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PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy

Acta Pharmacologica Sinica volume 45, pages 1809–1820 (2024)Cite this article

Abstract

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 μM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

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Fig. 1: PHLDA1 upregulation in hippocampal tissue and primary hippocampal neurons after neonatal HI in vivo and in vitro.
Fig. 2: PHLDA1 aggravates acute brain injury and impairs long-term neurological outcomes after neonatal HI.
Fig. 3: PHLDA1 exacerbates neuronal damage and mitochondrial dysfunction after OGD/R in vitro.
Fig. 4: PHLDA1 inhibition improves OGD/R-induced neuronal damage by activating FUNDC1-mediated mitophagy in vitro.
Fig. 5: PHLDA1 inhibition exerts protective effects against neonatal HI brain injury by promoting FUNDC1-mediated mitophagy.

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Acknowledgements

This work was funded by the National Natural Science Foundation of China (82271405, 82071379, 82171703, 82371719), Interdisciplinary Basic Frontier Innovation Program of Suzhou Medical College of Soochow University (YXY2304072); The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (22KJB320021); The Training Program Foundation for health talents of Gusu (GSWS2020052, GSWS2019049); And the work is also funded by the Project of Suzhou Science and Technology Development Plan (SKY2021008; SYS2020154).

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  1. These authors contributed equally: Xiao-lu Jiang, Zu-bin Zhang, Chen-xi Feng, Chen-jie Lin

Authors and Affiliations

  1. Pediatrics Research Institute, Children’s Hospital of Soochow University, Suzhou, 215025, China

    Xiao-lu Jiang, Zu-bin Zhang, Chen-xi Feng, Chen-jie Lin, Hui Yang, Lan-lan Tan, Li-xiao Xu, Gen Li, Tao Pan, Bin Sun, Xing Feng & Mei Li

  2. Department of Neonatology, Children’s Hospital of Soochow University, Suzhou, 215025, China

    Xiao-lu Jiang, Xin Ding & Xing Feng

  3. Soochow Key Laboratory of Prevention and Treatment of Child Brain Injury, Children’s Hospital of Soochow University, Suzhou, 215025, China

    Xiao-lu Jiang & Xing Feng

  4. Jiangsu Key Laboratory for Translational Research and Therapeutics of NeuroPsycho Diseases, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China

    Zu-bin Zhang

  5. Institute of Health Technology, Global Institute of Software Technology, Qingshan Road, Suzhou Science & Technology Tower, Hi-Tech Area, Suzhou, 215163, China

    Zheng-hong Qin

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Contributions

ML and XF conceived and designed the project; XLJ and ZBZ performed the animal model and the cell experiments; CXF and CJL performed the behavioral testing experiments; HY, LLT and XD conducted data acquisition and analyses; LXX, GL, TP and BS contributed essential reagents or tools.ML, XF and ZHQ prepared, wrote, reviewed, and/ or revised the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript. The authors have no conflicts of interest to declare.

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Correspondence to Zu-bin Zhang, Xing Feng or Mei Li.

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Jiang, Xl., Zhang, Zb., Feng, Cx. et al. PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy. Acta Pharmacol Sin 45, 1809–1820 (2024). https://doi.org/10.1038/s41401-024-01292-x

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