Limits...
Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Fontes JD, Ramsey J, Polk JM, Koop A, Denisova JV, Belousov AB - PLoS ONE (2015)

Bottom Line: In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect.Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons.A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT
Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

No MeSH data available.


Related in: MedlinePlus

The role of gap junctions in neuronal death is connexin type-independent and requires channel activity.A-D, Statistical data from MTT assay experiments with the use of NMDAR-excitotoxicity (A, B) and OGD (C, D) in neuronal cortical cultures obtained from Cx36 knockout mice are shown. Cultures were transduced with the indicated lentiviruses on DIV6; NMDA and OGD injuries were conducted 48 hours post-transduction; neuronal death analysis was performed 24 hours post-injury. Functional gap junctions made from neuronal and non-neuronal connexins all support neuronal death (A, C). Gap junction channels that are communication-deficient do not support neuronal death (B, D). Statistical analysis: ANOVA with post hoc Tukey; shown relative to (i) pCDH-LUC plus injury (NMDA or OGD) and (ii) pCDH-LUC (without injury); in all figures, n = 8–12 per group; mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4446213&req=5

pone.0125395.g007: The role of gap junctions in neuronal death is connexin type-independent and requires channel activity.A-D, Statistical data from MTT assay experiments with the use of NMDAR-excitotoxicity (A, B) and OGD (C, D) in neuronal cortical cultures obtained from Cx36 knockout mice are shown. Cultures were transduced with the indicated lentiviruses on DIV6; NMDA and OGD injuries were conducted 48 hours post-transduction; neuronal death analysis was performed 24 hours post-injury. Functional gap junctions made from neuronal and non-neuronal connexins all support neuronal death (A, C). Gap junction channels that are communication-deficient do not support neuronal death (B, D). Statistical analysis: ANOVA with post hoc Tukey; shown relative to (i) pCDH-LUC plus injury (NMDA or OGD) and (ii) pCDH-LUC (without injury); in all figures, n = 8–12 per group; mean ± SEM.

Mentions: The conductance of ions or small molecules by gap junctions so far measured is roughly within one order of magnitude [38], though more significant differences have been reported for larger permeants and some metabolites, with selective permeability based upon charge and size [39, 40]. It is unlikely, though not impossible, that gap junctions resulting from connexins from distinct subfamilies, with significant amino acid differences, would participate in common protein-protein interactions and signaling pathways. Therefore, if multiple, disparate gap junctions support neuronal death in the same cell death paradigm, the most likely mechanism is channel function, where the molecule or ion “death signal” has sufficient permeability through the tested gap junctions. In our experiments, we used neuronal cell transductions with lentivirus expressing neuronal Cx36 or non neuronal Cx43 or Cx31, which belong to three distinct connexin subfamilies with significant differences in amino acid composition in their intracellular domains [41]. To isolate the effects of transduced gap junctions from the contribution of endogenous gap junctions, the experiments were conducted in neuronal cortical cultures prepared from Cx36 knockout mice. On DIV6, cultures were singly transduced with lentivirus expressing WT Cx36, Cx43, Cx31 or Cypridina luciferase (control). As demonstrated in control tests (Fig 2B,2C and 2E), all WT connexin lentiviruses resulted in expression of the corresponding connexins and functional gap junctions. Forty-eight hours after transduction, one group of cultures was treated for 30 minutes with 10 μM NMDA followed by wash out. Another group was subjected to OGD for 30 minutes. Twenty-four hours later, MTT assay was conducted. No neuronal death was observed in NMDA-treated cultures that were infected with the control vector (pCDH-LUC; Fig 7A). This supports the previous observations that NMDAR-mediated excitotoxicity is substantially reduced (or absent) in the absence of neuronal gap junction coupling [14, 18, 19]. However, significant NMDAR-dependent neuronal death was detected in cultures expressing either neuronal (Cx36-) or non-neuronal (Cx43- or Cx31-containing) gap junctions (Fig 7A). Following OGD, some neuronal death was detected in the control, but it was augmented in cultures expressing the various WT connexin channels (Fig 7C). Together, these data suggest that the role of gap junctions in neuronal death is connexin type-independent and point to channel function as the primary mechanism for the contribution of gap junctions to neuronal death.


Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Fontes JD, Ramsey J, Polk JM, Koop A, Denisova JV, Belousov AB - PLoS ONE (2015)

The role of gap junctions in neuronal death is connexin type-independent and requires channel activity.A-D, Statistical data from MTT assay experiments with the use of NMDAR-excitotoxicity (A, B) and OGD (C, D) in neuronal cortical cultures obtained from Cx36 knockout mice are shown. Cultures were transduced with the indicated lentiviruses on DIV6; NMDA and OGD injuries were conducted 48 hours post-transduction; neuronal death analysis was performed 24 hours post-injury. Functional gap junctions made from neuronal and non-neuronal connexins all support neuronal death (A, C). Gap junction channels that are communication-deficient do not support neuronal death (B, D). Statistical analysis: ANOVA with post hoc Tukey; shown relative to (i) pCDH-LUC plus injury (NMDA or OGD) and (ii) pCDH-LUC (without injury); in all figures, n = 8–12 per group; mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446213&req=5

pone.0125395.g007: The role of gap junctions in neuronal death is connexin type-independent and requires channel activity.A-D, Statistical data from MTT assay experiments with the use of NMDAR-excitotoxicity (A, B) and OGD (C, D) in neuronal cortical cultures obtained from Cx36 knockout mice are shown. Cultures were transduced with the indicated lentiviruses on DIV6; NMDA and OGD injuries were conducted 48 hours post-transduction; neuronal death analysis was performed 24 hours post-injury. Functional gap junctions made from neuronal and non-neuronal connexins all support neuronal death (A, C). Gap junction channels that are communication-deficient do not support neuronal death (B, D). Statistical analysis: ANOVA with post hoc Tukey; shown relative to (i) pCDH-LUC plus injury (NMDA or OGD) and (ii) pCDH-LUC (without injury); in all figures, n = 8–12 per group; mean ± SEM.
Mentions: The conductance of ions or small molecules by gap junctions so far measured is roughly within one order of magnitude [38], though more significant differences have been reported for larger permeants and some metabolites, with selective permeability based upon charge and size [39, 40]. It is unlikely, though not impossible, that gap junctions resulting from connexins from distinct subfamilies, with significant amino acid differences, would participate in common protein-protein interactions and signaling pathways. Therefore, if multiple, disparate gap junctions support neuronal death in the same cell death paradigm, the most likely mechanism is channel function, where the molecule or ion “death signal” has sufficient permeability through the tested gap junctions. In our experiments, we used neuronal cell transductions with lentivirus expressing neuronal Cx36 or non neuronal Cx43 or Cx31, which belong to three distinct connexin subfamilies with significant differences in amino acid composition in their intracellular domains [41]. To isolate the effects of transduced gap junctions from the contribution of endogenous gap junctions, the experiments were conducted in neuronal cortical cultures prepared from Cx36 knockout mice. On DIV6, cultures were singly transduced with lentivirus expressing WT Cx36, Cx43, Cx31 or Cypridina luciferase (control). As demonstrated in control tests (Fig 2B,2C and 2E), all WT connexin lentiviruses resulted in expression of the corresponding connexins and functional gap junctions. Forty-eight hours after transduction, one group of cultures was treated for 30 minutes with 10 μM NMDA followed by wash out. Another group was subjected to OGD for 30 minutes. Twenty-four hours later, MTT assay was conducted. No neuronal death was observed in NMDA-treated cultures that were infected with the control vector (pCDH-LUC; Fig 7A). This supports the previous observations that NMDAR-mediated excitotoxicity is substantially reduced (or absent) in the absence of neuronal gap junction coupling [14, 18, 19]. However, significant NMDAR-dependent neuronal death was detected in cultures expressing either neuronal (Cx36-) or non-neuronal (Cx43- or Cx31-containing) gap junctions (Fig 7A). Following OGD, some neuronal death was detected in the control, but it was augmented in cultures expressing the various WT connexin channels (Fig 7C). Together, these data suggest that the role of gap junctions in neuronal death is connexin type-independent and point to channel function as the primary mechanism for the contribution of gap junctions to neuronal death.

Bottom Line: In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect.Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons.A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT
Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

No MeSH data available.


Related in: MedlinePlus