and astrocyte subcluster snRNA-seq analysis.

a, UMAP visualization of all ependymal nuclei colored by subcluster populations. b, Dot plot of ependymal subcluster markers. c, Violin boxplot of gene expression in key differentially expressed genes. Single-cell differential expression was tested using a robust linear model implemented in the R package ‘MAST’. P values were corrected for multiple testing using the Bonferroni procedure (Prrt1 **P = 0.006; Foxp2 *P = 0.013; Rnf220 *P = 0.042. Nuclei obtained from N = 4 mice/group. d, UMAP visualization of all astrocyte nuclei colored by subcluster populations. e, Dot plot of astrocyte subcluster markers. f, Comparison of significant ligand-receptor pairs between ependymal and astrocyte subpopulations in SCI+veh vs SCI+CX546 groups based on cell-cell communication inference analysis. Nuclei obtained from N = 4 mice/group. g, Gene set enrichment analysis of biological processes upregulated in CX546 treatment compared to vehicle treatment in the reactive astrocyte subcluster (based on pseudo-bulk DEG analysis). Pathways were considered statistically significant if permutation test FDR < 0.05. h, Quantification of tdT-GFAP surface contact as a percent of total tdT surface area. Data are mean ± s.e.m., n = 8 mice in veh, n = 10 mice in CX546. Two way ANOVA (P = 0.0181 between treatments). Right panel shows 3D reconstruction of confocal Z-stack on IMARIS. *P < 0.05, **P ≤ 0.01.

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Extended Data Fig. 8 Behavioral analysis in Gria1-3 cKO mice.

a, BMS score in Gria1-3 cKO mice showed a trend towards increased recovery with CX546 treatment compared to vehicle controls. Data are mean ± s.e.m., n = 10 mice in cKO+vehicle, n = 11 in cKO + CX546. Two way ANOVA. b, There was no significant improvement in FLAS testing in cKO mice treated with CX546 compared to vehicle control. Data are mean ± s.e.m., n = 10 mice in cKO+vehicle, n = 11 cKO + CX546. Two tailed Mann Whitney test. c, Catwalk metrics in Gria1-3 cKO mice treated with vehicle or CX546 showed minimal differences. Data are mean ± s.e.m., n = 10 mice in cKO+vehicle, n = 11 in cKO + CX546. Body speed two-tailed t-test, forelimb swing speed two-tailed t-test, hindlimb step cycle two-tailed Mann Whitney test.

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Extended Data Fig. 9 Neuron sub-clustering snRNA-seq analysis.

a, UMAP visualization of all neuron nuclei colored by subcluster populations. b, Dot plot of neuronal subcluster markers. c, Cell type prioritization score of neuronal subpopulations using Augur in uninjured vs SCI groups. d, Cell type prioritization score of neuronal subpopulations using Augur in SCI+veh vs SCI+CX546 groups. e, Comparison of significant ligand-receptor pairs between ependymal and neuron subpopulations in SCI+veh vs SCI+CX546 groups based on cell-cell communication inference analysis. Nuclei obtained from N = 4 mice/group.

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Hachem, L.D., Moradi Chameh, H., Balbinot, G. et al. Augmenting AMPA receptor signaling after spinal cord injury increases ependymal-derived neural stem/progenitor cell migration and promotes functional recovery. Nat Neurosci 28, 2054–2066 (2025). https://doi.org/10.1038/s41593-025-02044-8

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