POLDIP2 Regulates Cerebral Vascular Permeability by Modulating ROS-Mediated ZO-1 Phosphorylation and Localization

Background

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS), composed of brain endothelial cells, astrocytes, pericytes, and neurons. Brain endothelial cells form a continuous physical barrier through tight junctions (TJs), regulating the permeability of substances from the blood to the brain. The main components of TJs include transmembrane proteins (such as occludin and claudins) and scaffolding proteins (such as ZO-1, ZO-2, and ZO-3), with ZO-1 playing a key role in maintaining the structure and function of TJs. Studies have shown that oxidative stress induces tyrosine phosphorylation of ZO-1, leading to its dissociation from intercellular junctions and thereby disrupting BBB integrity.

POLDIP2 (Polymerase Delta-Interacting Protein 2) is a multifunctional protein initially identified for its interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA). Recent studies have demonstrated that POLDIP2 plays an important role in regulating mitochondrial reactive oxygen species (ROS) production and cytoskeletal dynamics. However, the role of POLDIP2 in brain endothelial cells and its regulatory mechanisms on BBB permeability remain unclear. This study aims to elucidate the molecular mechanisms by which POLDIP2 regulates cerebral endothelial cell permeability through the phosphorylation and localization of ZO-1.

Source of the Paper

This paper was co-authored by Keke Wang, Hongyan Qu, Ruinan Hu, and others, with the research team affiliated with Emory University School of Medicine, Xi’an Jiaotong University, and other institutions. The paper was published in 2025 in the journal Cell Communication and Signaling, titled “Polymerase delta-interacting protein 2 mediates brain vascular permeability by regulating ROS-mediated ZO-1 phosphorylation and localization at the interendothelial border.”

Research Process and Results

1. Construction and Validation of Endothelial-Specific POLDIP2 Knockout Mouse Model

The research team first constructed endothelial-specific POLDIP2 knockout mice (POLDIP2 EC-/-) and evaluated their impact on BBB permeability using a cerebral ischemia model. Experimental results showed that Evans Blue dye extravasation was significantly reduced in POLDIP2 EC-/- mice after cerebral ischemia, indicating that POLDIP2 knockout protects BBB integrity.

2. Effect of POLDIP2 Knockdown on Cerebral Endothelial Cell Permeability

In in vitro experiments, the research team used rat brain microvascular endothelial cells (RBMECs) as a model, knocked down POLDIP2 expression using siRNA, and assessed its impact on tumor necrosis factor α (TNF-α)-induced endothelial cell permeability. The results showed that POLDIP2 knockdown significantly inhibited TNF-α-induced increases in endothelial cell permeability. Further immunofluorescence experiments demonstrated that POLDIP2 knockdown prevented TNF-α-induced dissociation of ZO-1 from intercellular junctions.

3. Effect of POLDIP2 Overexpression on Cerebral Endothelial Cell Permeability and ZO-1 Localization

To further validate the role of POLDIP2, the research team overexpressed POLDIP2 in RBMECs using an adenoviral vector. Experimental results showed that POLDIP2 overexpression significantly increased endothelial cell permeability and reduced ZO-1 localization at intercellular junctions. Additionally, POLDIP2 overexpression significantly increased intracellular H2O2 production.

4. Inhibition of POLDIP2-Induced ZO-1 Dissociation by the Antioxidant NAC

To verify the role of ROS in POLDIP2-mediated regulation of ZO-1, the research team treated POLDIP2-overexpressing RBMECs with the antioxidant N-acetylcysteine (NAC). Experimental results showed that NAC treatment significantly inhibited POLDIP2-induced ZO-1 dissociation, indicating that POLDIP2 regulates ZO-1 localization through a ROS-mediated mechanism.

5. POLDIP2-Induced Tyrosine Phosphorylation of ZO-1

Through co-immunoprecipitation experiments, the research team found that POLDIP2 overexpression induced tyrosine phosphorylation of ZO-1, and this process could be inhibited by NAC or the mitochondrial ROS scavenger Mitotempo. These results suggest that POLDIP2 regulates endothelial cell permeability through mitochondrial ROS-mediated tyrosine phosphorylation of ZO-1.

Conclusions and Significance

This study is the first to reveal the molecular mechanism by which POLDIP2 regulates cerebral endothelial cell permeability through mitochondrial ROS production and ZO-1 tyrosine phosphorylation. The findings indicate that POLDIP2 disrupts BBB integrity by increasing ROS production and inducing ZO-1 phosphorylation and dissociation under pathological conditions such as cerebral ischemia. This discovery provides a theoretical basis for developing novel therapeutic strategies targeting POLDIP2 and its downstream signaling pathways, which may have significant implications for the treatment of neurological diseases such as cerebral ischemia and brain edema.

Research Highlights

1. Innovative Mechanism: First to reveal that POLDIP2 regulates cerebral endothelial cell permeability through mitochondrial ROS-mediated ZO-1 phosphorylation.
2. Comprehensive Validation: Combined in vivo and in vitro experiments to comprehensively validate the role of POLDIP2 in BBB regulation.
3. Potential Therapeutic Target: POLDIP2 and its downstream signaling pathways may serve as new targets for treating cerebral ischemia, brain edema, and other diseases.

Additional Valuable Information

This study also provides detailed experimental methods and data analysis procedures, including RNA extraction and qPCR, immunofluorescence staining, and co-immunoprecipitation techniques, offering important references for researchers in related fields. Additionally, the research team developed an adenovirus-based POLDIP2 overexpression system, providing a powerful tool for subsequent studies.