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

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.

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE), a subset of neonatal encephalopathy, is caused by perinatal hypoxia-ischemia (HI). The global incidence of HIE is approximately 0.1%–0.8%, with the incidence in developed countries estimated to be 0.15%, and as high as 2.6% in less developed countries [1–3]. Neonatal hypoxic-ischemic brain injury (HIBI) is a significant concern in newborns, often leading to long-term neurological deficits. And approximately 25% of surviving children develop permanent neurological deficits, such as cerebral palsy, epilepsy, hyperactivity, cognitive impairment and learning difficulties. The degree of brain injury can worsen over time, substantial burden on affected families and the society as a whole [4].

Therapeutic hypothermia (TH) is often used for brain protection after HIBI; however, more than one-quarter of survivors still suffer from severe developmental disorders [5]. In particular, there are different opinions regarding the prognosis of brain injury after rewarming following TH, such as a short treatment window and limited efficacy [6]. A recent retrospective cohort study by Szakmar et al. from the Harvard Medical School found that cerebral oxygen saturation (CrSO₂) was significantly increased in infants with brain injury from the first to the second day of TH, and a significant increase in CrSO₂ after rewarming was correlated with MRI brain injury parameters [7], indicating that brain injury during the rewarming period after TH may be related to excessive CrSO₂. An observational study conducted by the Department of Pediatrics at Harvard Medical School and the Department of Neonatal Medicine at Boston Children’s Hospital investigated the effects of TH treatment on cerebral blood flow and metabolism. The results showed that during TH, the hemoglobin oxygen saturation (SO₂) of newborns with HIE was significantly higher than that of age-matched control newborns. Furthermore, compared with the TH period, SO₂ remained unchanged after TH [8], indicating the presence of excessive SO₂ during the rewarming period after TH treatment for neonatal HIE.

Donadello et al. [9] reported a significant increase in cerebral oxygenation following hypothermic treatment in healthy pigs, which elevated the lactate/pyruvate ratio and metabolic levels, indicating that high oxygen levels during the rewarming phase may exacerbate brain damage. Therefore, it is important to identify new treatment strategies to decrease or prevent hypoxia caused by microcirculatory damage rather than inducing high oxygen levels. And ferroptosis has been reported to play a crucial role in HIE and serves as a potential therapeutic target [10, 11].

The recently identified anti-hypoxic property of nicotinamide adenine dinucleotide (NAD) has attracted much attention as a potential treatment strategy for HI brain injury and AD [12, 13]. Mechanistically, the two cellular forms of NAD, oxidized (NAD⁺) and reduced (NADH), play a coenzymatic role in redox reactions, and their imbalanced ratio can damage the synthesis of adenosine triphosphate (ATP) in the electron transport chain (ETC), leading to dysregulated cellular metabolism [14]. NAD inhibits mitochondrial division and reactive oxygen species (ROS) production by regulating cellular redox reactions and metabolic homeostasis, thereby exerting neuroprotective effects against cerebral ischemia [15]. Supplementation with exogenous NAD⁺ or its precursors improves the mitochondrial function [16] and prevents the development of neurodegenerative diseases [13, 17]. We recently demonstrated in a transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) model of adult mice that NAD⁺ alleviates infarct volume, brain edema, and neurological deficits by upregulating ATP levels and reducing lactate content [18]. However, there are significant differences in the distribution of neuronal receptors and excitotoxicity mechanisms between the neonatal and adult brains. Permanent MCAO ischemia in adult mice and neonatal hypoxia/ischemia may result in different cell death mechanisms and injury phenotypes, and drug interventions may have different effects on neonatal and adult brain injuries [19, 20]. Therefore, the effects of NAD⁺ on neonatal HIBI and its potential molecular mechanisms require further investigation.

Supplementation with NMN, the NAD⁺ precursor, inhibits mitochondrial division and ROS production in adult ischemic mice, thereby playing a neuroprotective role in cerebral ischemia [15]. This indicates that supplementation with exogenous NAD⁺ or its precursors can improve mitochondrial function [16] and prevent the development of neurodegenerative diseases [17]. We recently found that intravenous injection of NAD⁺ within the MCAO/R in adult mice significantly reduced the volume of infarction, cerebral edema, and neurological deficits, and effectively reversed the decrease in ATP and increase in lactate after ischemia/reperfusion [18]. However, the unique development and distribution of neuronal receptors in the neonatal brain, compared to those in the adult brain, may result in significant differences in the phenotype and mechanism of excitotoxicity. Thus, permanent MCAO ischemia in adult mice and neonatal hypoxia/ischemia may involve different mechanisms of cell death and the choice of intervention drugs may vary based on these mechanisms [19, 20]. Further studies are required to explore whether NAD⁺ exerts a protective effect against neonatal HIBI.

