Gap Junctions Mediate Neuronal Cell Death in Mammalian Retina

Purpose: : There is compelling evidence that propagation of toxic/rescue messengers through gap junctions (gj) plays a major, but controversial, role in neuronal death and survivability. The objective of this study was to determine whether gjs increase the vulnerability of coupled retinal neurons to injury under pathological conditions and to identify possible messengers.

Methods: : To induce single cell apoptosis and assess cell-to-cell coupling, cytochrome C and Neurobiotin were injected intracellularly into retinal ganglion cells (RGCs) in wild-type (WT) or connexin (Cx) knockout (KO) mice. For population studies of excitotoxic cell death, retinas were incubated with NMDA, then washed and assessed for cell death using a live/dead viability assay, TUNEL staining, or labeling for activated caspase-3.

Results: : Apoptosis induced in single RGCs resulted in the death of adjacent amacrine cells (ACs) and RGCs coupled through gjs. Similar effects were observed after induction of apoptosis in single Muller cells and assessment of cell death in coupled glia. Application of NMDA caused excitotoxic cell death in both the INL and GCL in WT retinas. The number of NMDA-induced dead cells was significantly lower in retinas pretreated with the gj blockers 18β–glycerrhetinic acid (GA;80 ± 6%) or meclofenamic acid (MFA; 74 ± 2%). To determine whether the connexin makeup of a gj is a factor in the extent of secondary cell death, we applied NMDA to retinas of WT, Cx36 KO, Cx45 KO, and Cx36/45 dKO mice. The loss of Cx45 increased cell survivability by 25±3%, whereas there was a dramatic 53±3% increase in cell survivability in the Cx36 KO mouse. The reduction in cell death seen following ablation of Cx36 and Cx45 individually was not additive (50±5%) in the Cx36/45 dKO. IP3 is a possible toxic messenger propagated through gjs. In support of this idea, inhibition of IP3 receptors by xestosponginC (XesC) reduced NMDA-induced cell death in WT, but not in Cx36 KO retinas. Similarly, blockade of gjs with MFA, in addition to XesC, produced only a minor reduction in cell death in WT retinas.

Conclusions: : Our results indicate that gjs form a conduit for the spread of deleterious signals, such as IP3, resulting in the death of coupled retinal neurons. Our data suggest a link between connexin makeup, cell subtype and neuronal vulnerability to injury under pathological conditions. Reducing the permeability of gjs, particularly those expressing Cx36, significantly increased neuronal survivability, suggesting a unique strategy for targeted therapeutic intervention.