283 - Hypoxic-Ischemic Injury Augments Inter-α Inhibitor Protein Interactions with GAPDH and Annexin A1 in Cerebral Cortical Nuclei

Publication Number: 4279.283

Xiaodi F. Chen, Women & Infants Hospital of Rhode Island, Providence, RI, United States; Carla A.. Chen, ProThera Biologics, Inc., Barrington, RI, United States; Bingqing Wang, Women & Infants Hospital of Rhode Island, PROVIDENCE, RI, United States; Andre R. Santoso, ProThera Biologics, Barrington, RI, United States; Benjamin Lee, Women & Infants Hospital of Rhode Island, White Plains, NY, United States; Joseph Qiu, ProThera Biologics, Providence, RI, United States; Hang Xing, Women & Infants Hospital of Rhode Island, Providence, RI, United States; Yow-Pin P. Lim, ProThera Biologics, Inc., Providence, RI, United States; Barbara S.. Stonestreet, Brown Univ, Barrington, RI, United States

Background: Hypoxic-ischemic encephalopathy is a major cause of mortality and neurodevelopmental disability. Inter-α inhibitor proteins (IAIPs) are glycoproteins with anti-inflammatory and cytoprotective properties, expressed throughout the brain in neurons and glia. However, their subcellular localization and regulation have not been entirely elucidated. We showed that endogenous IAIPs translocate into neuronal nuclei after ischemia, which suggests critical intracellular functions. We identified GAPDH and Annexin A1 (ANXA1) as potential in vitro binding partners of IAIPs in vitro by LC-MS. GAPDH and ANXA1 are stress-responsive proteins that translocate into the nucleus and regulate oxidative injury, inflammation, and cell death. Interactions between IAIPs and these proteins have not been determined after exposure to HI in the brain.

Objective: To examine endogenous IAIP expression and interactions with GAPDH and ANXA1 after HI in the brain.

Design/Methods: P7 rats were randomly assigned to Sham and HI groups. HI was induced by right carotid artery ligation and 8% O₂ for 90 min. Ipsilateral cortices were collected after cold saline perfusion 72 h after HI for IAIPs by ELISA (pR-21 antibody). Brains were perfused and fixed for histological analysis. Parietal cortical sections were examined by double IHC and proximity ligation assays (PLA) using pR-21 for IAIP expression and IAIP-GAPDH or IAIP-ANXA1 interactions. The PLA signal was quantified as total puncta area (µm2)/DAPI+ cell (Fiji).

Results: Endogenous IAIPs were higher in the brains of HI than Sham rats (P < 0.05; Fig. A-II). IAIPs colocalized mainly with GAPDH in the cytoplasm of Sham rats (Fig. B, arrows, top panel). IAIP-GAPDH colocalization shifted to the nucleus after HI (Fig. B, arrowheads, top panel). A similar pattern was observed for ANXA1 with colocalization of IAIP-ANXA1 mainly in the cytoplasm of Sham and nucleus after HI (Fig. B, arrows vs. arrowheads, lower panel). IAIP-GAPDH PLA puncta were symmetrical and low in Sham hemispheres (Fig. C-I, left panel). PLA puncta were higher in the ipsilateral than contralateral cortex after HI (Fig. C-I, middle panel) and higher in HI than Sham rats (Fig. C-I, right panel; *P < 0.05). IAIP-ANXA1 PLA interactions showed similar patterns, without hemispheric differences in Sham, but with increased ipsilateral puncta and overall increases after HI (Fig. C-II; *P < 0.05).

Conclusion(s): Endogenous IAIPs increase after exposure to HI in neonates and exhibit increased nuclear interactions with GAPDH and ANXA1. These findings suggest novel mechanisms for intracellular IAIPs in HI-related brain injury.

Endogenous IAIP Expression, Subcellular Colocalization, and Nuclear Interactions with GAPDH and Annexin A1 after HI Brain Injury in Neonatal Rats
Figure A. IAIP expression in neonatal brain. (I) Representative immuno-fluorescence images show endogenous IAIP expression at P10 in the rat brain 72 h after exposure to HI. IAIP fluorescence intensity was higher in the ipsilateral (right) brain after HI compared with the contralateral non-ischemic (left) side (open arrows, top and bottom panels). (II) ELISA quantification demonstrates significant increases in IAIP levels 72 h after exposure to HI vs. Sham brains (*P <0.05). Statistical analysis: Mann-Whitney test.

Figure B. Subcellular colocalization of IAIPs with GAPDH and Annexin A1. In the Sham cortex, IAIPs (green) co-localize with GAPDH (red) in the cytoplasm (arrows, top panels). After HI, IAIP-GAPDH co-localization shifts into the nucleus (arrowheads, top panel), confirmed by DAPI (blue). In the Sham cortex, IAIPs also co-localize with Annexin A1 (red) in the cytoplasm (arrow, bottom panel). After HI, IAIP-Annexin A1 co-localization shifts into the nucleus (arrowheads, bottom panel), confirmed by DAPI (blue).

Figure C. Nuclear IAIP-GAPDH and IAIP-Annexin A1 interactions by PLA. (I) PLA puncta representing IAIP-GAPDH proximity ( <40 nm) were minimal and symmetric between hemispheres in Sham rats (left panel) but markedly increased in the ipsilateral cortex after HI (right panel). Quantification shows significantly higher right/left PLA ratios in HI vs. Sham rats (*P <0.05, right panel). (II) Similarly, PLA puncta representing IAIP-Annexin A1 proximity were minimal and symmetric between hemispheres in Sham rats (left panel) but substantially increased in the ipsilateral cortex after HI (right panel). Quantification demonstrates a significantly higher right/left PLA ratio in HI compared with Sham rats (*P < 0.05, right panel). Statistical analysis: Mann-Whitney test.

Note: Sample sizes (males and females combined) are shown. Data are presented as box plots (open box = Sham; red box = HI) with medians, interquartile ranges (25th/75th), 10th/90th percentiles, and individual data points; n values are indicated in the panels. Statistical analyses and plotting were performed using GraphPad Prism 10 and R. Scale bars are shown on images.