Recently, we explored the neuroprotective effects of exogenous NAD⁺ in an HI model and observed that it significantly improved the neurobehavioral outcomes in neonatal mice following HI. Further analysis using tandem mass tag proteomics combined with liquid chromatography tandem mass spectrometry confirmed that complexin 2 (CPLX2) exhibited significant variations in expression between HI and NAD⁺ intervention groups [21]. Western blotting showed that CPLX2 protein levels were significantly reduced in the HI model group, which could be reversed by NAD⁺ supplementation, confirming that CPLX2 is mainly expressed in neurons. However, the effects of overexpression and downregulation of CPLX2 on HIBI phenotype have not yet been reported. Moreover, the effect of different CPLX2 expression levels on the efficacy of NAD⁺ treatment has not yet been evaluated.

This study is the first to test whether pharmacologically regulated CPLX2 expression preserves the brain morphological integrity and neurobehavioral function in a mouse model of HIBI. We first investigated the effects of intraventricular injection of a lentiviral vector with knocked-down or overexpressed CPLX2, or completely knocked-out CPLX2 on brain injury pathology, behavioral phenotype, and NAD⁺ neuroprotective effects. Subsequently, we investigated the effects of CPLX2 regulation on redox activity, ferrous iron (Fe²⁺) metabolism, and NAD⁺ protection in the HT22 cell line OGD/R model.

Materials and methods

The flowchart of the study was shown in Fig. 1.

Fig. 1. The flowchart of the study.

a, b Experiments in vivo: knockdown and knockout mice. c, d Experiments in vitro: HT22 cell.

Animal model of HI

All animal experiments were conducted in accordance with institutional guidelines for the care and use of animals. This study was approved by the Animal Ethics Committee of Soochow University of China. Neonatal C57BL/6 male mice were purchased from Zhaoyan New Drug Research Center Co., Ltd. (Suzhou, China). CPLX2 knockout mice were conducted in the Nanjing Biomedicine Research Institute of Nanjing University. Mice were anesthetized with 1.5%–2% isoflurane (RWD, Shenzhen, China) and its artery was ligated using two 5-0 surgical sutures and cut in the middle using surgical scissors. Next, the subcutaneous tissue was sutured and the skin was disinfected with 70% ethanol. Overall, permanent ligation of the left common carotid artery in each mouse was performed within 5 min. After each mouse had recovered with its mother for 1 h, all experimental mice were placed in a chamber at 37 °C and exposed to hypoxic conditions (92% N₂/8% O₂) for 2 h. The mice were allowed to recover alone for 1 h being returned to their mothers.

NAD treatment

NAD (N7004, Sigma, Missouri, USA) was intraperitoneally injected (5 mg/kg body weight) 30 min before surgery, and once every 24 h thereafter until death. Mice in non-NAD group were injected with an equal volume of saline (Huarena, Shenzhen, China).

Viral infection in vivo

For viral infection in vivo, knockdown lentivirus of CPLX2 and its control lentivirus were infused into the left lateral ventricle (3 μL/injection site) of 1-day-old mice.

Quantitative real-time PCR and Western blot

Quantitative real-time PCR was performed as previously described [22]. And primers used here were as follows:

CPLX2: forward 5’-CGAGACAAGTATGGGCTGAAGAA-3’,

   reverse 5’-GGAGGAGGATGGGTTACTTCTTG-3’.

GPX4: forward 5’-CAAGTTTGACATGTACAGCAAGATCT-3’,

   reverse 5’-CACGGCAGGTCCTTCTCTATCA-3’.

SLC7A11: forward 5’-TCTTCGATACAAACGCCCAGATA-3’,

   reverse 5’-GTGCTGAATGGGTCCGAGTAAAG-3’.

β-actin: forward 5’-ACTGTCGAGTCGCGTCC-3’,

   reverse 5’-CTGACCCATTCCCACCATCA-3’.

Western blotting was performed as previously described [23]. Briefly, cells were collected 6 h after OGD/R and proteins were extracted using radioimmunoprecipitation assay buffer (P0013C, Beyotime, Shanghai, China). The extracted proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% milk powder for 1 h, followed by overnight incubation at 4 °C with the following primary antibodies: rabbit anti-CPLX2 (1:1000, 18149-1-AP, Proteintech, Chicago, USA), mouse anti-GPX4 (1:1000, 67763-1-Ig, Proteintech, Chicago, USA), rabbit anti-SLC7A11 (1:1000, 98051, Cell Signaling Technology, Massachusetts, USA), and mouse anti-β-actin (1:10,000, Abcam, ab6276, Cambridge, UK). Next, the membranes were incubated with anti-rabbit or anti-mouse IgG (1:10,000, Abclonal, AS014, AS003, Wuhan, China) secondary antibodies for 1 h. The blots were scanned using an Imaging System (Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence

Male 10-day-old C57BL/6 mice were continuously anesthetized via isoflurane inhalation, and their brains were removed after cardiac perfusion. The tissues were then fixed in 4% paraformaldehyde for 24 h to prepare paraffin sections. After incubation with primary and secondary antibodies and DAPI (C1002, Beyotime, Shanghai, China), images were collected under a fluorescence microscope.

