Curcumin Attenuates Cuproptosis via Activating Autophagy Through Inhibition of the AKT/mTOR/P70S6K-Signaling Pathway in Parkinson’s Disease Models

Abstract

This research surveyed the therapeutic potential of curcumin (Cur) in Parkinson’s disease (PD), focusing on its effects on cuproptosis and underlying molecular mechanisms. A MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse model and a MPP⁺ (1-methyl-4-phenylpyridinium)-treated PC12 cell model were used in this study. In vivo, Cur treatment significantly mitigated MPTP-treated dyskinesia and lessened the damage of dopaminergic (DA) neurons in SNpc. Additionally, Cur reversed MPTP-induced changes by increasing TH (tyrosine hydroxylase) expression and decreasing α-syn (α-synuclein) accumulation in the SN. In vitro, Cur mitigated MPP⁺-treated apoptosis and the cytotoxicity of differentiated PC12 cells. Furthermore, Cur reversed MPTP/MPP⁺-induced changes in the cuproptosis-related protein expression, including DLAT (dihydrolipoamide S-acetyltransferase), FDX1 (ferredoxin 1), and upregulating SLC31A1 (solute carrier family 31 member 1) and HSP70 (heat shock protein 70). 3-MA (3-methyladenine) reversed Cur-mediated expression levels of DLAT, FDX1, SLC31A1, and HSP70 in the PD models. Mechanistically, Cur decreased the expression of p-AKT (p-protein kinase B), p-mTOR (p-mammalian target of rapamycin), and p-P70S6K (p-70 KDa ribosomal protein S6 kinase‌) in the PD models, suggesting it has an inhibitory effect on the AKT/mTOR/P70S6K signaling pathway. Furthermore, pretreatment with SC79 (an AKT activator) reversed Cur-induced autophagy activation, supporting the role of this pathway in Cur-mediated neuroprotection. Cur protected against DA neuronal loss by modulating the interplay between cuproptosis and autophagy via the suppression of the AKT/mTOR/P70S6K. The study findings provide novel insights into the mechanism of Cur’s neuroprotective effect, highlighting the AKT/mTOR/autophagy/cuproptosis axis as a potential target and Cur as a medicant for PD management.

Introduction

Parkinson’s disease (PD) is a global neurodegenerative disease that, according to recent epidemiological data, affects more than 6 million individuals. This number is projected to double within the next three decades [1, 2]. Tyrosine hydroxylase (TH) has been established as a unique marker of dopaminergic (DA) neurons, and α-synuclein (α-syn) aggregation has been recognized as a hallmark of PD [3, 4]. Although the precise etiology of PD remains still uncertain, it is widely acknowledged to be a multifactorial disorder resulting from the complicated interrelationship of environmental factors and genetic predispositions that disrupt critical cellular processes [5]. Despite advancements in symptomatic treatments [6], including dopaminergic therapies, no available interventions can effectively halt or slow the progressive neurodegeneration in PD, underscoring the urgent need to identify novel treatment targets and strategies for disease management.

Studies have implicated copper ion (Cu²⁺) dyshomeostasis in the pathogenesis of various central nervous system (CNS) disorders, including PD [7, 8]. Excessive copper accumulation has been shown to induce “cuproptosis,” a recently acknowledged modality of programmed cell death [9]. The copper ion concentration and cuproptosis-associated proteins, including DLAT (dihydrolipoamide S-acetyltransferase), ferredoxin 1 (FDX1), the solute carrier family 31 member 1 (SLC31A1), and heat shock protein 70 (HSP70), serve as the unique markers of cuproptosis [10]. Elevated Cu²⁺ levels promote α-syn aggregation, induce mitochondrial dysfunction, and exacerbate dopaminergic (DA) neuronal degeneration, thereby accelerating PD progression [11]. Nevertheless, the precise role of cuproptosis in PD remains poorly understood. Thus, regulating copper homeostasis and inhibiting cuproptosis represent a promising approach for mitigating DA neuronal injury.

Curcumin (Cur) has demonstrated neuroprotective potential through its anti-inflammatory, antioxidant, and autophagy-regulating properties [12–14]. Notably, Cur can chelate free copper ions and enhance copper metabolism in murine models, potentially inhibiting cuproptosis-related pathways and attenuating copper-induced cellular damage [15]. Although studies suggest that Cur plays a neuroprotective role against copper-mediated cytotoxicity, the precise mechanisms by which Cur regulates cuproptosis in PD remain poorly understood, warranting their further investigation.

