Spinal cord ischemia reperfusion injury induces cuproptosis in neurons

Abstract

Background

Spinal cord ischemia reperfusion injury (SCIRI) is a serious disease that can result in irreversible neuronal damage, leading to the loss of sensory and motor function. Cuproptosis, a novel form of regulated cell death, has been studied in various diseases. However, the role and mechanism of cuproptosis in SCIRI remain to be elucidated.

Results

The results of transcriptome analysis showed significant downregulation of ATP7B, which regulates copper ion efflux. Concurrently, another key cuproptosis-related gene, FDX1, was significantly altered. Thus, we performed qPCR and Western blot assays in vivo and in vitro to detect changes in cuproptosis-related genes. The results indicated that cuproptosis was indeed activated by SCIRI or OGD/R. Moreover, immunofluorescence/immunohistochemitry staining and neuronal activity tests were consistent with the above results. Furthermore, we also proved that ammonium tetrathiomolybdate, a copper chelator and cuproptosis inhibitor, could not only ameliorate neuronal damage and promote neuronal survival but also improve lower limb motor dysfunction.

Conclusions

SCIRI caused ATP7B downregulation, which blocked copper ion efflux, leading to copper ion accumulation, DLAT oligomerization, degradation of iron-sulfur cluster proteins and ultimately cuproptosis in neurons.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13578-025-01463-1.

Introduction

Spinal cord ischemia reperfusion injury (SCIRI) is a serious disease that could result in irreversible damage to neurons, leading to the loss of sensory and motor function [1]. It is a prevalent complication of thoracic and abdominal aortic surgery despite advances in surgical technology and skills, and also occurs in other spinal disorders such as trauma, degeneration, and tumors [2, 3]. This imposes an onerous financial burden on patients and society at large [4]. Advances in therapeutic modalities have improved the survival rate and partially relieved neurological dysfunction after SCIRI [5–7]. However, despite decades of investigations culminating in large-scale clinical trials, no safe and effective therapy for SCIRI has been found [3, 7]. Therefore, studying the molecular and cellular mechanisms of SCIRI and identifying feasible and applicable treatments are particularly important.

Copper is one of the essential micronutrients and a key substance in cellular biological processes [8]. Not only does an intracellular copper deficiency hinder biological processes, but intracellular copper overload can also lead to toxicity in cells [9]. Therefore, copper levels need to be finely regulated within the cell to achieve a dynamic equilibrium that ensures an adequate supply for biological processes, but not so much as to produce toxicity [10, 11]. Humans have evolved elaborate and complex mechanisms to maintain homeostasis of copper levels at the systemic and cellular levels [12]. Absorption of copper from food by the intestine is via the SLC31A1 transporter [13]. Additionally, the process of cellular copper uptake also requires SLC31A1 in conjunction with metal reductase, and efflux from the cell then requires the involvement of ATP7A/B transporters [14, 15]. Cell membrane chaperones and glutathione (GSH) bind copper to protect against toxicity of free copper [16]. It has been found that mitochondrial copper overload triggers a novel form of regulated cell death, which is termed cuproptosis [17].

Cuproptosis is distinct from apoptosis, ferroptosis, and necrosis in terms of morphology, biochemistry, and genetics [18]. It is not mediated by oxidative stress but by mitochondrial proteotoxic stress [12]. More specifically, the normal lipid acylation of tricarboxylic acid cycle enzymes, such as DLAT, is impaired by copper ions, leading to their oligomerization. Copper ions also bind and destabilize iron-sulfur (Fe-S) cluster proteins via ferredoxin-1 (FDX1), which acts as an upstream regulator of the lipoylation process [17, 19]. Cuproptosis is triggered by combined dysregulation of lipoylated and Fe-S proteins, which results in serious metabolic disorders and proteotoxic stress [17, 20]. There is mounting evidence corroborating that cuproptosis, which is distinctive among all main cell death modalities discovered to date, is a pivotal mediator of numerous pathological processes [21–23]. Nevertheless, the role and mechanism of cuproptosis in SCIRI remain to be elucidated. More research could contribute to refining therapeutic approaches targeting cuproptosis.

In the current study, we provide the first clarification of the molecular mechanism of cuproptosis in SCIRI. We found that cuproptosis plays a role in SCIRI. Transcriptomic analysis and phenotype experiments revealed that ATP7B was inhibited. Mechanistically, SCIRI-induced down-regulation of ATP7B triggers copper efflux dysfunction and copper ion overaccumulation, which leads to consumption of GSH, oligomerization of DLAT, dysregulation of iron-sulfur proteins, ultimately culminating in cuproptosis. In brief, our findings suggest that cuproptosis palys a pivotal role in the pathogenesis of SCIRI. It provides new insights into inhibiting cuproptosis for SCIRI treatment.