Staining with 2,3,5-triphenyl tetrazolium chloride

The redox indicator, 2,3,5-triphenyl tetrazolium chloride (TTC) is reduced to a fat-soluble compound by mitochondrial succinate dehydrogenase. Mice were euthanized 24 h after HI induction. The brain tissue was placed on a slide and frozen at −20 °C for 10 min. Five uniform coronal sections were incubated with 1% TTC (T8877, Sigma, Missouri, USA) at 37 °C for 20 min in the dark. Cerebral infarcts were measured using ImageJ software and the infarct volume was expressed as a percentage of the total brain hemisphere.

Behavioral evaluation of adolescent mice

The mice were subjected to behavioral tests in a quiet room with non-distracting homogenous lighting. To explore the long-term effects of NAD⁺ on behavior, behavioral tests were performed following HI induction. All behavioral tests were performed by two testers who were blinded to the experimental group descriptions and treatments.

Neurodevelopmental reflexes

For all neurological function tests, instruments were disinfected with 70% ethanol. The cliff-avoidance reflex was tested to evaluate the ability of each mouse to respond to an adverse environment [24]. The paws and nose of each mouse were placed over the bench edge. The time required by each mouse to return to the bench edge was recorded.

A negative geotaxis reflex test [24, 25] was performed to assess the proprioceptive and vestibular functions. Each mouse was blindfolded, placed head-down on a board that had been tilted at 45°, and transitioned to a head-up position. The time required for each mouse to transition to the head-up position was recorded [26].

The forelimb suspension reflex was used to evaluate locomotion and coordination in the mice. Each mouse was suspended by its forepaws and allowed to grasp a rod 50 cm above the ground. The duration of the rod grasping was recorded.

The surface righting reflex was analyzed to determine the reaction speed [21, 24]. Each mouse was placed in a supine position on a piece of cardboard and flipped to an upright position [27, 28]. The time required for the mice to move from the supine to the upright position was recorded using a stopwatch, and each mouse was tested thrice.

Open field test

The open field test was used to evaluate exploratory behavior and spontaneous activity in the mice [29]. The bottom of a square open-field arena was divided evenly into 16 small squares. The middle four squares were regarded as the central area, whereas the other areas were regarded as the surrounding area. Each mouse was placed in the center of the arena and allowed to move freely for 5 min. After 24 h, the following test was conducted: each mouse was allowed to explore freely for 5 min. The total number of squares crossed by the mice (number of grids), the number of times the mice groomed (number of groomings), and the amount of time that the mouse spent in the central area (center duration) were recorded.

Novel object recognition test

A novel object recognition test was used to evaluate the memory and cognitive abilities of mice [28]. On the first day, the mice were placed in the middle of the test chamber and allowed to explore freely for 5 min. On the second day, two identical square building blocks were placed diagonally at the bottom of the test box, and the mice were allowed to explore freely for 5 min. On the third day, one of the blocks was changed to a triangular block, and the other block was unchanged. Mice were placed in the middle of the test box and allowed to explore freely for 5 min. The time spent in the two blocks was recorded. Normally, mice are curious, and spend more time with new objects. The cognitive indices of each group were recorded.

Foot fault test

The foot fault test was conducted to assess sensorimotor function [28]. The test was conducted on a lab-prepared grid with 2 cm long and 2 cm wide holes. Each mouse was allowed to walk freely on the grid for 5 min during the training session. One hour later, the test was conducted as follows: An incorrect step was regarded as a limb entry into the hole. We recorded the number of foot faults for over 50 steps [30].

Step-through test

The step-through test was performed to evaluate the memory and learning abilities of mice using a DC pulse-stimulating current device. On the first day, mice were trained for 5 min. Initially, mice were placed in the center of a bright room with their backs facing the hole. When the mice entered the dark room, they were electrically stimulated and then returned to the bright room. On the second day, the formal experiment began by recording the time at which the mice entered the dark room for the first time within 5 min (latent period).

Determination of seizure threshold

After the behavioral tests, each mouse received an intraperitoneal injection of penicillin (5.1 × 10⁶ U/kg, Sangon Biotech, Shanghai, China). We recorded the seizure latency over 90 min. Grade IV or higher seizures were defined as described previously [31]. The seizure latency was observed for 90 min. If no seizures occurred, the latency was recorded as 90 min.

Identification of knockout mice

Genotypes of the knockout mice were identified via PCR from tail biopsies. Experiments were conducted as described previously [28]. The following primers were amplified using the PCR fragments:

F1: 5’-AAACCAGCCTGGCAGGATTCTCT-3’.

R1: 5’-ATTCTTAGAGGGTACAAGGAGGGGC-3’.

F2: 5’-TGTGCCATGAGGCTCCTCATT-3’.

R2: 5’-CAGGAAGCCTTGACAAGGTGGTA-3’.

The product was then identified by agarose electrophoresis. And the expression of CPLX2 in the cortical of 10-day-old mice was detected by qPCR and Western blotting.

Histopathological analysis

Three days after HI induction, the mice were anesthetized using 1.5%–2% isoflurane (RWD, Shenzhen, China) and brain tissues were harvested for hematoxylin and eosin (H&E) staining (G1120, Solarbio, Beijing, China) as previously described [21]. Images were captured under a light microscope (Nikon, Tokyo, Japan).