Cuproptosis has been associated with autophagy, with evidence suggesting that Cu²⁺ accumulation activates autophagic signaling [16]. Thus, cuproptosis is considered a form of autophagy-dependent cell death [17]. Mechanistically, copper-induced autophagy involves increasing autophagy-related proteins, including LC3, SQSTM1, and ATG5 [18], regulating the AMPK-mTOR signaling pathway [19], or inducing oxidative stress [20, 21]. However, the regulatory role of autophagy in copper-driven neuronal death in PD remains elusive.

The dynamic interplay between cuproptosis and autophagy in PD is not understood. Additionally, the potential molecular mechanisms underlying the neuroprotective effects of Cur, particularly through modulating cuproptosis and autophagy, require further investigation. In this study, a PD model was established in C57BL/6J mice using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the corresponding in vitro PD model was generated by treating PC12 cells (differentiated to exhibit DA neuron-like properties) with 1-methyl-4-phenylpyridinium (MPP⁺). These models were used to evaluate the neuroprotective effects of Cur, with a specific focus on its effect on cuproptosis in DA neurons and the underlying molecular mechanisms. This study provides experimental evidence supporting Cur as a potential disease-modifying agent and offers valuable insight into therapeutic strategies targeting copper-mediated neurotoxicity in PD.

Materials and Methods

Animals

A total of 80 specific pathogen-free (SPF)-grade male C57BL/6J mice (8 weeks old) were obtained from Hubei University of Medicine Animal Research Center: SCXK [E] 2019-0008; SYXK [E] 2019-0031 (experimental animal use license). All experimental protocols were approved by the Institutional Ethics Committee on Animal Care and Use, and all experimental protocol was approved by the Ethics Committee of Hubei University of Medicine (No. 2021–059, 20/10/2021). A maximum of five mice were housed per cage and raised under standard conditions (22 ± 2 °C room temperature, a 12-h light/dark cycle, and 50–70% relative humidity).

Animal Model Grouping

The mice were subjected to adaptive behavioral training using pole-climbing and suspended rope tests once daily for 3 consecutive days after a 1-week acclimatization period. They were randomly assigned to three primary cohorts, each comprising four groups (n = 10 per group). The first cohort included the control, MPTP group, Cur group (MPTP + Cur), and ES group (MPTP + Cur + ES) (elesclomol, a cuproptosis activator), while the second cohort comprised the control, MPTP group, Cur group (MPTP + Cur), and 3MA (3-methyladenine) group (MPTP + Cur + 3MA). The third cohort comprised the control, MPTP group, Cur group (MPTP + Cur), and SC79 group (MPTP + Cur + SC79). The treatment regimens were administered as follows: the control group received intraperitoneal injections of sterile physiological saline every day; the MPTP group was administered intraperitoneal injections of MPTP (25 mg/kg, 23007-85-4, Sigma-Aldrich, MO, USA) for 7 days to induce DA neurotoxicity; the MPTP + Cur group received Cur (80 mg/kg, C1386, Sigma-Aldrich, MO, USA) by oral gavage 1 h before MPTP administration for 7 consecutive days; the MPTP + Cur + ES (elesclomol) group was administered Cur (80 mg/kg) by oral gavage 1 h before MPTP administration, followed by intraperitoneal injection of ES (10 mg/kg, SML2651, Sigma-Aldrich, MO, USA) 30 min later; the MPTP + Cur + 3MA group was administered Cur (80 mg/kg) by oral gavage 1 h before MPTP administration, followed by intraperitoneal injection of 3MA (HY-19312, MedChemExpress, NJ, USA) at 15 mg/kg 30 min later; the MPTP + Cur + SC79 group was administered Cur (80 mg/kg) by oral gavage 1 h before MPTP administration, followed by intraperitoneal injection of SC79 (10 mg/kg, 123871, Sigma-Aldrich, MO, USA) 30 min later. Behavioral assessments were conducted on day 8, following completion of the 7-day treatment protocol, as shown in Fig. 1A.

Fig. 1.

Cur inhibited body weight loss and improved MPTP-induced behavioral deficits in C57BL/6J mice. The mice were randomly assigned included control, MPTP group, Cur group (MPTP + Cur), and ES group (MPTP + Cur + ES). The MPTP group received daily intraperitoneal injections of MPTP (25 mg/kg/day). The Cur group received daily intraperitoneal injections of Cur (80 mg/kg/day) 1 h before MPTP administration. The ES group received daily injections of ES (10 mg/kg/day) and Cur (80 mg/kg/day) 1 h before MPTP. Control mice received intraperitoneal injections of normal saline (25 mL/kg). A Graphical timeline of the experimental procedure. B Cur inhibited MPTP-induced weight loss in mice. Pole-climbing time (C) and wire-hanging time (D) were assessed. E The representative trajectory of NOR of mice in each group. F The result of new object recognition experiment of mice in each group. n = 5 mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Pole-Climbing Assay

The motor coordination and exercise tolerance of mice were evaluated using the pole-climbing test [22]. A rough-surfaced cylindrical wooden pole (approximately 60 cm in length and 2 cm in diameter) was positioned at a 60° angle from the horizontal plane. Each mouse was placed head-downward on the top, and the time required to descend from the top to the bottom of the pole was recorded. Each mouse performed three tests, and the average descent time was recorded and used as the climbing performance score. Shorter times indicated better motor function.