Materials and methods

Animals

All male Sprague‒Dawley (SD) rats (188–200 g in weight, 8 weeks old) in this study were obtained from the Animal Laboratory of Beijing Charles River Corporation (Beijing, China). The rats were housed with water and commercial diet available at will in an air-conditioned room with constant temperature and humidity and a 12-hour light/dark cycle and were acclimated to their surroundings for one week prior to experiments. The above animal study was performed in accordance with the Basel Declaration and the approval of the Laboratory Animal Welfare Ethics Committee of Qingdao University (approval number: 20230917SD20231225065).

Spinal cord ischemia reperfusion injury (SCIRI) model

The model was built as previously reported [24]. Briefly, the rats were anesthetized and the abdominal aorta was clamped under the right renal artery near the heart using a 50 g aneurysm clip for 60 min. After the operation, the rat was placed in a box at 28 °C to recover and subsequently placed in a separate cage with food and water available at will. All rats were neurologically intact before the experiment.

Cells and culturing conditions

The rat ventral spinal cord cells (VSC4.1) were sourced from the Shanghai Jinyuan Biotechnology Corporation (JY737). These cell lines were cultured in DMEM medium (PM150210B, Procell) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). The cells were cultured at 37 ℃ in a 5% CO₂ atmosphere and were passaged at 80–90% confluence.

Oxygen and glucose deprivation/reoxygenation (OGD/R) model

In order to mimic the ischemia and hypoxia state of neurons in SCIRI, the VSC4.1 cells were exposed to OGD/R as reported previously [25, 26]. Briefly, the cells were cultured in serum-free, glucose-free DMEM medium (PM150270, Procell), and placed in a closed chamber, which was flushed with a continuous flow of mixed gas (95% N₂/5% CO₂) for 15 min. The chamber was then sealed and placed in a 37 °C incubator. After culturing for 60 min, the medium was replaced with complete medium and then placed in a normal incubator for 24 h.

Drug preparation and procedures

Ammonium tetrathiomolybdate (ATTM) was purchased from Sigma-Aldrich (323446, Sigma). ATTM was dissolved in dimethyl sulfoxide (DMSO) to yield a stock solution and further diluted in saline or complete medium before administration.

The cells in the ATTM group were treated with renewed normal medium with different concentrations of ATTM after reoxygenation. The rats in the ATTM group also received the same procedure as the SCIRI group but were treated with 10 mg/kg ATTM [27] immediately after reperfusion.

Neurological function analysis

The Basso, Beattie, and Bresnahan (BBB) open-field locomotor scale ranges from 0 (complete paralysis) to 21 (normal locomotion) and was used to measure locomotor recovery after SCIRI [28]. Two experienced investigators, blinded to the experimental design, recorded the BBB scores at 1, 6, 12, and 24 h after reperfusion. Any discrepancies were discussed and resolved to achieve a consensus.

Cell viability assay

Cell viability was determined by CCK-8 assay (C0037, Beyotime) in vitro. Briefly, Cells were seeded uniformly in a 96-well plate and cultured at 37 °C with 5% CO₂ atmosphere. Different treatments were performed according to experimental needs. Discard the medium at the observation time point, and 100 µl of medium containing 10% CCK-8 solution was added. Then, the plate was then incubated at 37 °C for 60 min. Lastly, absorbance was measured at 450 nm.

RNA sequencing and bioinformatics analysis

The spinal cords of approximately 2 mm centered on L2 segment 24 h after reperfusion were rapidly collected and flash-frozen in liquid nitrogen. The samples were immediately sent to Wekemo Tech (Shenzhen, China) for transcriptomic RNA sequencing, with three rats in each group. RNA was harvested using the RNeasy mini plus kit. Total RNA was used for the construction of sequencing libraries. RNA libraries for RNA-seq were prepared using the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, USA) following the manufacturer’s protocols. The library strategy was RNA-Seq, the library source was transcriptomic and the library selection was cDNA. The sequencing was performed using the Illumina NovaSeq 6000 instrument. Differentially expressed genes (DEGs) were determined by DESeq2 (|log2 fold change (FC)| >0; p < 0.05). P < 0.05 was considered to indicate a significant difference.