Behavioral evaluation and seizure threshold of adolescent mice

Viral infection and drug treatment in vitro

HT22 cells were seeded in six-well plates. Lentiviral transfection was performed after 24 h. Knockdown lentivirus with an MOI value of 20 or overexpression lentivirus with an MOI value of 50 was added to the cells. Then CPLX2 knockdown or overexpressed stable HT22 cells were established with purinomycin (2 μg/mL). Cells in NAD⁺-treated groups were pretreated with NAD⁺ (100 μM) for 12 h.

Oxygen-glucose deprivation cell model

When the degree of fusion of HT22 cells reached 80%, the OGD/R model was established by culturing the cells in six-well plates for 24 h. The cells were washed three times with PBS buffer, and the residual medium was removed. Sugar-free medium (PM150270, Pricella, Wuhan, China) was added to the culture plate, the cells were placed in an oxygen deficiency chamber, and a mixture of nitrogen and carbon dioxide was injected into the chamber (10:1). The oxygen-deficient chamber was closed and the cell culture chamber was incubated for 6 h under hypoxic conditions. After induction of hypoxia, fresh cell medium was added.

Cell viability

HT22 neuronal cells were prepared as a cell suspension and inoculated into 96-well culture plates (inoculation density of 5 × 10³ cells/hole). Cells in each group were treated similarly. After 24 h of culture, the medium was replaced with a CCK8 mixture (CK04, Dojindo, Kumamoto, Japan), gently mixed, and placed in a cell incubator. The absorbance at 450 nm was measured every hour for 4 h using an enzyme-labeled instrument.

Lactate dehydrogenase

HT22 neuronal cells were prepared as cell suspensions and seeded in 96-well culture plates (inoculation density of 5 × 10³ cells/well). The cells in each group were treated similarly and lactate dehydrogenase (LDH) levels were evaluated 24 h after treatment with LDH kit (4744926001, Roche, Basel, Switzerland). First, a reaction solution (250 μL catalyst + 11.2 mL stain) was prepared and mixed by upside-down movement. From this reaction solution, 200 μL was added to each well, incubated for 30 min, then 50 μL termination solution was added and mixed well. Optical density at 490 nm was measured.

Ferrous iron

Procedures were performed according to the instructions of the assay kits for ferrous iron (I291, Dojindo). Twenty-four hours after HI modeling in newborn mice, cerebral cortex tissues were extracted and suspended in 1 mL of pre-cooled PBS, blown 20 times with a pipette, and shaken for 10 s using a vortex. This suspension was centrifuged at 12,000× g for 4 min and the supernatant was discarded. Assay buffer was added, and the tissue was homogenized with an ultrasonic crusher for 5 min. After centrifugation at 16,000× g for 10 min, the supernatant was mixed well with detection probe incubated at 37 °C for 1 h and the absorbance was measured at 593 nm using an enzyme-labeled instrument. Fe²⁺ content was calculated from the standard curve and protein concentration.

The cells were washed with serum-free culture medium three times, and 1 μmol/L FerroOrange working liquid (F347, Dojindo) was added. After culturing in an incubator at 37 °C and 5% CO₂ for 30 min, fluorescence was observed under a fluorescence microscope and the value calculated using ImageJ.

Glutathione content detection

Following 24 h after HI modeling in newborn mice, the cerebral cortex tissue homogenate from each mouse group was removed according to the instructions of GSH assay kit (S0053, Beyotime). The protein removal reagent M solution was added and vortexed vigorously, and the supernatant was mixed with the diluted GSH removal auxiliary solution and GSH cleaning reagent working solution. The samples or standards were mixed with the total GSH test solution for 5 min at room temperature, after which reduced NADP⁺ (NADPH) solution was added and mixed well. The absorbance at 412 nm was measured immediately, and the GSH content of the sample was calculated according to the standard curve and protein concentration.

The cell culture medium was then discarded. The cells were washed once with pre-cooled PBS and the cell pellets were collected via centrifugation. The other procedures were the same as those used for mouse tissue measurements.

Malondialdehyde

Experiments were peformed according to the instructions of malondialdehyde (MDA) assay kit (S0131, Beyotime). After 24 h of HI modeling of newborn mice, the cerebral cortex tissues of each group were removed and homogenized in PBS before centrifugation. The supernatant was collected. The TBA storage and MDA working solutions were prepared according to the manufacturer’s instructions. PBS was used as a blank control. And 0.1 mL of standard solutions or samples were added for MDA determination. Next, 0.2 mL of detection fluid was added, mixed thoroughly, and heated in a boiling water bath for 15 min. The mixture was centrifuged and the supernatant was added to a 96-well plate. Absorbance was measured at 532 nm. The MDA content was calculated according to the standard curve and protein concentration.

The cells were washed three times with pre-cooled PBS and several times with PBS before centrifugation. Supernatants were collected for subsequent experiments. Other procedures were the same as those used for mouse tissue measurements.