Suspended Rope Test

Motor coordination and muscular strength were assessed using the suspended rope test [23]. A hemp rope, 80 cm in length and 25 cm above the ground, was horizontally secured between two platforms. Each mouse was placed at the midpoint of the rope, gripping the rope with its front paws. Timing was initiated once the mouse firmly gripped the rope, and the time it took for the mouse to reach either platform was recorded. Each mouse performed three tests. The mean time was calculated and used as the final performance score. Shorter traversal times indicated better motor coordination and muscle strength.

The Novel Object Recognition (NOR) Assay

Recognition memory and cognitive function which are often impaired in PD [24] were assessed using the NOR test. Mice were tested in a non-transparent cubic box measuring 25 cm × 25 cm at the base and 40 cm in height. Object A and object B were symmetrically laid in the apparatus, 10 cm away from the side and rear walls, and fixed to prevent displacement. Each mouse was gently placed in the apparatus equidistant between the objects, during which the interaction time with each object was recorded. Each apparatus was disinfected before testing the next mouse. Following a 1-h inter-trial interval, object B was in place of object C, differing in color and shape. The same procedure was used to record exploration behavior and contact time with objects A and C. The discrimination index (DI) value for each mouse was computed using the formula: DI (%) = [(time exploring novel object − time exploring familiar object)/(total exploration time)] × 100%.

Quantification of Copper Content in Mouse Midbrain Tissues

The copper content assay kit (BC5755, Solarbio, Beijing, China) was used to assess the copper ion concentration in the SN. Approximately 0.1 g of mouse midbrain tissue was accurately weighed and homogenized in an Eppendorf tube containing 1 mL of distilled water. The homogenate was then centrifuged at 4 °C for 10 min at 10,000 × g, and the resulting supernatant was collected. A visible spectrophotometer (TU-1901, Persee, Beijing, China) was used to determine the copper content. The spectrophotometer was pre-warmed for 30 min, set to a wavelength of 580 nm, and zeroed with distilled water. Blank, standard, and test samples were prepared. Distilled water, tissue supernatant, copper standard solution, reagent 1, and reagent 2 were added sequentially to an Eppendorf tube. The mixtures were incubated in the dark at 37 °C for 5 min. Then, the reaction mixtures were transferred to 1-mL glass cuvettes, and the absorbance was immediately measured at 580 nm and recorded as A_blank, A_standard, and A_test. The blank and standard tubes were measured 1 to 2 times to ensure consistency. The tissue copper content was calculated using the formula: tissue copper content (μM/g) = 0.08 × (Δ_test/Δ_standard)/W, Δ_test = A_test − A_blank, Δ_standard = A_standard − A_blank, where W represents the tissue weight in grams.