Live/dead staining

According to the manufacturer’s instructions, cells were gently washed and incubated with Calcein and PI staining dye of the live/dead viability/cytotoxicity kit (C2015, Beyotime) at 37 °C for 30 min. Then, the fluorescent images were captured on a Nikon microscope (Ti2-A).

Mitochondrial membrane potential detection

The mitochondrial membrane potential was detected by the JC-1 detection kit (C2006, Beyotime). According to the instructions, cells were gently washed and incubated with JC-1 working solutions at 37 °C for 20 min. After incubation, a portion was used for fluorescent images were captured on a Nikon microscope (Ti2-A). The other portion was washed and resuspended with 100 µl PBS containing 1% FBS. Subsequently, the samples were run on BECKMAN CytoFLEX S and results were analyzed using the FlowJo Software.

Glutathione (GSH) assay

In accordance with the instructions provided by the manufacturer, the concentration of GSH was determined in the spinal cord lysates and cell lysates using a total glutathione assay kit (S0052, Beyotime).

Immunofluorescence staining

For section immunofluorescence, the L2 spinal cord frozen sections were washed, permeabilized, blocked and incubated in primary antibody overnight at 4 °C. After rinsing with PBS, then the sections were incubated with the corresponding secondary antibodies conjugated with Alexa Fluor-labeled. Finally, the sections were mounted with ProLong Gold antifade reagent with DAPI to label the nuclei (P36935, Invitrogen). Traced sections were examined with a Zeiss microscope (Axioscope 5).

For cell immunofluorescence, cells were seeded uniformly in a 96-well plate, with different treatments according to experimental needs. Discard the medium at the observation time point and wash with PBS. Then, fixation using paraformaldehyde, permeabilizing, and subsequent blocking. Primary antibodies were applied to cells, followed by the appropriate fluorescent secondary antibodies. Nuclei were counterstained with DAPI (C1006, Beyotime). Using a Nikon microscope (Ti2-A), images were captured.

Immunohistochemical staining

The staining protocol was based on our previous report [29]. Briefly, the L2 spinal cord paraffin sections were deparaffinized, rehydrated and antigen retrieval. Then, sections were incubated in hydrogen peroxide, blocked, and then incubated in primary antibody overnight at 4 °C. The corresponding SABC-HRP kit (P0615, Beyotime) was used. Positive staining was visualized with DAB (P0202, Beyotime). Sections were counterstained with hematoxylin and dipped in acid alcohol as needed before being dehydrated and mounted on coverslips. Traced sections were examined with a Nikon microscope (NI-U).

Nissl body staining

The staining protocol was based on manufacturer’s instructions for Nissl staining solution (Beyotime, C0117). Briefly, the L2 spinal cord paraffin sections were deparaffinized, rehydrated, incubated in Nissl staining solution and bleached using 95% ethanol. Then, sections were dehydrated, transparent and mounted. Traced sections were examined with a Nikon microscope (NI-U).

Western blotting (WB) and qPCR

The protocol was based on previously described methods [30]. In brief, the cells and L2 spinal cords were treated with ice-cold RIPA (Beyotime, P0013C) containing a protease and phosphatase inhibitor cocktail (Beyotime, P1050), and the protein concentration was measured using BCA assay kit (Beyotime, P0012). Equal protein amounts were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride filter membrane (ISEQ00010, Millipore) and analyzed by WB using antibodies against DLAT (#12362, CST), FDX1 (12592-1-AP, Proteintech), HSP70 (25405-1-AP, Proteintech), LIAS (CSB-PA861526LA01HU, CUSABIO), DLST (A13297, Abclonal), SLC31A1 (T510261, Abmart), ATP7B (19786-1-AP, Proteintech), and with β-actin (60008, Proteintech) as the loading control. Blots were imaged on an imaging system (FUSION SOLO S, VILBER).

