The 20 Kilodalton Isoform of Connexin 43: A Multifaceted Contributor to Cerebral Cavernous Malformation Type 3 Pathogenesis

Abstract

Familial cerebral cavernous malformation type III (fCCM3) is a cerebrovascular disease associated with loss-of-function mutations in programmed cell death 10 (PDCD10/CCM3). Vessel dilation, decreased contact between endothelial cells, and loss of junctional complexes result in loss of blood-brain barrier (BBB) integrity and hemorrhagic lesion formation. Ultimately, patient symptoms and complications arise from the leakage of hemorrhagic lesions. While BBB permeability is a key factor driving fCCM3 pathogenesis, the molecular mechanisms driving the loss of BBB integrity in fCCM3 remain unknown.

Our previously published research identifies increased connexin 43 (Cx43) gap junction (GJ) plaque formation as a contributor to fCCM3 pathogenesis. Loss of CCM3 also leads to increased expression of an N-terminally truncated isoform of Cx43 (GJA1-20k), leading us to hypothesize that this Cx43 isoform has a key role in fCCM3 disease progression through its unique signaling roles. To test our hypothesis, we investigated the ability of GJA1-20k to alter the barrier properties and phenotype of endothelial cells. We identify GJA1-20k as a driver of CCM3 brain endothelial barrier permeability, as GJA1-20k expression positively correlates with CCM3 lesion leakage and increases endothelial monolayer permeability through tight junction destabilization. We also characterize GJA1-20k as a novel mediator of transcriptomic and epigenetic changes. GJA1-20k modifies methylation patterns and transcript expression of genes involved in various cell processes, such as angiogenesis and cell adhesion, with gene methylation negatively correlating with the expression of specific genes. Our work also highlights that GJA1-20k mediates comparable transcriptomic and epigenetic profiles to those observed in endothelial cells depleted of CCM3. Finally, we demonstrate that increased ERK1/2 activity following loss of CCM3 leads to increased GJA1-20k expression, with activator protein 1 (AP-1) likely linking ERK1/2 activity to GJA1-20k expression.

Ultimately, this dissertation identifies GJA1-20k as a key contributor to fCCM3 disease pathogenesis, as GJA1-20k promotes brain endothelial barrier permeability in CCM3 and modifies the endothelial cell transcriptome and epigenome. Our work posits GJA1-20k as a promising target in the development of fCCM3 disease treatments.