Immunohistochemical Staining

Mice were given an intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg, P3761, Sigma-Aldrich, MO, USA). Systemic perfusion was initiated with 35–50 mL of ice-cold phosphate-buffered saline (PBS, G4207, Servicebio, Wuhan, China) until hepatic blanching was observed, indicating effective circulatory clearance. This clearance was followed by perfusion with pre-cooled 4% paraformaldehyde (PFA, BC539A, Biosharp, BJ, China) until signs of tissue fixation, such as tail elevation, muscle tremors, and body stiffness, were evident. All mice were decapitated, the cranial vaults were opened, and the intact brains were carefully harvested. A 4% PFA solution was used to post-fix the brains for 48–72 h using a fixative volume at least five times the tissue volume. The fixed specimens were labeled with group identifiers and fixation dates and stored at 4 °C until processing. The midbrain was grossly dissected, transferred into labeled embedding cassettes, and rinsed in ultrapure water for 8–12 h to remove residual fixatives. Tissue dehydration was accomplished utilizing 75%, 85%, 95%, and anhydrous ethanol I and II, followed by clearing in a 1:1 solution of anhydrous ethanol and xylene (20230502, Tianli, Tianjin, China) and subsequently in xylene I and II. Tissues were then infiltrated with paraffin (wax immersion I and II) and embedded in paraffin blocks, and then were cooled to allow solidification and stored at room temperature. The blocks were equilibrated in an ice-water mixture for 10 min after pre-cooling at −20 °C for 4 h. The blocks were cut into 4-μm-thick sections using a microtome (Leica, HDB, GER, Leica RM2245). Tissue sections were treated in xylene I, II, and III (15 min each), followed by rehydration in a 100%, 95%, 85%, 75%, and 50% graded ethanol series. The slides were then immersed in deionized water for 5 min heated in a microwave oven to 92–96 °C for optimal antigen exposure, and then cooled in retrieval solution. Then, the sections were washed three times with PBS for 5 min each. The sections were incubated in 3% hydrogen peroxide (15 min), followed by washing three times with PBS for 5 min each. The sections were blocked by incubating with 50 μL of 5% donkey serum (ANT030, AntGene, Wuhan, China) for 1 h at 37 °C. Then, 20 μL of a rabbit primary antibody TH (1:200, 58844S, Cell Signaling Technology, MA, USA), or else a sufficient volume to fully cover the tissue was applied to each section, followed by incubation overnight at 4 °C. The sections were washed with PBS-Tween three times for 10 min each and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (1:200, P8002, NCM Biotech, Suzhou, China) at room temperature for 1 h in a humidified chamber. The slides were washed twice with PBS (G4207, Servicebio, Wuhan, China) for 10 min each. Diaminobenzidine (DBA, 20230502, Tianli, Tianjin, China) substrate solution was freshly prepared at a 1:20 dilution under light-protected conditions and applied (approximately 20 μL) to each tissue section for chromogenic detection. Color development was observed using a microscope (IX53 + DP73, Olympus, Japan). A positive signal appeared as brown-yellow staining against a clear background. When optimal staining was achieved, the chromogenic time of the tissue was recorded. Hydration of the sections was maintained throughout the staining process. Following staining, the slides were washed three times with PBS for 5 min each, and the nuclei were counterstained with hematoxylin (G1004, Servicebio, Wuhan, China). The slides were washed with distilled water, followed by a brief differentiation (2 s) in 1% hydrochloric acid and additional washes. Nuclear staining was assessed using a microscope. The sections were then dehydrated through two 5-min immersions in 100% anhydrous ethanol and cleared with xylene for 10 min each. Following this, the sections were coverslipped using neutral resin. The immunostained sections were visualized under a microscope, and the data were analyzed.

Cell and Treatments

PC12 cells (differentiated rat adrenal pheochromocytoma cells, Catalog No. SCSP-517) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. RPMI-1640 (72400047, Gibco, NY, USA) was supplemented with 10% fetal bovine serum (FBS, 11011-8611, Gibco, NY, USA). The cells were randomly assigned to three primary cohorts, each comprising four groups. The first cohort included the control, MPP⁺ (1 mM, 144783, Abcam, Cambridge, UK) group, Cur (10 μM) (MPP⁺ + Cur) group, and ES (200 nM) (MPP⁺ + Cur + ES) group; the second cohort comprised the control, MPP⁺ group, Cur (MPP⁺ + Cur) group, and 3MA (2 mM) (MPP⁺ + Cur + 3MA) group; and the third cohort comprised the control, MPP⁺ group, Cur (MPP⁺ + Cur) group, and SC79 (10 μM) (MPP⁺ + Cur + SC79) group.

The Cell Counting Kit (CCK)−8 Assay

The CCK-8 assay (96992, Sigma-Aldrich, MO, USA) was used to assess cell viability. PC12 cells were seeded in 96-well plates at a density of 5 × 10³ cells in 100 μL of complete medium per well. MPP⁺ (0, 0.25, 0.5, 1, 2, or 4 mM) or Cur (0, 5, 10, 15, 20, 25, and 30 μM) was added, with four replicate wells per group. After 24 h of incubation, 100 μL of fresh medium containing 10 μL of CCK-8 reagent was added in 96-well plates. The plates were protected from light and incubated for 1–2 h, ensuring that the color development was not excessively dark. Absorbance was recorded using a microplate reader (Thermofisher, MA, USA) at 450 nm. To determine the protective effect of Cur against MPP⁺-induced cytotoxicity, the following experimental groups were established: blank control, negative control, MPP⁺ (1 mM), and MPP⁺ (1 mM) + Cur (at final concentrations of 0, 5, 10 μM) groups. Cell viability was determined by recording the absorbance at 450 nm using a microplate reader.