Hydrogen peroxide

After 24 h of HI modeling, the cerebral cortex tissue of each group of mice was removed and lysate was added for homogenization before centrifugation according to the instructions of H₂O₂ assay kit (S0038, Beyotime). The supernatant was used for subsequent analyses. The sample or standard was added to the 96-well plate, followed by 100 μL of detection reagent, and left for 30 min at room temperature. The absorbance was measured at 560 nm using an enzyme-labeled instrument. The concentration of hydrogen peroxide in the samples was calculated based on a standard curve and the protein concentration.

The cells were washed three times with pre-cooled PBS and centrifuged to collect cell precipitates, and the lysate was added before centrifugation. The supernatant was used for subsequent analyses. Other procedures were the same as those used for mouse tissue measurements.

Statistical analysis

Data are reported as mean ± SD. Statistical analyses were performed using GraphPad Prism software version 9. The data between the two groups were analyzed using Student t test. Data among three or more groups were analyzed using a one-way analysis of variance (ANOVA). All analyses were subjected to Tukey’s correction for multiple comparisons and statistical significance was accepted at P < 0.05.

Results

CPLX2 expression in mice

Western blotting results showed that CPLX2 expression was highest in the cortex and hippocampus, followed by the striatum, cerebellum, and olfactory bulb (Fig. 2a, b). Immunofluorescence results also showed that CPLX2 was highly expressed in the cortex and hippocampus (Fig. 2c), which was consistent with the Western blotting results. CPLX2 was mainly expressed in neurons, with low expression in microglia and astrocytes (Fig. 2d).

Fig. 2. Expression of CPLX2 protein in brain tissues of 10-day-old mice.

a Band representation of CPLX2 protein expression in different brain regions of mice detected by Western blotting. b Western blotting analysis of CPLX2 protein expression levels in brain regions of 10-day-old mice (n = 4). c CPLX2 protein expression in brain regions of 10-day-old mice was detected by immunofluorescence (n = 4, bar = 100 μm). d Expression of CPLX2 in different nerve cells was detected by immunofluorescence (n = 6, bar = 20 μm). Data was reported as mean ± SD. One-way ANOVA was used for statistical analysis. ^*P < 0.05.

CPLX2 editing efficiency

The mRNA and protein levels were significantly lower in the cortex of cells transfected with the CPLX2 knockdown lentivirus than in those transfected with the control lentivirus (Supplementary Fig. S1).

CPLX2 knockdown reversed the impact of NAD⁺ in brain infarction

The TTC results revealed that the region of brain infarction in the NAD⁺ intervention model group transfected with the knockdown control lentivirus was significantly smaller than that in the LV-sh-NC + HI group. However, CPLX2 knockdown reversed the effects of NAD⁺ on brain infarction (Fig. 3a, b).

Fig. 3. Knockdown of CPLX2 reversed the effect of NAD on inhibiting pathological morphological damage in HI mice.

a, b TTC staining (n = 5). c Timeline of neurobehavioral experiments. d Cliff avoidance reflex. e Negative geotaxis reflex. f Forelimb suspension reflex. g Surface righting reflex. h Open field test: number of grids. i Open field test: center duration time. j Open field test: number of groomings. k Novel object recognition test: cognitive index. l Foot fault test: number of foot faults. m Step-through test: Latent period. n Seizure threshold. Data were reported as mean ± SD. n = 10. One-way ANOVA was used for statistical analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

CPLX2 knockdown reversed the impact of NAD⁺ in neurobehavioral and seizure threshold evaluation

The long-term prognosis of mice in each group was evaluated using neurobehavioral assessments. In the NAD⁺ intervention model group, the cliff avoidance time, negative ground time, and plane righting time were decreased, whereas the forelimb suspension time and total grid number were increased. Moreover, the center duration time and number of grooming decreased, recognition index increased, foot error number decreased, and latency time of entering the dark box increased compared to those in the LV-sh-NC + HI group (Fig. 3c–n). CPLX2 knockdown reversed the effects of NAD⁺ on cliff avoidance, negative ground increase, plane righting, forelimb suspension, total grid number, center duration time, grooming, recognition index, foot error number, and latency to enter the dark box (Fig. 3c–n).

CPLX2 editing efficiency in knockout mice

Agarose gel electrophoresis was used to identify the genotypes of transgenic mice (Supplementary Fig. S2). Levels of mRNA and protein in the cortex of CPLX2-KO mice were significantly lower than those in wild-type mice (Supplementary Fig. S2).

CPLX2 knockout reversed the effect of NAD⁺ on histopathological morphology

TTC results revealed that the region of brain infarction in the NAD⁺ intervention model group was significantly smaller than that in the WT + HI group. CPLX2 knockout reversed the effects of NAD⁺ on brain infarction (Fig. 4a, b). Results of H&E and Nissl staining showed that the cells of the CA1, CA3, and DG in the left cortex and hippocampus of the WT + HI + NAD group were regularly arranged, wrinkling of the nucleus was reduced, nuclear color was darker, and the number of Nissl bodies was significantly higher than that in the WT + HI group. While CPLX2 knockout reversed the effect of NAD⁺ on brain morphology (Fig. 4c–h).

