Gap Junction Protein Connexin 43 - an Unexpected Regulator of Brain Endothelial Permeability in Familial Cerebral Cavernous Malformations Type III

Abstract

Familial Cerebral Cavernous Malformations type III (fCCM3) is a disease of the cerebrovascular system caused by loss-of-function mutations in pdcd10 (ccm3) that result in dilated capillary beds susceptible to hemorrhage. No effective pharmacologic treatment is available. Prior to hemorrhage, fCCM3 lesions are characterized by a hyperpermeable blood brain barrier (BBB) that is the key pathologic feature of fCCM3. We have identified that connexin 43 (Cx43), a gap junction (GJ) protein incorporated into the BBB junction complex, is upregulated in lesions of murine model of fCCM3 (ccm3+/-p53-/-). Importantly, we have found that Cx43 is highly expressed in developing lesions permeable to gadolinium diethylenetriaminepentacetate (Gd-DTPA) prior to hemorrhage. In murine brain endothelial cells (mBECs) in vitro, siRNA-mediated ccm3 knockdown (CCM3KD) upregulated Cx43 protein expression, increased GJ plaque size and GJ intracellular communication (GJIC) and increased barrier permeability. Barrier hyperpermeability was blocked by GAP27, a peptide inhibitor of Cx43 GJIC. Cx43 GJs appear to regulate permeability via effects on tight junction (TJ) formation. In particular, TJ protein ZO-1 preferentially accumulates as plaque-like structures at Cx43 GJs, only exhibiting fragmented ZO-1 staining at the TJs along the cell border. Inhibition of Cx43 GJs with GAP27 in CCM3KD cells restored ZO-1 to TJ structures and reduced plaque accumulation at Cx43 GJs. The TJ protein claudin-5 was also fragmented at TJs in CCM3KD cells, and GAP27 treatment lengthened TJ-associated fragments and increased transcellular claudin 5-claudin 5 interaction. We have additionally identified that overexpression of Cx43 and its 20kDa isoform (20-Cx43) in mBECs alone recapitulates many aspects of CCM3KD cells, including increased GJIC, increased hemichannel activity and increased permeability. Full length and 20-Cx43-overexpressing cells also display fragmented ZO-1 TJ staining. Intriguingly we observe that mBECs overexpressing 20-Cx43 have altered endogenous Cx43 and TJ gene transcription as well as a depletion of full length Cx43 from the chromatin-bound cellular compartment that mirrors CCM3KD cells. Overall, we demonstrate that Cx43 expression and GJIC in fCCM3 and Cx43 overexpressing cells regulate barrier permeability by a TJ-dependent mechanism. Importantly, Cx43 GJIC represents a potential target to pursue further in the search for pharmacologic fCCM3 treatments.