Flow Cytometry

An Annexin V-FITC/PI Apoptosis Detection Kit (MA0220, MeilunBio, Dalian, China) was used to assess apoptosis. The cell culture medium was collected into pre-labeled 15-mL centrifuge tubes. Adherent cells were detached using trypsin without EDTA. Once the cells became rounded, the trypsin solution was aspirated, and residual trypsin activity was neutralized by adding the original culture medium. The cells were gently resuspended, and approximately 1 × 10⁶ cells were collected and centrifuged at 1100 rpm at 4 °C for 3 min to obtain cell pellets. The pelleted cells were washed twice with pre-cooled PBS. Then, the cells were resuspended, and the corresponding fluorescent dyes were added to each group according to the kit’s instructions. After mixing, 400 μL of 1 × binding buffer was added to the cells and incubated for 10 min at room temperature in the dark. Flow cytometric analysis was performed to assess the percentage of apoptotic cells.

Transmission Electron Microscopy (TEM)

PC12 cells were divided into four groups: the control, MPP⁺ (1 mM) group, Cur (10 μM) (MPP⁺ + Cur) group, and ES (200 nM) (MPP⁺ + Cur + ES) group. These group were seeded in 6-well plates. After treatment, the cells were fixed with 2.5% glutaraldehyde (P1127, Solarbio, Beijing, China) in PBS overnight at 4 °C. The cells were then post-fixed in 1% osmium tetroxide (201030, Sigma-Aldrich, MO, USA) for 1 h, dehydrated through a graded series of ethanol (30%, 50%, 70%, 90%, and 100%), and embedded in epon resin (Head-EPON™ 812, Headbio, Beijing, China). Ultrathin sections (60–80 nm) were prepared using an ultramicrotome, stained with uranyl acetate and lead citrate, and observed via TEM (Talos F200X, Thermo Fisher Scientific, MA, USA). Images were acquired digitally.

Immunofluorescence Staining

PC12 cells were seeded in 24-well plates according to the experimental grouping. The cells were treated with designated compounds for 24 h and then fixed with 4% PFA for 20 min with gentle agitation at room temperature. After fixation, the cells were washed three times with PBS. The cell membranes were permeabilized by incubating for 20 min at room temperature with 0.5% Triton X-100, followed by three washes with PBS. Non-specific antibody binding was blocked by incubation with 5% goat serum for 1 h at room temperature. Without removing the blocking solution, the primary antibody (1:100, T5106718, Abmart, Shanghai, China) was added at the appropriate dilutions, and the cells were incubated at 4 °C overnight. The next day, the primary antibody was removed, and the cells were washed five times with PBS for 5 min each on a shaker. Fluorescently labeled secondary antibody (1:200, ANT045, AntGene, Wuhan, China) was then added to the cells and incubated in the dark at room temperature for 1 h. The cells were washed three times with PBS for 5 min each. Nuclei were counterstained with DAPI for 10 min, followed by three PBS washes of 5 min each. The fluorescence was observed using an inverted fluorescence microscope.

Monodansylcadaverine (MDC) Staining

Autophagosomes were visualized using MDC staining kit (C3018, Beyotime, Shanghai, China). PC12 cells were incubated in laser confocal culture dishes. The cells were treated with the appropriate agents and incubated further, according to the experiment protocol. The following day, the cells were incubated with 1 mL of MDC staining solution per well for 30 min in the dark. The incubation period was adjusted between 10 and 60 min based on the observed staining intensity. After removing the MDC staining solution, the cells were washed three times with 1 × assay buffer. Following aspiration of the buffer, 1 mL of assay buffer was added. MDC-labeled autophagosomes were visualized using a laser confocal microscope with appropriate excitation wavelengths to stimulate the cells and to visualize the green fluorescence emitted by MDC. Images were captured and recorded for further analysis.

