Blocking connexin43 expression reduces inflammation and improves functional recovery after spinal cord injury.

After traumatic CNS injury, a cascade of secondary events expands the initial lesion. The gap-junction protein connexin43 (Cx43), which is transiently up-regulated, has been implicated in the spread of 'bystander' damage. We have used an antisense oligodeoxynucleotide (asODN) to suppress Cx43 up-regulation in two rat models of spinal cord injury. Within 24 h of compression injury, rats treated with Cx43-asODN scored higher than sense-ODN and vehicle-treated controls on behavioural tests of locomotion. Their spinal cords showed less swelling and tissue disruption, less up-regulation of astrocytic GFAP, and less extravasation of fluorescently-labelled bovine serum albumin and neutrophils. The locomotor improvement was sustained over at least 4 weeks. Following partial spinal cord transection, Cx43-asODN treatment reduced GFAP immunoreactivity, neutrophil recruitment, and the activity of OX42(+) microglia in and around the lesion site. Cx43 has many potential roles in the pathophysiology of CNS injury and may be a valuable target for therapeutic intervention.

Spontaneous Activity as a Determinant of Axonal Connections.

To investigate the role of spontaneous retinal activity in map refinement, we studied goldfish kept in darkness during regeneration of a cut optic nerve. In one experiment, such fish (with lenses ablated to blur vision) were maintained for 70 days in stroboscopic light, diurnal light, or total darkness interrupted daily by 15 minutes of stroboscopic light. The retinotectal projection was then assessed for retinotopy by standard methods, using retrograde transport of wheat germ agglutinin-horseradish peroxidase. As in previous work, significantly more refinement was found in diurnal than in stroboscopic light. In darkness, refinement was as complete as in diurnal light. In a second experiment, similar fish were kept in stroboscopic light for 63 days. Some were then assessed to confirm that refinement had been delayed, while others were transferred to darkness or diurnal light for assessment later. After 7 days in either environment, no further refinement was seen; but after 21 days, substantial and significant refinement has occurred in both. Thus the effects of darkness and diurnal light were indistinguishable, and very different from those of stroboscopic light and (in previous studies) tetrodotoxin. Map refinement is evidently activity-dependent but not experience-dependent, and can effectively use the correlated spontaneous firing of neighbouring ganglion cells as its basis. Locally correlated spontaneous activity, which appears also to drive eye- and class-specific axon segregation in mammals, occurs widely in the nervous system. It could potentially generate systematic interconnection patterns even between neuronal populations without an overtly topographic organization.

Gap-junction proteins in retinal development: new roles for the "nexus".

Gap-junction channels, the cytoplasmic proteins that associate with them, and the transcriptional networks that regulate them are increasingly being viewed as critical communications hubs for cell signaling in health and disease. As a result, the term "nexus," which was the original structural name for these focal intercellular links, is coming back into use with new proteomic and transcriptomic meanings. The retina is better understood than any other part of the vertebrate central nervous system in respect of its developmental patterning, its diverse neuronal types and circuits, and the emergence of its definitive structure-function correlations. Thus, studies of the junctional and nonjunctional nexus roles of gap-junction proteins in coordinating retinal development should throw useful light on cell signaling in other developing nervous tissues.

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures.

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.

Abnormal connexin expression underlies delayed wound healing in diabetic skin.

OBJECTIVE: Dynamically regulated expression of the gap junction protein connexin (Cx)43 plays pivotal roles in wound healing. Cx43 is normally downregulated and Cx26 upregulated in keratinocytes at the edge of the wound as they adopt a migratory phenotype. We have examined the dynamics of Cx expression during wound healing in diabetic rats, which is known to be slow. RESEARCH DESIGN AND METHODS: We induced diabetes with streptozotocin and examined Cx expression and communication in intact and healing skin. RESULTS: We found that diabetes decreased Cx43 and Cx26 protein and communication in the intact epidermis and increased Cx43 protein and communication in the intact dermis. Diabetes also altered the dynamic changes of Cxs associated with wound healing. Within 24 h, Cx43 was upregulated in a thickened bulb of keratinocytes at the wound edge (rather than downregulated as in controls, which formed a thin process of migratory cells). Cx43 decline was delayed until 48 h, when reepithelialization began. Although Cx26 was upregulated as normal after wounding in diabetic skin, its distribution at the wound edge was abnormal, being more widespread. Application of Cx43-specific antisense gel to diabetic wounds prevented the abnormal upregulation of Cx43 and doubled the rate of reepithelialization, which exceeded control levels. CONCLUSIONS: Cx expression in diabetic skin is abnormal, as is the dynamic response of Cx43 to injury, which may underlie the delayed healing of diabetic wounds. Preventing the upregulation of Cx43 in diabetic wounds significantly improves the rate of healing and clearly has potential therapeutic value.

Multiphoton imaging of chick retinal development in relation to gap junctional communication.

Neural progenitor cells in the developing retina extend processes that stretch from the basal vitread surface to the apical ventricular surface. During the cell cycle, the nucleus undergoes interkinetic nuclear migration (INM), moving in a vitread direction during G1, passing through S-phase at its peak and then, on entering G2, returning towards the ventricular surface where it enters M-phase and divides. We have previously shown that individual saltatory movements of the nucleus correlate with transient changes in cytosolic calcium concentration within these progenitor cells and that these events spread to neighbouring progenitors through connexin43 (Cx43) gap junction channels, thereby coordinating the migration of coupled clusters of cells. Disrupting coupling with pharmacological agents, Cx43-specific antisense oligodeoxynucleotides (asODNs) or dominant negative Cx43 (dnCx43) inhibits the sharing of calcium events, reducing the number that each cell experiences and significantly slowing INM. We have developed protocols for imaging migrating progenitor cells by confocal microscopy over relatively short periods, and by multiphoton microscopy over more extended periods that include complete cell cycles. We find that perturbing gap junctional communication not only slows the INM of progenitor cells but also apparently prevents them from changing direction at critical phases of the cell cycle. It also disrupts the migration of young neurons to their appropriate layers after terminal division and leads to their ectopic differentiation. The ability to perform extended time-lapse imaging over 3D volumes in living retina using multiphoton microscopy should now allow fundamental mechanisms governing development of the retinal neuroepithelium to be probed in detail.