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

Expression of connexins with amino acid substitutions results in formation of communication-deficient gap junction channels.Data from MTT assay (A), western blot (B), immunostaining (C) and scrape-loading dye transfer (D) experiments in transduced Cx36 knockout mouse neuronal cortical cultures (A, B) and stably-transduced HeLa cells (C, D) are shown. A, Lentiviral transductions reduced neuronal survival over a three day period (statistical analysis: Student's t-test, shown relative to non-infected {Control} cultures; n = 4 per group; mean ± SEM). However, no statistical difference in neuronal survival between the control lentivirus and each of the other transductions was detected (P>0.05, not shown; Student's t-test). B, C, Lentiviral transductions induce mutant Cx36 and Cx43 proteins (B) that are expressed in the cell’s plasma membrane (C). The mutant connexins are recognized by the WT connexin antibodies. D, Cells expressing mutant connexin do not pass Neurobiotin. In all experiments, cell transduction conditions, timing, analyses, antibodies (for Cx36 and Cx43) and image presentations are as in Fig 2. In B-D, the images are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0125395.g004: Expression of connexins with amino acid substitutions results in formation of communication-deficient gap junction channels.Data from MTT assay (A), western blot (B), immunostaining (C) and scrape-loading dye transfer (D) experiments in transduced Cx36 knockout mouse neuronal cortical cultures (A, B) and stably-transduced HeLa cells (C, D) are shown. A, Lentiviral transductions reduced neuronal survival over a three day period (statistical analysis: Student's t-test, shown relative to non-infected {Control} cultures; n = 4 per group; mean ± SEM). However, no statistical difference in neuronal survival between the control lentivirus and each of the other transductions was detected (P>0.05, not shown; Student's t-test). B, C, Lentiviral transductions induce mutant Cx36 and Cx43 proteins (B) that are expressed in the cell’s plasma membrane (C). The mutant connexins are recognized by the WT connexin antibodies. D, Cells expressing mutant connexin do not pass Neurobiotin. In all experiments, cell transduction conditions, timing, analyses, antibodies (for Cx36 and Cx43) and image presentations are as in Fig 2. In B-D, the images are representative of three independent experiments.

Mentions: Next we characterized the cells expressing Cx36 (L10A/Q15A, L10P/Q15P or I22R) or Cx43 (G21R) containing amino acid substitutions. As with the WT proteins, each of the four mutant connexin expressing lentiviruses effectively transduced neurons in Cx36 knockout neuronal cortical cultures (>90% transduction efficiency; Fig 3). The transductions reduced neuronal survival during a three day period, however, there was no statistical difference in neuronal survival between cultures transduced with the control and mutant connexin lentiviruses (Fig 4A). Western blots showed expression of the mutant connexins in neuronal cultures (Fig 4B) and their stable expression in HeLa cells (not shown) (the amino acid substitutions did not affect antibody recognition of the proteins). In stably-transduced HeLa cells, the mutant connexins successfully reached the plasma membrane (Fig 4C). In scrape scrape-loading tests, the number Neurobiotin-positive, but Alexa Fluor 594-negative cells per microscope field was 2.3±0.7 for pCDH-Cx36(AA) cultures, 3.3±1.2 for pCDH-Cx36(PP), 2.3±1.9 for pCDH-Cx36(I22R) and 3.3±0.9 for pCDH-Cx43(G21R) group (Fig 4D). None of these values was statistically different from that of the control group, i.e., transduced with pCDH-LUC (5.0±1.6; analyzed using two-tailed unpaired Student's t-test; n = 3 in each group). The data indicate that the mutant gap junction channels are impermeable to Neurobiotin and therefore do not support gap junctional communication between cells.


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)

Expression of connexins with amino acid substitutions results in formation of communication-deficient gap junction channels.Data from MTT assay (A), western blot (B), immunostaining (C) and scrape-loading dye transfer (D) experiments in transduced Cx36 knockout mouse neuronal cortical cultures (A, B) and stably-transduced HeLa cells (C, D) are shown. A, Lentiviral transductions reduced neuronal survival over a three day period (statistical analysis: Student's t-test, shown relative to non-infected {Control} cultures; n = 4 per group; mean ± SEM). However, no statistical difference in neuronal survival between the control lentivirus and each of the other transductions was detected (P>0.05, not shown; Student's t-test). B, C, Lentiviral transductions induce mutant Cx36 and Cx43 proteins (B) that are expressed in the cell’s plasma membrane (C). The mutant connexins are recognized by the WT connexin antibodies. D, Cells expressing mutant connexin do not pass Neurobiotin. In all experiments, cell transduction conditions, timing, analyses, antibodies (for Cx36 and Cx43) and image presentations are as in Fig 2. In B-D, the images are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0125395.g004: Expression of connexins with amino acid substitutions results in formation of communication-deficient gap junction channels.Data from MTT assay (A), western blot (B), immunostaining (C) and scrape-loading dye transfer (D) experiments in transduced Cx36 knockout mouse neuronal cortical cultures (A, B) and stably-transduced HeLa cells (C, D) are shown. A, Lentiviral transductions reduced neuronal survival over a three day period (statistical analysis: Student's t-test, shown relative to non-infected {Control} cultures; n = 4 per group; mean ± SEM). However, no statistical difference in neuronal survival between the control lentivirus and each of the other transductions was detected (P>0.05, not shown; Student's t-test). B, C, Lentiviral transductions induce mutant Cx36 and Cx43 proteins (B) that are expressed in the cell’s plasma membrane (C). The mutant connexins are recognized by the WT connexin antibodies. D, Cells expressing mutant connexin do not pass Neurobiotin. In all experiments, cell transduction conditions, timing, analyses, antibodies (for Cx36 and Cx43) and image presentations are as in Fig 2. In B-D, the images are representative of three independent experiments.
Mentions: Next we characterized the cells expressing Cx36 (L10A/Q15A, L10P/Q15P or I22R) or Cx43 (G21R) containing amino acid substitutions. As with the WT proteins, each of the four mutant connexin expressing lentiviruses effectively transduced neurons in Cx36 knockout neuronal cortical cultures (>90% transduction efficiency; Fig 3). The transductions reduced neuronal survival during a three day period, however, there was no statistical difference in neuronal survival between cultures transduced with the control and mutant connexin lentiviruses (Fig 4A). Western blots showed expression of the mutant connexins in neuronal cultures (Fig 4B) and their stable expression in HeLa cells (not shown) (the amino acid substitutions did not affect antibody recognition of the proteins). In stably-transduced HeLa cells, the mutant connexins successfully reached the plasma membrane (Fig 4C). In scrape scrape-loading tests, the number Neurobiotin-positive, but Alexa Fluor 594-negative cells per microscope field was 2.3±0.7 for pCDH-Cx36(AA) cultures, 3.3±1.2 for pCDH-Cx36(PP), 2.3±1.9 for pCDH-Cx36(I22R) and 3.3±0.9 for pCDH-Cx43(G21R) group (Fig 4D). None of these values was statistically different from that of the control group, i.e., transduced with pCDH-LUC (5.0±1.6; analyzed using two-tailed unpaired Student's t-test; n = 3 in each group). The data indicate that the mutant gap junction channels are impermeable to Neurobiotin and therefore do not support gap junctional communication between cells.

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