Western Blot (WB) Analysis

Protein samples were lysed in RIPA buffer (C1053+, APPLYGEN, Shanghai, China). The BCA (BCA1, Sigma-Aldrich, MO, USA) working reagent was used to assess the protein concentrations. Protein samples were boiled at 100 °C for 10 min to ensure complete denaturation and stored at −20 °C until electrophoresis. The protein samples were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, P0675, Beyotime, Shanghai, China), then transferred to polyvinylidene difluoride membranes (PVDF membranes, ISEQ00010, Merck Millipore, Darmstadt, Germany). The samples were blocked with in TBST (5% milk) at room temperature for 1 h and then incubated with primary antibodies at 4 °C overnight. The primary rabbit antibodies were as follows: anti-α-syn (1:1000, 4179S, Cell Signaling Technology, MA, USA), anti-TH (1:1000, 58844S, Cell Signaling Technology, MA, USA), anti-FDX1 (1:1000, T510671S, Abmart, Shanghai, China), anti-GAPDH (1:1000, 13038S, Cell Signaling Technology, MA, USA), anti-LC3B (1:1000, ab192890, Abcam, Cambridge, UK), anti-DLAT (1:1000, 13426-1-AP, Sanying, Wuhan, China), anti-HSP70 (1:1000, T55150F, Abmart, Shanghai, China), anti-SLC31A1 (1:1000, T510261S, Abmart, Shanghai, China), anti-SQSTM1/p62 (1:1000, 5114S, Cell Signaling Technology, MA, USA), anti-P-AKT (1:1000, 4060 T, Cell Signaling Technology, MA, USA), anti-AKT (1:1000, 4691 T, Cell Signaling Technology, MA, USA), anti-P-mTOR (1:1000, 2971 T, Cell Signaling Technology, MA, USA), anti-mTOR (1:1000, 2972S, Cell Signaling Technology, MA, USA), anti-P-P70S6K (1:1000, 9208 T, Cell Signaling Technology, MA, USA), anti-P70S6K (1:1000, 2708S, Cell Signaling Technology, MA, USA). The following day, the samples were washed with 1 × TBST (8 min × 5) and incubated with a secondary antibody (1:3000, 7074S, Cell Signaling Technology, MA, USA) for 1 h. The samples were then washed again with TBST (8 min each). The samples were visualized using an enhanced chemiluminescence (ECL; BL520B, Biosharp, Beijing, China) detection system. Equal volumes of ECL ultra-sensitive solutions A and B were mixed in a light-protected environment to prepare the chemiluminescent working solution. The membranes were briefly drained and incubated with the working solution. Chemiluminescent signals were captured using a gel imaging system.

Statistical Analysis

Data are expressed as the mean ± standard deviation (SD). All statistical data were analyzed using SPSS 20.0 software (IBM, Armonk, NY, USA) or GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance followed by Tukey’s post hoc test was used to assess statistical comparisons between means. A P value of <0.05 was considered statistically significant.

Results

Cur Ameliorates Motor Impairment and Weight Loss in MPTP-Treated PD Mice

Weight loss, which is commonly observed in MPTP-induced PD mouse models, is associated with motor impairment, DA neuron degeneration, gastrointestinal dysfunction, and oxidative stress [25]. The daily body weight measurements are shown in Fig. 1B. In this study, MPTP-treated mice exhibited significantly greater weight loss than the control group, although during pretreatment with Cur (80 mg/kg) evidently mitigated this weight loss (Fig. 1B). Pole-climbing and rope suspension tests were used to evaluate motor function. MPTP treatment significantly prolonged rope suspension (Fig. 1C) and pole-climbing (Fig. 1D) times compared with the controls. Conversely, Cur-treated mice demonstrated significantly shortened rope suspension (Fig. 1C) and pole-climbing (Fig. 1D) times compared with the MPTP group, indicating improvement in motor dysfunction. Cognitive function was assessed using the NOR test, a standard behavioral assay used for evaluating memory and learning in rodents [22]. The result of NOR index results revealed a significant decrease in the MPTP group compared with the control group, displaying cognitive impairment (Fig. 1E, F). In contrast, Cur pretreatment significantly increased NOR index values compared with the MPTP group, suggesting a protective effect against cognitive deficits (Fig. 1E, F).

Cur Reduces DA Neuron Loss and Inhibits α-syn Aggregation in the SNpc of MPTP-Induced PD Mouse Models

Progressive DA neuron loss in the SNpc, often accompanied by abnormal α-syn aggregation, is a hallmark of PD [26]. TH serves as a unique marker of DA neurons [27]. In our research, immunohistochemistry was used to quantify TH-positive neurons, and WB was employed to assess α-syn and TH protein expressions in midbrain tissues of mice and to assess whether Cur mitigates α-syn aggregation and DA neuron loss in MPTP-treated PD models. The results revealed that MPTP treatment significantly decreased TH-positive neurons in the SNpc compared with the controls (Fig. 2A, B). In contrast, Cur pretreatment increased TH-positive neurons compared with the MPTP group (Fig. 2A, B). WB showed a remarkable increase in a α-syn expression (Fig. 2C, D) and a decrease in TH expression (Fig. 2C, D) in the MPTP group compared with the control group. Conversely, Cur-pretreated mice exhibited downregulated α-syn (Fig. 2C, D) and upregulated TH expression (Fig. 2C, D) levels compared with MPTP-treated mice. These findings suggest that Cur protects DA neurons by preserving TH expression and inhibiting α-syn aggregation in MPTP-induced PD mice.

Fig. 2.

