CONNEXIN 43 HEMICHANNELS MODULATE THE EARLY RESPONSE OF EPENDYMAL CELLS TO SPINAL CORD INJURY

Spinal cord injury (SCI) reactivates a latent population of progenitor-like cells lining the central canal that contribute to glial scar formation. However, the early signaling mechanisms driving this response remain unclear. Because connexin (Cx) 43 is expressed in ependymal cells (EC) of the intact spinal cord, we first assessed the presence of Cx hemichannels in spinal cord (SC) slices of neonatal mice (P4–P7), a developmental stage in which the ependyma is constitutively active. Ethidium bromide (EtBr) uptake was robust under control conditions and significantly reduced by meclofenamic acid (MFA; p < 0.0001). Genetic deletion of Cx43 in adult ependymal cells reduced EtBr uptake suggesting Cx43 is the main isoform accounting for the functional hemichannels. We next investigated whether Cxs shape Ca²⁺ signaling induced by P2X7 receptor activation. In SC slices from FoxJ1CreER-GCaMP3 mice, BzATP -a selective P2X7 receptor agonist-evoked propagating Ca²⁺ waves within the ependyma. MFA reduced Ca²⁺ wave propagation (p<0.005) and peak ΔF/F₀ values (p<0.0001), while the blockade of Cx43 hemichannels with TAT-Gap19 attenuated maximal Ca²⁺ responses (p<0.0001) without abolishing wave propagation. We then explored the role of Cx43 hemichannels in injury-induced ependymal cell proliferation in vivo. Pre-emptive blockade with the selective Cx43 hemichannel antagonist TAT-Gap19 significantly reduced injury-induced EC proliferation compared to the scrambled peptide (p<0.001), suggesting hemichannels are required during the early signaling that takes place in response to injury. Together, these results identify Cx43 hemichannels as key elements for the early signaling needed to awake the dormant ependymal stem cell niche.