Graduate Student Chelsea Phillips Research Recognitions

By Lynn McCain | January 26 2022

Congratulations to Chelsea Phillips, a graduate student in the Neurosciences graduate program. Mentored by Dr. Anuska Andjelkovic-Zochowski, Chelsea received honors on work presented at two different meetings in late 2021. The first was a Junior Scientist Poster award for her poster “A 20kDa isoform of Connexin-43 (Cx43-20kDa) mediates transcriptomic and epigenetic changes in brain endothelial cells" presented at the 23rd International Brain Barriers SocietyChelsea Phillips Meeting, held virtually on September 22-24, 2021.  The second was an oral presentation of her work, “The role of a 20kDa isoform of Connexin-43 (Cx43-20kDa) in Cerebral Cavernous Malformation 3” presented at the Cerebral Cavernous Malformation (CCM) Scientific Meeting, also held virtually on November 11-12, 2021. Phillips began her graduate studies in 2017 and joined the Andjelkovic-Zochowski lab in 2018.  Her long-term goal is to continue in academics as a researcher within the field of cerebrovascular biology, specifically combining molecular biology with bioinformatics to study the molecular mechanisms driving cerebrovascular injury. 

As Phillips explained the research presented in the award-winning poster, connexin-43 (Cx43) is a gap junction protein that has six N-terminally truncated isoforms. The 20 kilodalton isoform (Cx43-20kDa) has the highest expression and is involved in various cellular processes. Previous research from the Andjelkovic-Zochowski lab demonstrates that Cx43-20kDa alters the phenotype of mouse brain endothelial cells (mBECs); for example, Cx43-20kDa overexpression alters junctional complex protein expression and organization. This study’s objective was to determine the mechanism through which Cx43-20kDa alters the phenotype of brain endothelial cells.

In this project, Chelsea and other experts within the lab conducted mRNA-sequencing (RNA-seq) with control mBECs and Cx43-20kDa-overexpressing mBECs , and found that Cx43-20kDa overexpression alters the endothelial cell transcriptome. Differentially expressed genes are involved in immune processes, cell-cell junctions, and the extracellular matrix. Interestingly, RNA-seq analysis demonstrated that Cx43-20kDa overexpression decreases transcript expression of an epigenetic enzyme, DNA methyltransferase 3A (DNMT3A), which was confirmed through western blot. She investigated DNA methylation patterns in Cx43-20kDa-overexpressing mBECs through reduced representation bisulfite sequencing (RRBS), and found that Cx43-20kDa overexpression alters the endothelial cell methylome, with differently methylated cytosine residues located on genes encoding junction proteins. Collectively, these data demonstrate that Cx43-20kDa mediates transcriptomic and epigenetic changes in brain endothelial cells. 

Chelsea then detailed the important work she presented at the CCM meeting. Cerebral cavernous malformation type III (CCM3), a cerebrovascular disease caused by loss-of-function mutations in the gene encoding programmed cell death 10 (PDCD10/CCM3), is characterized by loss of blood-brain barrier integrity, resulting in hemorrhagic lesions. Previous research from the Andjelkovic-Zochowski lab demonstrates that loss of CCM3 leads to increased expression of connexin-43 (Cx43), a gap junction protein, and a 20 kilodalton isoform of Cx43 (Cx43-20kDa). Cx43-20kDa is a N-terminally truncated Cx43 isoform that is involved in various cellular processes. Overexpression of Cx43-20kDa in mouse brain endothelial cells (mBECs) recapitulates the phenotype of CCM3-depleted mBECs, including increased Cx43 expression, increased gap junction intercellular communication, and tight junction (TJ) complex reorganization. The aim of our study was to investigate the role of Cx43-20kDa in brain endothelial barrier permeability and CCM3 lesion leakage.

The role of Cx43-20kDa in brain endothelial barrier permeability and CCM3 lesion leakage was studied with in vitro and in vivo CCM3 models. They discovered that Cx43-20kDa overexpression results in TJ complex reorganization, as PLA and STORM microscopy demonstrate aberrant TJ complex interactions, localization, and membrane-associated fragment length. RNA-seq analysis reveals five differentially expressed genes (DEGs) common across experimental groups: Serpinb9c, Gpr137b, Trib3, Ccn3, and Plat. MRI and western blot demonstrates a positive correlation between lesion permeability and Cx43-20kDa expression, along with lesion volume and Cx43-20kDa expression. Together, these data demonstrate that Cx43-20kDa overexpression reorganizes TJ complexes, alters the mBEC transcriptome, and positively correlates with CCM3 lesion leakage, highlighting a potential molecular target for novel CCM3 treatments.