Cx43 carboxyl terminal domain determines AQP4 and Cx30 endfoot organization and blood brain barrier permeability

Abstract

The neurovascular unit (NVU) consists of cells intrinsic to the vessel wall, the endothelial cells and pericytes, and astrocyte endfeet that surround the vessel but are separated from it by basement membrane. Endothelial cells are primarily responsible for creating and maintaining blood–brain-barrier (BBB) tightness, but astrocytes contribute to the barrier through paracrine signaling to the endothelial cells and by forming the glia limitans. Gap junctions (GJs) between astrocyte endfeet are composed of connexin 43 (Cx43) and Cx30, which form plaques between cells. GJ plaques formed of Cx43 do not diffuse laterally in the plasma membrane and thus potentially provide stable organizational features to the endfoot domain, whereas GJ plaques formed of other connexins and of Cx43 lacking a large portion of its cytoplasmic carboxyl terminus are quite mobile. In order to examine the organizational features that immobile GJs impose on the endfoot, we have used super-resolution confocal microscopy to map number and sizes of GJ plaques and aquaporin (AQP)-4 channel clusters in the perivascular endfeet of mice in which astrocyte GJs (Cx30, Cx43) were deleted or the carboxyl terminus of Cx43 was truncated. To determine if BBB integrity was compromised in these transgenic mice, we conducted perfusion studies under elevated hydrostatic pressure using horseradish peroxide as a molecular probe enabling detection of micro-hemorrhages in brain sections. These studies revealed that microhemorrhages were more numerous in mice lacking Cx43 or its carboxyl terminus. In perivascular domains of cerebral vessels, we found that density of Cx43 GJs was higher in the truncation mutant, while GJ size was smaller. Density of perivascular particles formed by AQP4 and its extended isoform AQP4ex was inversely related to the presence of full length Cx43, whereas the ratio of sizes of the particles of the AQP4ex isoform to total AQP4 was directly related to the presence of full length Cx43. Confocal analysis showed that Cx43 and Cx30 were substantially colocalized in astrocyte domains near vasculature of truncation mutant mice. These results showing altered distribution of some astrocyte nexus components (AQP4 and Cx30) in Cx43 null mice and in a truncation mutant, together with leakier cerebral vasculature, support the hypothesis that localization and mobility of gap junction proteins and their binding partners influences organization of astrocyte endfeet which in turn impacts BBB integrity of the NVU.

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

“…; Cx30 KO GFAP-Cre + littermates: 31,692 ± 5681 a.u. ; n = 3–5), as reported by others ( 6 , 32 ); under high-pressure conditions leakage may occur, however ( 6 , 33 ).…”

Connexins Signatures of the Neurovascular Unit and Their Physio-Pathological Functions

“…Astrocytic end feet are connected by GJs composed of Cx43 and Cx30 both temporally and spatially expressed [ 39 , 56 , 57 ]. The consequential homo-cellular (i.e., astrocyte–astrocyte) or hetero-cellular communication represents one of the crucial interaction checkpoints available to modulate NVU functions and CNS homeostasis [ 58 ].…”

“…The high capability of endothelial cells to dynamically modulate their Cxs expression profile and the permeability of the channels they are forming is significant. Indeed, even if no direct contact between astrocytes and endothelial cells occurs in the NVU, these cells can interact in an in vitro coculture system [ 57 , 118 , 119 , 120 ]. The capability of endothelial and glial cells to effect such plastic changes, shaped the idea that they are able to modulate intercellular communication, ion homeostasis, and paracrine mediators release in a context dependent manner [ 113 , 114 , 115 ].…”

Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function

“…Interestingly, Cx43 downregulation is also induced by SARS-CoV-2 Spike and is accompanied by decreased AJs proteins, resulting in hyperpermeability in primary brain vascular ECs, and is more pronounced in diabetic brain vascular ECs [194]. Since the endothelial barrier also depends on other cells, the perivascular organization of Cx43 GJ channels in astrocytes contributes to maintaining vascular communication and the BBB equilibrium [195]. Cx43 hemichannel participation was evaluated by the short-time exposure of Cx43 peptide Gap27, inhibiting BK-triggered [Ca 2+ ] i oscillations and BBB permeability in mouse brain ECs [147] and in human brain endothelial cells [159].…”