Targeting gliovascular connexins prevents inflammatory blood-brain barrier leakage and astrogliosis
Marijke De Bock, Maarten De Smet, Stijn Verwaerde, Hanane Tahiri, Steffi Schumacher, Valérie Van Haver, Katja Witschas, Christian Steinhäuser, Nathalie Rouach, Roosmarijn E. Vandenbroucke, Luc Leybaert
Marijke De Bock, Maarten De Smet, Stijn Verwaerde, Hanane Tahiri, Steffi Schumacher, Valérie Van Haver, Katja Witschas, Christian Steinhäuser, Nathalie Rouach, Roosmarijn E. Vandenbroucke, Luc Leybaert
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Research Article Inflammation Neuroscience

Targeting gliovascular connexins prevents inflammatory blood-brain barrier leakage and astrogliosis

Abstract

The blood-brain barrier is formed by capillary endothelial cells expressing connexin 37 (Cx37), Cx40, and Cx43 and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS administration increased endothelial and astrocytic Cx43 expression; elevated TNF-α, IL-1β, IFN-γ, and IL-6 in plasma and IL-6 in the brain; and induced barrier leakage recorded over 24 hours. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double knockout in astrocytes, slightly diminished by endothelial Cx43 knockout, and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 peptides just before LPS also prevented barrier leakage, and intravenously administered BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19, and BAPTA. LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL-6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.

Authors

Marijke De Bock, Maarten De Smet, Stijn Verwaerde, Hanane Tahiri, Steffi Schumacher, Valérie Van Haver, Katja Witschas, Christian Steinhäuser, Nathalie Rouach, Roosmarijn E. Vandenbroucke, Luc Leybaert

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Figure 6

Voltage-, [Ca2+]i-, and LPS-dependent activation of Cx43 hemichannels in RBE4 cells.

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Voltage-, [Ca2+]i-, and LPS-dependent activation of Cx43 hemichannels in...
(A) Example traces depicting unitary current activities during 30-second voltage steps under conditions of 50 and 250 nM [Ca2+]i. At 250 nM, activity was present at both positive and negative voltages, which was inhibited by Gap19 and Gap27. (B) I-V plot for 250 nM unitary currents, demonstrating a slope conductance of 228 ± 1 pS (ncells = 48, 5 independent experiments), characteristic for Cx43 hemichannels. (C) All-point histograms for unitary activities and Qm summary data at –60 and +60 mV (color codes as in A; ncells = 10–31 per condition, 5 independent experiments), showing [Ca2+]i-dependent current activation that is blocked by Gap19 and Gap27 (1-way ANOVA, Bonferroni test). (D and E) Consecutive current traces obtained at –50 mV (60 seconds) with and without 1 μg/mL LPS and corresponding all-point histogram (E). LPS induced periodic burst opening of about 210 pS unitary current activity (O1 in histogram), including a longer lasting substate of about 60 pS (S in histogram). The activity was blocked by Gap19 or 10 mM BAPTA in the patch pipette or bath-applied Gap27 (30 minutes’ preincubation). C, closed state. (F) Qm summary data of D (ncells = 8 per condition; 4 independent experiments). Stars compare LPS time points versus baseline at 60 seconds (repeated measures ANOVA, Dunnett test). Colored symbols compare LPS with control without LPS (green) and interventions with Gap27 (purple), Gap19 (red), or BAPTA (blue) at the last 600-second time point (1-way ANOVA, Dunnett test). *P < 0.05, **P < 0.01, ***P < 0.001, ##P < 0.01, $$P < 0.01, §§P < 0.01, &&P < 0.01.