Total RNA of cells and spinal cords was extracted using the FastPure Cell/Tissue Total RNA isolation kit (RC112-01, Vazyme) and reverse transcribed (R323, Vazyme). Quantitative PCR (Q711, Vazyme) was performed on a QuantStudio™ 3 Real-Time PCR system (Applied Biosystems). Gene expression was calculated from Ct values and normalized to the expression of the β-actin reference gene. Primers were synthesized by Sangon Biotech (Shanghai, China). The detailed sequences were listed below: ATP7B (Forward: 5′-CGT GTT GTT CGC CTT GAT GTC-3′; Reverse: 5′-ACT GTG TTG TCC TCC ATG ATT GC-3′), FDX1 (Forward: 5′-CAG TCC ACT TCA AGA ACC GAG ATG-3′; Reverse: 5′-CCT CAC ACG CAC CAA ATC CAT C-3′), SLC31A1 (Forward: 5′-GAA CCA CAC GGA CGA CAA CAT C-3′; Reverse: 5′-CCA AAG TAG AAG GTC ATA GGC ATC ATC-3′), β-actin (Forward: 5′- ACG GTC AGG TCA TCA CAT TCG-3′; Reverse: 5′- GGC ATA GAG GTC TTT ACG GAT G-3′).

Statistical analysis

The data are presented as the mean ± standard deviation (mean ± SD) of at least three independent experiments in this study. Analyzes of two groups were performed using two-tailed Student’s t-test, and more than groups was performed using one-way or two-way ANOVA with Tukey’s post hoc test in GraphPad Prism version 9.5.1. For all statistical tests, a P value < 0.05 was considered statistically significant.

Results

SCIRI caused neuron loss and lower limb motor dysfunction

The BBB score was used to assess the locomotor function after SCIRI. Motor function of the lower extremities was significantly impaired in the SCIRI rats compared to the Sham rats (Fig. 1a). To evaluate the morphometry of neurons after SCIRI, Nissl staining was performed (Fig. 1b). The results revealed that the number of Nissl body in the spinal cords was significantly decreased in the SCIRI rats compared to the Sham group (Fig. 1c). The results of the BBB score were consistent with Nissl staining.

Fig. 1.

SCIRI caused neuron loss and lower limb motor dysfunction. a. The BBB scores of different groups at each time point (n = 6). b. Representative Nissl staining of spinal cord cross-section from L2 segment (100×, scale bar = 400 μm). c. Quantitative analyses of Nissl body in the ventral horn regions of the spinal cord (n = 3). d. Schematic illustration of the experimental design and sample preparation for transcriptomic analysis. e. Heatmap showed differentially expressed genes (DEGs) between the spinal cord (L2 segment, 2 mm, reperfusion 24 h) from the Sham and the SCIRI group (n = 3) Red: up-regulated expression levels; Blue: down-regulated expression levels. f. Volcano map and histogram showed the DEGs. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001. BBB, Basso, Beattie & Bresnahan locomotor rating scale; SCIRI, spinal cord ischemia reperfusion injury

To further reveal the underlying mechanism of SCIRI induced neuronal injury, RNA sequencing was performed on the spinal cord approximately 2 mm centered on the L2 segment 24 h after reperfusion (Fig. 1d and e). A total of 1091 differentially expressed genes (DEGs, Supplementary Table S1A) were identified after SCIRI, of which 529 genes were upregulated and 562 genes were downregulated (Fig. 1f).

Transcriptomic analysis and phenotype verification revealed SCIRI-induced cuproptosis in the spinal cord

Based on the Gene Ontology (GO) analysis, “electron transport chain”, “copper ion binding”, “secondary active transmembrane transporter activity”, “response to endoplasmic reticulum stress”, “flavin adenine dinucleotide binding”, “oxidoreductase activity”, “glutathione metabolic process”, and “cellular response to oxidative stress” showed significant difference. These GO terms are associated with lipoylation, copper metabolism, and oxidative stress response, suggesting that cuproptosis might play a very critical role in the damage of neurons caused by SCIRI (Fig. 2a). The results of transcriptomic analysis indicated that significant downregulation of ATP7B, which regulates copper ion efflux, and of FDX1, which regulates the lipoylation pathway. Both genes are the most dominant and critically relevant genes for cuproptosis [17]. We also screened other cuproptosis-related genes [17, 31], and the results showed that many of these were altered, though not statistically significant (Fig. 2b, Supplementary Table S1B).

Fig. 2.