Fig. 4. CPLX2 knockout reversed the effect of NAD⁺ on neurobehavioral evaluation and seizure threshold evaluation.

a, b TTC staining (n = 5). c, d HE and Nissl staining (n = 6, bar = 20 μm). e–h Number of Nissl bodies in CA1, CA3 and DG regions of the cortex and hippocampus. Data were reported as mean ± SD. One-way ANOVA was used for statistical analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

CPLX2 knockout reversed the effect of NAD⁺ on neurobehavioral evaluation and seizure threshold evaluation

The long-term prognosis of mice in each group was evaluated using neurobehavioral assessments. In the NAD⁺ intervention model group, the cliff avoidance time, negative ground time, and plane righting time were decreased, whereas the forelimb suspension time and total grid number were increased. Moreover, the center duration time and number of grooming decreased, recognition index increased, foot error number decreased, and latency time of entering the dark box increased compared to those in the WT + HI group (Fig. 5a–j). CPLX2 knockout reversed the effects of NAD⁺ on cliff avoidance, negative ground increase, plane righting, forelimb suspension, total grid number, center duration time and grooming, recognition index, foot error number, and latency to enter the dark box (Fig. 5a–j). The convulsive threshold was higher in the NAD⁺ intervention group than in the WT + HI group (Fig. 5k). However, CPLX2 knockout reversed the effect of NAD⁺ on the convulsive threshold (Fig. 5k).

Fig. 5. Neurobehavioral evaluation in mice of CPLX2 knockout mice.

a Cliff avoidance reflex. b Negative geotaxis reflex. c Forelimb suspension reflex. d Surface righting reflex. e Open field test: number of grids. f Open field test: center duration time. g Open field test: number of groomings. h Novel object recognition test: cognitive index. i Foot fault test: number of foot faults. j Step-through test: Latent period. k Seizure threshold. n = 10. Data were reported as mean ± SD. One-way ANOVA was used for statistical analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

CPLX2 knockdown reversed the impact of NAD⁺ on ferroptosis in HT22 cells

The mRNA and protein levels in CPLX2 knockdown cells (LV-sh-CPLX2-1 and LV-sh-CPLX2-3) were significantly lower than those in the control lentivirus group (Supplementary Fig. S3). The CCK8 value was significantly increased, whereas the LDH value was significantly decreased in the NAD⁺-treated model cells compared to those in the LV-sh-NC + OGD/R group. However, the CPLX2 knockdown reversed the effects of NAD⁺ on cell viability (Fig. 6a, b). The concentrations of Fe²⁺, MDA and H₂O₂ were significantly decreased and GSH, the expression of GPX4 and SLC7A11 were significantly increased in the NAD⁺-treated model cells compared to those in the LV-sh-NC + OGD/R group (Fig. 6c–k). However, the CPLX2 knockdown reversed the inhibitory effects of NAD⁺ on ferroptosis (Fig. 6c–k).

Fig. 6. CPLX2 knockdown reversed the impact of NAD⁺ on ferroptosis in HT22 cells.

a Cell viability (n = 6). b LDH content (n = 6). c Level of ferrous iron (n = 5). d–f Levels of GSH, MDA and H₂O₂ (n = 6). g–k Levels of mRNA and protein expression of GPX4 and SLC7A11 (n = 3). One-way ANOVA was used for statistical analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^**** P < 0.0001.

CPLX2 overexpression enhanced the inhibitory effects of NAD⁺ on ferroptosis in HT22 cells

The mRNA and protein levels in the CPLX2 overexpression lentivirus cells were significantly higher than those in the control lentivirus group (Supplementary Fig. S3). CCK8 value was significantly increased, whereas the LDH value was significantly decreased in the NAD-treated model cells compared to those in the LV-OE-NC + OGD/R group. CPLX2 overexpression enhanced the effect of NAD⁺ on cell viability (Fig. 7a, b). Concentrations of Fe²⁺, MDA, and H₂O₂ were significantly decreased, and GSH, the expression of GPX4 and SLC7A11 were significantly increased in the NAD-treated model cells compared to those in the LV-OE-NC + OGD/R group (Fig. 7c–k). CPLX2 overexpression partially enhanced the inhibitory effects of NAD⁺ on ferroptosis (Fig. 7c–k).