Cur protected against dopaminergic neuron loss in the SN. The MPTP group received daily intraperitoneal injections of MPTP (25 mg/kg/day). The Cur group received daily intraperitoneal injections of Cur (80 mg/kg/day) 1 h before MPTP administration. The ES group received daily injections of ES (10 mg/kg/day) and Cur (80 mg/kg/day) 1 h before MPTP. Control mice received intraperitoneal injections of normal saline (25 mL/kg). A TH-positive cells were stained by IHC and observed through a fluorescence microscope. The scale bar represents 200 μm (top) and 50 μm (bottom). B Number of TH-positive cells. C and D Western blot analysis of TH and α-synuclein protein levels. n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Cur Protects Against DA Neuron Loss in MPTP-Induced PD Mice via Inhibiting Cuproptosis

Studies have suggested that a copper-dependent form of regulated cell death, called cuproptosis, is closely related to PD pathogenesis [28, 29]. Copper concentrations in the SNpc of the midbrain were quantified in all treatment groups to determine the function of Cur in modulating cuproptosis. Copper concentrations were evidently reduced in MPTP-treated mice compared with the control group (Fig. 3A). Copper levels were significantly higher in Cur-pretreated mice than in the MPTP group (Fig. 3A). In contrast, mice co-treated with the cuproptosis inducer ES and Cur exhibited significantly lower copper levels compared with the group treated with Cur alone (Fig. 3A). WB was used to evaluate the cuproptosis-related protein expression in midbrain tissues, including DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70, in midbrain tissues. MPTP treatment downregulated FDX1 and DLAT and upregulated SLC31A1 and HSP70 protein expression compared with the controls (Fig. 3B, D). In Cur-pretreated mice, FDX1 and DLAT protein expression was significantly increased, while SLC31A1 and HSP70 protein expressions were evidently reduced compared with the MPTP-induced mice (Fig. 3B, D). However, combined treatment with ES and Cur resulted in downregulated FDX1 and DLAT and upregulated SLC31A1 and HSP70 expression levels compared with mice treated with Cur alone (Fig. 3B, D). Treatment with MPTP significantly induced DLAT oligomerization, whereas Cur treatment markedly reversed this effect (Fig. 3C, D). Meanwhile, the result revealed that ES administration significantly exacerbated weight loss compared with Cur treatment, as shown in Fig. 1B. Behavioral assessments revealed that ES significantly increased pole-climbing and wire-hanging time and decreased the NOR index values compared with Cur treatment alone, indicative of worsened motor dysfunction (Fig. 1C–F). Immunohistochemical result displayed a remarkable decrease in TH-positive neurons in ES-treated mice compared with the Cur-treated mice (Fig. 2A, B). Additionally, ES treatment reversed the Cur-induced effects on TH and α-syn protein expression levels (Fig. 2C, D). The results collectively show that Cur exerts neuroprotective effects in MPTP-induced PD mice by alleviating cuproptosis-associated DA neuronal injury.

Fig. 3.

Cur reduced cuproptosis in MPTP-treated mice. The MPTP group received daily intraperitoneal injections of MPTP (25 mg/kg/day). The Cur group received daily intraperitoneal injections of Cur (80 mg/kg/day) 1 h before MPTP administration. The ES group received daily injections of ES (10 mg/kg/day) and Cur (80 mg/kg/day) 1 h before MPTP. Control mice received intraperitoneal injections of normal saline (25 mL/kg). A Copper content in the brain tissue. B and C Western blot of DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70. D Western blot analysis of DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70. n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Cur Attenuates Cuproptosis in MPTP-Induced PD Mice in an Autophagy-Dependent Manner

Several studies have suggested that cuproptosis may be an autophagy-dependent form of cell death, and impaired autophagy may exacerbate neuronal damage associated with dysregulated copper metabolism [30, 31]. TH-positive neurons were quantified using immunohistochemistry to investigate whether Cur mediates its protective effect through autophagy. TH-positive neurons of MPTP-induced group were evidently reduced compared with the control group (Fig. 4A, B), and Cur pretreatment significantly increased the number of TH-positive neurons (Fig. 4A, B) compared with the MPTP-induced group. Co-treatment with Cur and the autophagy inhibitor 3-MA significantly decreased TH-positive neurons compared with Cur treatment alone (Fig. 4A, B), indicating that autophagy inhibition negates Cur-mediated neuroprotection.

Fig. 4.

