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Complexin 2 contributes to the protective effect of NAD+ on neuronal survival following neonatal hypoxia-ischemia

Acta Pharmacologica Sinica volume 46, pages 2363–2375 (2025)Cite this article

Abstract

Nicotinamide adenine dinucleotide (NAD) is a key coenzyme involved in cell metabolism associated with aging, cancer, neurodegenerative diseases and metabolic disorders. We recently showed that NAD+ therapy significantly improved neurobehavioral outcomes in neonatal mice after hypoxia-ischemia (HI), and bioinformatics analysis revealed that the expression of complexin 2 (CPLX2) in the injured cerebral cortex was significantly decreased 24 h after HI injury but could be reversed by NAD+ intervention. In this study we explored the role of CPLX2 in the survival and function of neonatal hypoxic-ischemic cortical neurons. HI models were established by permanent ligation of the left common carotid artery in mice. CPLX2-knockdown lentiviral vector was injected intraventricularly on postnatal day 1 (P1); CPLX2 knockout mice were also used. NAD+ (5 mg·kg−1·d−1, i.p.) was administered before HI surgery, thereafter once a day until sampling. We showed that NAD+ administration significantly ameliorated the morphological damages and neurobehavioral defects, and elevated the seizure thresholds in HI mice. All the beneficial effects of NAD+ were abolished by CPLX2 knockdown or knockout. In HT22 neuronal cells subjected to OGD/R, pretreated with NAD+ (100 μM) for 12 h significantly increased the cell viability, decreased the LDH levels, and inhibited the ferroptosis evidenced by the changes in redox-related parameters including concentrations of Fe2+, GSH, MDA, H2O2 as well as the expression of GPX4 and SLC7A11. CPLX2 knockdown in HT22 neuronal cells blocked the protective effects of NAD+ as in HI mice, whereas CPLX2 overexpression enhanced the inhibitory effects of NAD+ on ferroptosis in HT22 neuronal cells.

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Fig. 1: The flowchart of the study.
Fig. 2: Expression of CPLX2 protein in brain tissues of 10-day-old mice.
Fig. 3: Knockdown of CPLX2 reversed the effect of NAD on inhibiting pathological morphological damage in HI mice.
Fig. 4: CPLX2 knockout reversed the effect of NAD+ on neurobehavioral evaluation and seizure threshold evaluation.
Fig. 5: Neurobehavioral evaluation in mice of CPLX2 knockout mice.
Fig. 6: CPLX2 knockdown reversed the impact of NAD+ on ferroptosis in HT22 cells.
Fig. 7: CPLX2 overexpression enhanced the inhibitory effects of NAD+ on ferroptosis in HT22 cells.
Fig. 8: CPLX2 contributes to the protective effect of NAD+ on neuron survival following neonatal hypoxia-ischemia knockdown or knockout of CPLX2 significantly inhibited the protective effects of NAD+ on the pathological morphology and long-term neurobehavioral injury phenotype caused by HI.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 62127810, 81871024 and 82471742).

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  1. These authors contributed equally: Xiao-wen Xu, Xiu-wen Zhou, Li Zhang

Authors and Affiliations

  1. Department of Brain Science, Institute of Pediatric Research, Children’s Hospital of Soochow University, Suzhou, 215025, China

    Xiao-wen Xu, Xiu-wen Zhou, Li Zhang, Qing Wang, Xin-xin Wang, Yi-ming Jin, Li-li Li, Mei-fang Jin & Hong Ni

  2. Department of Endocrinology, Genetics and Metabolism, Children’s Hospital of Soochow University, Suzhou, 215025, China

    Xiao-wen Xu & Hai-ying Wu

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

    Xin Ding

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Contributions

HN conceived the whole study and designed experiments. HN, XD and HYW jointly supervised this work. XWX, XWZ and LZ designed and performed most of the experiments and analyzed data. XWX and XWZ performed experiments on mice. XWX and LZ performed experiments on cells. QW, XXW, YMJ, LLL and MFJ helped with the analysis of data. XWX wrote and revised the manuscript. HN revised the manuscript.

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Correspondence to Hai-ying Wu, Xin Ding or Hong Ni.

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Xu, Xw., Zhou, Xw., Zhang, L. et al. Complexin 2 contributes to the protective effect of NAD+ on neuronal survival following neonatal hypoxia-ischemia. Acta Pharmacol Sin 46, 2363–2375 (2025). https://doi.org/10.1038/s41401-025-01555-1

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