Fig. 7. CPLX2 overexpression enhanced the inhibitory effects of NAD⁺ on ferroptosis in HT22 cells.

a Cell viability (n = 6). b LDH content (n = 6). c Level of ferrous iron (n = 5). d–f Levels of GSH, MDA and H₂O₂ (n = 6). g–k Levels of mRNA and protein expression of GPX4 and SLC7A11 (n = 3). Data were reported as mean ± SD. One-way ANOVA was used for statistical analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Discussion

This study indicated that NAD⁺ has a significant protective effect on the brain injury phenotype after neonatal HI (a cerebral palsy animal model). This effect is related to the activation of CPLX2, and in vitro studies have shown that the knockdown of CPLX2 can exacerbate neuronal ferroptosis injury following OGD/R. Researches showed the potential of targeted treatment of ferroptosis in HIBI. For example, it was found that pathological damage to brain tissue increased in the HIBI model group around the ventricles. Anemia can reduce the increase rate of ferrous ion in the brain after HI and prevent HIBI [32]. And study also showed that HI injury significantly increased reactive oxygen species levels and proteins related to ferroptosis. While ferrostatin-1, an ferroptosis inhibitor, effectively alleviates the brain injury behavioral phenotype [33]. These results suggest that ferroptosis is involved in HI pathophysiology and inhibiting ferroptosis exerts neuroprotection. Ferroptosis has been identified as a possible key injury mechanism of HIBI [34]. Understanding how NAD⁺-activated CPLX2 modifies neuronal ferroptosis in acute neuronal injury may lead to the development of novel therapeutic strategies.

Mechanistically, supplementing NAD⁺ has potential therapeutic effects on HIBI. In vivo studies of hypoxia have suggested its possible effects on the redox state of brain cells. Two redox couples in the brain, NAD⁺/NADH and NADP⁺/NADPH, are crucial for maintaining cellular redox homeostasis and survival. Garofaro et al. [35] investigated the in vitro effects of hypoxia on redox status, wherein NADH and NAD⁺ were simultaneously extracted from cerebral cortex slices and measured by bioluminescence. The results showed that severe hypoxia increased NADH levels and NADH/NAD⁺ ratios by more than 200%. Yager et al. [36] investigated cerebral oxidative metabolism and redox status during HI and early recovery in immature rats using a model of unilateral carotid artery ligation and exposure to 8% O₂ for 3 h. They reported that the cytoplasmic NAD⁺/NADH ratio decreased (increased reduction) to 50% of the control during HI and remained in a reduced state throughout the 4 h of recovery. Therefore, NAD⁺ supplementation may have therapeutic effects in HIBI.

To address this issue, we recently conducted the first study on the interventional effects of NAD⁺ on the HIBI phenotype and demonstrated that NAD⁺ treatment can significantly improve neurobehavioral damage in newborn mice after HI [21]. Subsequent proteomics combined with spectrometry confirmed that the expression of CPLX2 in the damaged cortex was significantly reduced compared to that in the control group but could be reversed by NAD⁺ intervention. CPLX2 has various roles in neurological diseases and is associated with ferroptosis [37–42]. There are currently no research reports directly linking CPLX2 in the brain to ferroptosis. However, it has been demonstrated in other tissue specimens that CPLX2 is associated with inhibiting ferroptosis. For example, Li et al. recently investigated the relationship between CPLX2 and ferroptosis in hepatocellular carcinoma (HCC). QPCR and Western blotting were used to detect CPLX2 levels. The contents of iron, ROS, MDA, and GSH were used to evaluate the function of CPLX2 on ferroptosis. The results showed that the downregulation of CPLX2 significantly promotes the levels of iron, ROS, and MDA, while inhibiting the GSH levels of HCC cell lysate, indicating that CPLX2 is involved in ferroptosis inhibition [37]. Ferroptosis is a form of programmed cell death driven by iron-dependent lipid peroxidation and tightly controlled by oxidation and antioxidant systems, primarily including GPX4-dependent antioxidant system. Targeting ferroptosis by iron chelators, antioxidants, and inhibitors of lipid peroxidation holds potential as a therapeutic strategy to improve outcomes in HIE through maintaining cellular redox homeostasis of brain [11, 33, 43, 44]. Thus, CPLX2 may be an activation target to reduce the HIBI phenotype following NAD⁺ intervention.

In this study, we produced and analyzed CPLX2 deficient (CPLX2^-/-) mice for the first time. The effects of CPLX2 knockdown or knockout on the neuroprotective effect of NAD⁺ in an HI mouse model were evaluated, showing that knockdown or knockout of CPLX2 prevented the protective effect of NAD against HI, specifically manifested in morphological and neurobehavioral injury phenotypes (Figs. 3–5).

These adverse effects of CPLX2 downregulation on brain function, observed in vivo following HI, were in line with the functional and gene expression evaluations revealed in our cell models using downregulated or upregulated CPLX2 viral vectors, accompanied by changes in redox-related parameters, including Fe²⁺, GSH, MDA and H₂O₂ content, and, in particular, the expression of the ferroptosis molecules GPX4 and SLC7A11 (Figs. 6 and 7). Downregulation of CPLX2 expression inhibits GSH levels in hepatocellular carcinoma (HCC) cell lysates and increases the accumulation of lipid peroxides, suggesting that CPLX2 is involved in the inhibition of oxidative stress damage and ferroptosis [37]. Based on our recent findings in a mouse model of ischemic stroke (IS), which suggest that non-NADPH-dependent NAD⁺/NADH redox coupling plays a crucial role in resisting IS-induced oxidative stress damage and ferroptosis [45], we speculated that the upregulation of CPLX2 expression may contribute to the NAD⁺-induced HIBI phenotype by inhibiting neuronal ferroptosis.