Transcriptomic analysis and phenotype verifications revealed SCIRI-induced cuproptosis in the spinal cord. a. GO Chord plot according to GO enrichment analysis displayed the relationship between the significant difference terms and genes, including “electron transport chain”, “copper ion binding”, “secondary active transmembrane transporter activity”, “response to endoplasmic reticulum stress”, “flavin adenine dinucleotide binding”, “oxidoreductase activity”, “glutathione metabolic process”, and “cellular response to oxidative stress”. b. Overall expression heatmap of CuDEGs in the Sham and the SCIRI groups (n = 3) Red: upregulated expression levels; Cyan: downregulated expression levels. c. qPCR analysis of cuproptosis-related genes (ATP7B, FDX1, and SLC31A1, n = 3). d. WB showing expression levels of cuproptosis-related proteins (HSP70, ATP7B, DLST, FDX1, LIAS and SLC31A1). e. Densitometric analysis and quantification of proteins (n = 3). f. Representative images of the ventral horn regions of L2 spinal cord frozen sections labeled with FDX1 (green) and DAPI (blue) in each group (200×, scale bar = 100 μm). g. Quantitative fluorescence intensity of FDX1 (n = 3). Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; n.s., not significant. CuDEGs, differentially expressed cuproptosis-related genes; DAPI, 4′,6-diamidino‐2‐phenylindole

To verify the RNA-sequencing results, we performed qPCR and WB, which showed that the mRNA and protein expression of ATP7B and FDX1 were significantly down-regulated in the spinal cord of SCIRI rats compared with that of Sham rats, whereas there were no differences in mRNA and protein expression of SLC31A1 (Fig. 2c and e). In addition, we also investigated the protein HSP70 reflective of acute proteotoxic stress, and LIAS, which is an Fe-S cluster protein, and DLST, which is a lipoylated protein (Fig. 2d). The results indicated that decreased protein expression of DLST and LIAS, and increased expression of HSP70 in the SCIRI rats (Fig. 2e). In addition, we also performed immunofluorescence staining of FDX1 (Fig. 2f), and immunohistochemical staining for HSP70 and ATP7B (Supplementary Fig. S1a, S1c), all of which were consistent with the WB results (Fig. 2g, Supplementary Fig. S1b and S1d). Collectively, our findings indicated that SCIRI could induce cuproptosis in the spinal cord of rats.

Cuproptosis was activated by OGD/R

To further elucidate whether cuproptosis occurs in neurons after SCIRI, we performed in vitro phenotypic verifications. First, we performed OGD/R treatment on neuronal cells and used this to mimic neuronal damage from spinal cord ischemia reperfusion. CCK-8 and Calcein/PI staining results showed that OGD/R induced prominent neuronal death (Fig. 3a and c). Second, we further investigated mRNA of cuproptosis-related genes after OGD/R treatment. The results indicated that the mRNA expression of ATP7B and FDX1 was significantly down-regulated after OGD/R treatment, whereas there was no difference in the expression of SLC31A1 (Fig. 3d). Third, we also detected the protein expression of related genes (HSP70, ATP7B, DLST, FDX1, LIAS, and SLC31A1) (Fig. 3e). The results showed that decreased protein expression of ATP7B, DLST, FDX1, and LIAS, and increased protein expression of HSP70 after OGD/R treatment, whereas there was no difference in the expression of SLC31A1 (Fig. 3f). Finally, the immunofluorescence staining of FDX1 (Fig. 3g) and DLAT (Fig. 3i), all of which were consistent with the WB results (Fig. 3h and j). In addition, we tested the level of GSH, which can bind copper to prevent free copper toxicity. The results indicated that the GSH levels were significantly decreased after OGD/R treatment (Fig. 3k).

Fig. 3.

Cuproptosis was activated by OGD/R. a. Representative images of Calcein/PI staining labeled with Calcein (green) and PI (red) in each group (100×, scale bar = 50 μm). b. Cell survival rate in each group (n = 3). c. CCK8 assay was employed to analyze the cell’s viability in each group (n = 3). d. qPCR analysis of cuproptosis-related genes (ATP7B, FDX1, and SLC31A1; n = 3). e. WB showing expression levels of cuproptosis-related proteins (HSP70, ATP7B, DLST, FDX1, LIAS and SLC31A1). f. Densitometric analysis and quantification of proteins (n = 3). g. Representative images of cells labeled with FDX1 (green) and DAPI (blue) in each group (200×, scale bar = 100 μm). h. Quantitative fluorescence intensity of FDX1 (n = 3). i. Representative images of cells labeled with DLAT (red) and DAPI (blue) in each group (200×, scale bar = 100 μm). j. Quantitative fluorescence intensity of DLAT (n = 3). k. Relative levels of GSH in each group (n = 3). Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; n.s., not significant. OGD/R, oxygen and glucose deprivation/reoxygenation

Taken together, our results suggested that OGD/R could impair copper ion efflux, down-regulated Fe-S cluster proteins and lipoylated proteins, and decreased the level of GSH in neuronal cells after OGD/R treatment. In short, in neuronal cells, OGD/R damage can induce cuproptosis.

