UNRAVELING THE ROLE OF POLDIP2 IN BRAIN ENDOTHELIAL CELL CYTOSKELETAL ORGANIZATION

Background and aims: Polymerase δ-interacting protein 2 (Poldip2) has been implicated in increasing leukocyte trafficking into the central nervous system and promoting blood–brain barrier (BBB) permeability. However, how Poldip2 influences brain endothelial cells (ECs)—the principal component of the BBB—remains poorly understood. We hypothesize that Poldip2 controls cytoskeletal organization and intercellular junctions in brain ECs to regulate barrier integrity and BBB permeability.
Methods: [GK1] [ZZ2] Endogenous Poldip2 in rat brain microvascular endothelial cells (RBMECs) was silenced using siRNA, and endothelial barrier function was assessed by transendothelial electrical resistance (TEER) assays following TNF-α (10 ng/mL) stimulation. Phosphorylated myosin light chain (p-MLC), a marker of cytoskeletal contractile signaling, was analyzed by western blot. To evaluate how Poldip2 domains regulate cytoskeletal remodeling, RBMECs were transduced with adenoviruses encoding full-length Poldip2 or deletion mutants (AdΔ64, lacking the YccV-like domain; AdΔ231, lacking the DUF525 domain). After 48 h, stress fiber formation was visualized by phalloidin staining, and contractility signaling was quantified as above. Poldip2–MLC interaction sites were mapped by immunoprecipitation using the same constructs.
Results: siRNA-mediated Poldip2 knockdown in RBMECs reduced p-MLC levels and attenuated TNFα-induced barrier disruption measured by TEER. Conversely, overexpression of full-length Poldip2 increased basal p-MLC at 48 hours post-transduction, and phalloidin staining revealed enhanced stress fiber formation. Full-length Poldip2 and deletion mutants modulate total MLC expression and endothelial barrier function. Immunoprecipitation localized MLC binding to the YccV-like domain of Poldip2.
Conclusion: Poldip2 supports TNFα-mediated actin remodeling, a process linked to barrier dysfunction and heightened permeability.