Abstract
Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.
Highlights
Gap junction (GJ) channels are composed by two hexameric arrays of connexins (Cx), a multigene family of 20 integral membrane proteins described so far [1]
GJ Blockers Limit Apoptosis Spread In Vitro Since our results indicated that the use of GJ blockers increase cell viability in lesioned retinas, we further investigated whether these blockers endow changes in the distribution of apoptotic cells
Values obtained from bitmap pixel analysis (Fig.8N) indicated that the decrease of normalized pixel brightness is larger when retinas were maintained in quinine (222.3061.30, P,0.01) or CBX + quinine (219.1360.55, P,0.05), when compared to phosphate buffered saline (PBS) (26.3362.77). These results revealed that apoptosis spread is reduced when retinas are treated with GJ blockers
Summary
Gap junction (GJ) channels are composed by two hexameric arrays of connexins (Cx), a multigene family of 20 integral membrane proteins described so far [1]. The association of six Cx makes a hemichannel (connexon), and the docking of two hemichannels in apposed cell membranes forms GJ channels, where gating and permeation properties differ depending on the Cx combination involved [2]. Several studies have addressed this question using widespread apoptosis models, which do not create obvious molecular gradients between coupled cells. In these situations, experimental design may obscure whether spreading of apoptosis is related to GJ communication. In the retina ubiquitous cell coupling and high Cx expression were previously described. We focused on Cx36 and Cx43, which stand out as two of the most studied GJ proteins in the retina [4,5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