Cuproptosis was involved in OGD/R-induced neuronal cell death

To explore whether cuproptosis was involved in OGD/R-induced cell death, we used ammonium tetrathiomolybdate (ATTM), a well-known copper chelator, and a representative inhibitor for cuproptosis. We first tested the toxicity of ATTM. The CCK-8 results indicated that there was no difference in cell viability ranging from 0.25 µM to 20 µM treatment in normal neuronal cells (Fig. 4a). Next, the CCK-8 results showed that different concentrations of ATTM treatments all decreased OGD/R-induced cell death and 0.25 µM ATTM was chosen for subsequent experiments (Fig. 4a). Calcein/PI staining results showed that ATTM significantly decreased OGD/R-induced cell death (Fig. 4b and c).

Fig. 4.

ATTM ameliorated OGD/R-induced cells death. a. CCK8 assay was employed to analyze the cell’s viability in each group (n = 3). b. Representative images of Calcein/PI staining labeled with Calcein (green) and PI (red) in each group (100×, scale bar = 50 μm). c. Cell survival rate in each group (n = 3). d. Representative flow cytometry plots of JC-1 in each group, monomers (FITC) and aggregates (PE-A). e. Quantitative analysis ratio of aggregates to monomers in each group (n = 3). f. Representative images of JC-1 staining labeled with Monomers (green) and Aggregates (red) in each group (600×, scale bar = 300 μm). g. Quantification analysis ratio of aggregates in each group (n = 3). h. Relative levels of GSH in each group (n = 3). Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant. ATTM, ammonium tetrathiomolybdate

Cuproptosis occurs via the mitochondrial electron transport chain [17, 32]. Therefore, we applied JC-1 to assess the mitochondrial membrane potential and electron transport chain. The flow cytometry results indicated that JC-1 aggregates were largely decreased in the OGD/R group, but were reversed after ATTM treatment (Fig. 4d, e). The JC-1 fluorescence staining results were consistent with the flow cytometry results (Fig. 4f, g). In addition, ATTM treatment ameliorated OGD/R-induced GSH reduction (Fig. 4h).

Subsequently, we tested the protein level of cuproptosis-related genes (Fig. 5a). As expected, ATTM treatment downregulated HSP70 and the oligomerization of DLAT protein levels, and upregulated DLST, LIAS, and FDX1 protein levels (Fig. 5b). In addition, the immunofluorescence staining of FDX1 and DLAT was consistent with the above results (Fig. 5c and f). In summary, we speculated that excessive copper leads to irreversible cell death by inducing cuproptosis.

Fig. 5.

Cuproptosis was involved in OGD/R-induced neuronal cell death. a. WB showing expression levels of cuproptosis-related proteins (HSP70, DLST, FDX1, LIAS, DLAT, and SLC31A1). b. Densitometric analysis and quantification of proteins (n = 3). c. Representative images of cells labeled with FDX1 (red), Phalloidin (green) and DAPI (blue) in each group (200×, scale bar = 100 μm). d. Quantitative fluorescence intensity of FDX1 (n = 3). e. Representative images of cells labeled with DLAT (red), Mito-Tracker Green (green) and DAPI (blue) in each group (400×, scale bar = 200 μm). f. Quantitative fluorescence intensity of DLAT (n = 3). Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant

Cuproptosis inhibition ameliorated SCIRI-induced neuron death

We further explored the therapeutic impact of ATTM on the damaged neurons induced by SCIRI in rats. ATTM treatment could significantly reduce the SCIRI-induced neuronal loss according to the number of Nissl bodies (Fig. 6a and b), alleviate lower limb motor dysfunction based on BBB scores (Fig. 6c), and enhance GSH content compared to the SCIRI group (Fig. 6d). Moreover, WB results showed that the protein levels of DLST, FDX1, and LIAS were increased, and HSP70 and the oligomerization of DLAT were decreased in the ATTM treatment groups compared to the SCIRI group (Fig. 6e and f). To investigate the anti-cuproptosis effects of ATTM on neurons, DLAT was colocalized with NeuN, a neuronal marker (Fig. 6g). The fluorescence intensity of DLAT in neurons was decreased by ATTM administration compared to the SCIRI group (Fig. 6h). In addition, ATTM could improve the expression of FDX1 as shown by immunohistochemical staining compared to the SCIRI group (Fig. 6i and j). Altogether, our findings suggested that inhibition of cuproptosis is a feasible strategy for reversing neuronal injury in SCIRI rats.