To verify this hypothesis, we transfected HT22 cells with knocked-down and overexpressed CPLX2 lentiviral vectors and analyzed ferroptosis markers, including Fe²⁺, GSH, MDA, and H₂O₂, as well as the expression levels of GPX4 and SLC7A11. The results showed that NAD⁺ intervention inhibited OGD/R-induced ferroptosis by decreasing Fe²⁺and GSH contents, increasing MDA and H₂O₂, and downregulating the expression of GPX4 and SLC7A11(Figs. 6 and 7). When transfected with the knockdown CPLX2 lentivirus, this effect ceased (Fig. 6). However, when transfected with CPLX2 overexpression lentivirus, this effect persisted and was enhanced (Fig. 7). Interestingly, recent studies on HCC cell lines have found that the downregulation of CPLX2 significantly increases the content of iron, ROS, and MDA, while inhibiting GSH levels in cell lysates, indicating that CPLX2 is involved in ferroptosis. Additionally, CPLX2 knockdown promoted sorafenib-induced cell death [37]. These studies, combined with our current findings, suggest that the inhibitory effect of NAD⁺ on OGD/R-induced ferroptosis may be related to the upregulation of CPLX2. Therefore, CPLX2 may be an effective target for ferroptosis inhibition.

Indeed, it has been proven that CPLX2 plays important biological functions such as participating in synaptic vesicle fusion and regulating neurotransmitter release. In particular, Glynn et al. found through previous studies on the downregulation of CPLX2 expression in humans with depression, animal models of depression, and neurological diseases such as Huntington’s disease, where depression is the main symptom, that CPLX2 knockout (Cplx2^-/-) mice was used to investigate the effects of CPLX2 knockout on emotional disorder behavior. The results showed that Cplx2^-/- mice also exhibited emotional and behavioral disorders, including abnormal social interactions and depressive like behavior [41]. Our current study is precisely an extension of the work ideas and findings of Glynn et al. In order to better reveal the function of CPLX2 in neonatal hypoxic-ischemic brain injury, we established a CPLX2 knockdown or knockout mouse model and conducted a series of neurological experiments. We found that knockout or overexpression of CPLX2 eliminated or enhanced the protective effect of NAD on cell survival, as well as the link between CPLX2 and NAD in reversing ferroptosis in vitro.

The timing of NAD⁺ administration appears critical in determining its efficacy. NAD⁺ exerts a dual effect on mitochondrial activity. Short-term incubation with NAD⁺ boosts the Krebs cycle and ETC, and enhances pyrimidine biosynthesis. Extended incubation with NAD⁺ results in the depletion of pyrimidines, accumulation of purines, activation of the replication stress response, and cell cycle arrest [46]. Recently, Qu et al. [47] examined the effects of two methods, NAD⁺ precursor administration 20 min after reperfusion and daily administration 3 days before an ischemic attack, on middle cerebral artery occlusion brain injury in mice. They found that a single dose of NAM or NR after ischemia improved the tissue and neurological outcomes at 72 h. In contrast, pre-ischemic treatment for 3 days expanded the infarct and exacerbated the neurological deficits. Here, we explored for the first time the effect and molecular mechanism of NAD⁺ injection 30 min before neonatal HI on the HIBI phenotype. Administration of NAD⁺ 30 min before HI significantly improved the HIBI phenotype in newborns. These results have both translational and clinical significance. TH is currently the only approved treatment for protecting the brain after HI. However, rewarming after hypothermia may lead to secondary brain damage. Impaired blood flow and oxygen transport caused by the diffusion of microcirculatory convection in the brain are key pathological changes that lead to secondary brain injury after rewarming. Our recent study showed that NAD⁺ administration significantly alleviated ischemia/reperfusion brain injury [18]. The current findings emphasize the possibility that NAD⁺ supplementation before HI treatment combined with hypothermic treatment after HI may be the best opportunity to improve the prognosis of patients with HIBI.

However, there are several limitations in this study. This study confirmed the relationship between CPLX2 and ferroptosis in vitro and neuroprotective effects of CPLX2. However, the relationship between CPLX2 and ferroptosis should be identified in vivo. And neurotransmitters should be tested to exclude confounding factors due to the biological functions in synaptic vesicle fusion in future works.

In summary, 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. CPLX2 knockdown reversed the inhibitory effect of NAD⁺ on ferroptosis in the HT22 cell line after OGD/R, whereas overexpression of CPLX2 enhanced the protective effect of NAD⁺ (Fig. 8). This suggests that CPLX2 plays a crucial role in the neuroprotective effects of NAD⁺ in neonatal HIBI.

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.

CPLX2 knockdown reversed the inhibitory effect of NAD⁺ on ferroptosis in the HT22 cell line after OGD/R, whereas overexpression of CPLX2 enhanced the protective effect of NAD⁺.

Author 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.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-025-01555-1.