Fig. 6.

Cuproptosis inhibition ameliorated SCIRI-induced neuron death in rats. a. Representative Nissl staining of spinal cord cross-section from L2 segment (100×, scale bar = 400 μm). b. Quantitative analyses of Nissl body in the ventral horn regions of the spinal cord (n = 3). c. The BBB scores of different groups at each time point (n = 6). d. Relative levels of GSH in each group (n = 3). e. WB showing expression level of cuproptosis-related proteins (HSP70, DLST, FDX1, LIAS, DLAT, and SLC31A1). f. Densitometric analysis and quantification of proteins (n = 3). g. Representative images of the ventral horn regions of L2 spinal cord frozen sections labeled with DLAT (red), NeuN (green) and DAPI (blue) in each group (200×, scale bar = 100 μm). h. Quantitative fluorescence intensity of DLAT (n = 3). i. Representative images of the ventral horn regions of L2 spinal cord paraffin sections of immunohistochemistry staining with FDX1 in each group (200×, scale bar = 200 μm). j. Analysis of the mean integrated option density of FDX1 in each group (n = 3). Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant

Discussion

In the present study, the results showed that the SCIRI rats had significantly lower BBB scores and Nissl body counts. RNA sequencing indicated that cuproptosis was significantly activated. Subsequent phenotype experiments demonstrated that SCIRI induced cuproptosis in neuronal cells along with copper efflux impairment and degradation of Fe-S cluster proteins and lipoylated proteins, and this process was dependent on cuproptosis. Notably, the copper chelator ATTM significantly ameliorated SCIRI-induced neuron loss and lower limb motor dysfunction in rats. More generally, these findings provide a novel role for neuronal cuproptosis in the pathogenesis of SCIRI.

Copper is a double-edged sword: It is a cofactor necessary for a variety of enzymes, but it can be toxic even at moderate intracellular concentrations, leading to cell death [8]. Tumor proliferation, metastasis, angiogenesis, and immune evasion occur due to genetic variation in copper homeostasis [22, 23, 33, 34]. In the past, there was ambiguity about the mechanism by which copper leading to cell death. However, as research has progressed, it has gradually been realized that copper toxicity occurs via a unique mechanism. Therefore, it was subsequently termed cuproptosis, which is distinct from all other known mechanisms of regulated cell death, including apoptosis, ferroptosis, and necroptosis [17, 18].

Spinal trauma, degeneration, and tumors have been reported to cause SCIRI [2, 3]. Our results suggested that regulating cellular copper might be an effective therapy for SCIRI. Our RNA-sequence results strongly suggested that cellular copper homeostasis might play a crucial role in the pathogenesis of SCIRI. ATP7B and SLC31A1 are currently the most studied regulators cellular copper [14, 15]. In our study, SCIRI downregulated ATP7B protein levels but not SLC31A1. We speculated that SCIRI might not only impede copper ion efflux but also attack the mitochondrial electron transport chain and deplete GSH. Though SCIRI involves complex pathophysiologic processes that are difficult to attribute to a specific cell death [4], in the present study we clearly demonstrated that SCIRI-induced neuronal death is at least partially attributable to cuproptosis.

Cuproptosis is a mitochondrial cell death pathway that is triggered by copper overloading, leading to proteotoxic stress [12, 35]. In this process, mitochondrial copper directly interacts with lipoylated proteins like DLAT, inducing their aggregation [36]. HSP70 is a molecular chaperone protein, which is upregulated during stress (such as hypoxia, oxidative stress), indicating acute proteotoxic stress [37]. DLST is the core component of the mitochondrial α-ketoglutarate dehydrogenase complex, which affects ATP production and metabolic homeostasis [38]. Ischemia-reperfusion leads to mitochondrial dysfunction, and the decrease in DLST activity may inhibit the TCA cycle, reduce ATP supply, and aggravate neuronal death [39]. LIAS is an Fe-S cluster protein, which participates in energy metabolism and has antioxidant functions [40]. Lack of LIAS activity may lead to lipoic acid deficiency, mitochondrial enzyme complex dysfunction, and exacerbation cell death [41]. Our results indicated that copper overloading was mainly attributed to be the down-regulation of ATP7B not the regulation of SLC31A1. ATP7B supports the biosynthesis of copper-dependent enzymes by transporting copper and enables the efflux of copper from the cell [42, 43]. Recent studies demonstrated that deletion of ATP7B leads to copper overloading and cuproptosis in the liver of Wilson’s disease mouse models [17, 44]. Our results show that ATP7B expression was downregulated by OGD/R, which stimulates the onset of cuproptosis. The in vivo results were consistent with the in vitro results. Our findings suggest that targeting cuproptosis may be an effective therapy for SCIRI.

For genetic disorders of copper dysregulation syndromes (Wilson’s disease and Menkes disease), These mechanisms involve SLC31A1 (copper importer) or ATP7A and ATP7B (copper exporters), which are encoded by genes that are mutated, respectively, and copper chelation is an effective treatment [45]. Inhibiting cuproptosis using a copper ion chelator is a promising strategy for SCIRI therapy. Our results suggest that ATTM-treated cells have higher GSH levels and iron-sulfur proteins, and lower DLAT oligomers, which inhibit the occurrence of cuproptosis in vitro. Consistent with this, ATTM administration in SCIRI rats exhibited increased lower limb motor function and neural survival. These findings indicate that ATTM could modulate copper homeostasis and promote resistance to SCIRI-induced cuproptosis in neurons.

There are still limitations in our study. In this study, we primarily focused on neurons; however, other cells (such as astrocytes, microglia, and oligodendrocytes) are also among the major cells in the spinal cord. Previous studies have reported that glial cell viability and differentiation affect the outcome of SCIRI [46, 47]. However, the mechanism is still not fully elaborated and further evidence, such as knockdown experiments via AAV-shRNA [48], is needed to support the role of cuproptosis in SCIRI. On the other hand, glial cells ultimately have an effect on nerve cells. This further emphasizes the critical role of cuproptosis in the pathogenesis of SCIRI. Therefore, more studies should be designed for further elucidation.

Conclusions

In conclusion, our findings supported the concept that ATP7B-mediated copper homeostasis in neurons played a vital role in the pathogenesis of SCIRI. Impaired copper ion efflux leads to accumulation of copper ions, resulting in significant GSH depletion, DLAT oligomerization, degradation of lipoylated and Fe-S cluster proteins, mitochondrial electron transport chain dysregulation, and ultimately leading to cuproptosis. We also identified the involvement of cuproptosis and the therapeutic effects of ATTM (Fig. 7). Insights into the molecular mechanism of cuproptosis may provide a new treatment option for SCIRI patients.

Fig. 7.

Hypothesized schematic presentation of the protective mechanism of ATTM in SCIRI. ATTM promotes the neuronal survival and motor functional recovery by inhibiting cuproptosis induced by spinal cord ischemia reperfusion injury

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ATTM

Ammonium tetrathiomolybdate

BBB

Basso, Beattie & Bresnahan locomotor rating scale

CCK-8

Cell counting kit-8

CuDEGs

Differentially expressed cuproptosis-related genes

DAPI

4′,6-diamidino-2-phenylindole

DMEM

Dulbecco’s minimum essential medium

FBS

Fetal bovine serum

Fe-S

Iron-sulfur

GSH

Glutathione

JC-1

5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide

NeuN

Neuronal nuclei

OGD/R

Oxygen and glucose deprivation/reoxygenation

PBS

Phosphate-buffered saline

qPCR

Quantitative PCR

RIPA

Radio immunoprecipitation assay lysis buffer

SCIRI

Spinal cord ischemia reperfusion injury

WB

Western blot

Author contributions

Lei Xie and TengBo Yu conceived the study. Lei Xie and Xiao Xiao designed the research; Lei Xie, Hang Wu, Qiuping He performed the experiments; Lei Xie and Weipeng Shi contributed essential reagents or tools. Lei Xie, Hang Wu, and Qiuping He analyzed the data. Lei Xie and Xiao Xiao wrote the paper. TengBo Yu and Xiao Xiao provided suggestions. All authors have read and approved the article.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82472431, 82401552), Shandong Provincial Natural Science Foundation (No. ZR2024QH590), Shandong Province Major Scientific and Technical Innovation Project (No. 2021SFGC0502), and Qingdao Science and Technology Benefit People Demonstration Guide Special Project (22-3-7-smjk-5-nsh).

Data availability

The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

All animal operations and care procedures were approved by the Laboratory Animal Welfare Ethics Committee of Qingdao University (approval number: 20230917SD20231225065) and in accordance with the Basel Declaration.

Consent for publication

All authors have read the manuscript, and agree with the consent for publication.

Competing interests

The authors declare that they have no competing interests.