Cur reduces cuproptosis via inhibiting autophagy in MPTP-induced PD model. The mice were randomly assigned included control, MPTP group, Cur group (MPTP + Cur), and 3-MA group (MPTP + Cur + 3-MA). The MPTP group received daily intraperitoneal injections of MPTP (25 mg/kg/day). The Cur group received daily intraperitoneal injections of Cur (80 mg/kg/day) 1 h before MPTP administration. The 3-MA group was administered daily intraperitoneal injections of 3-MA (15 mg/kg/day) and Cur (80 mg/kg/day) 1 h before MPTP administration. Control mice received intraperitoneal injections of normal saline (25 mL/kg). A TH-positive cells were stained by IHC and observed through a fluorescence microscope. The scale bar represents 200 μm (top) and 50 μm (bottom). B Number of TH-positive cells. C and D Western blot analysis of LC3 and P62. E and F Western blot analysis of DLAT, FDX1, SLC31A1, and HSP70. n = 3–5 mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

The levels of autophagy-related protein expression were assessed by WB to further explore the role of autophagy. MPTP-treated mice showed significantly decreased LC3II/I expression and increased P62 levels compared with the controls (Fig. 4C, D), suggesting autophagy inhibition. In contrast, Cur-pretreated mice demonstrated significantly decreased P62 expression and increased LC3II/I expression compared with the MPTP group, indicative of autophagy activation (Fig. 4C, D). Co-treatment with Cur and 3-MA significantly decreased LC3II/I and increased P62 expression compared with Cur treatment alone (Fig. 4C, D). These findings indicate that MPTP suppressed autophagic flux, whereas Cur treatment restored it. Notably, pharmacological inhibition of autophagy via 3-MA partially abolished the neuroprotective effects of Cur, suggesting a functional involvement of autophagy in Cur-mediated protection.

The levels of cuproptosis-related protein expression were assessed in the midbrain using WB to determine whether Cur regulates cuproptosis by activating autophagy. The results showed an evident reduction in DLAT and FDX1 expression and an increase in SLC31A1 and HSP70 protein expression in the MPTP-induced group compared with the control (Fig. 4E, F). DLAT and FDX1 expression was significantly increased in Cur-pretreated mice, while SLC31A1 and HSP70 protein expression was decreased compared with the MPTP-induced mice (Fig. 4E, F). Co-treatment with 3-MA and Cur significantly decreased DLAT and FDX1 expression and increased SLC31A1 and HSP70 expression compared with Cur treatment alone (Fig. 4E, F). These findings suggest that in an MPTP-induced PD mouse model, Cur alleviates cuproptosis-like damage in DA neurons, which is associated with the activation of autophagy.

Cur Antagonizes Decreases in PC12 Cell Viability and Apoptosis in the MPP⁺-Induced Cell Model

PC12 cells, which exhibit DA neuron-like properties [32], were used as an in vitro model to ascertain the optimal non-toxic concentration of Cur. A CCK-8 assay was performed to determine the optimal cytotoxic MPP⁺ concentration in PC12 cells. Following 24-h exposure to 0, 0.25, 0.5, 1, 2, or 4 mM MPP⁺, the results showed that MPP⁺ treatment brought about a dose-dependent reduction in PC12 cytoactive, with 1 mM MPP⁺ decreasing cell viability by approximately 50% (Fig. 5A) and 1 mM MPP⁺ was selected for subsequent experiments. Consequently, cells were treated with different doses of Cur (0, 5, 10, 15, 20, 25, and 30 μM) for 24 h. The CCK-8 results revealed decreased cell viability at Cur concentrations of 15 μM and higher (Fig. 5B). Thus, Cur concentrations of 10 μM were selected for subsequent in vitro experiments. Pretreatment with 0, 5, or 10 μM Cur for 1 h followed by MPP⁺ (1 mM) treatment significantly prevented the MPP⁺-induced decreases in cell viability (Fig. 5C).

Fig. 5.

Cur inhibited the MPP⁺-induced decrease in PC12 cell viability. A PC12 cell viability was evaluated by CCK-8 assay after MPP⁺ (0, 0.25, 0.5, 1, 2, or 4 mM) treatment for 24 h. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with 0. B PC12 cell viability was evaluated by CCK-8 assay after Cur (0, 5, 10, 15, 20, 25, or 30 μM) treatment for 24 h. *P < 0.05, **P < 0.01 compared with 0. C PC12 cell viability was evaluated by CCK-8 assay after pretreatment of PC12 cells with 0, 5, or 10 μM Cur or 1 mM MPP⁺. **P < 0.01 and ***P < 0.001 compared with MPP⁺ (+) and Cur (−). D and E The results of PC12 cell apoptosis in each group were detected by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n = 3 in each group. The data are expressed as the mean ± standard deviation

The effect of Cur on apoptosis in the MPP⁺-induced PD cell model was analyzed by flow cytometry. The MPP⁺ group exhibited an evidently higher ratio of apoptotic cells than the control group (Fig. 5D, E). However, Cur treatment significantly decreased apoptotic cells compared with the MPP⁺ group (Fig. 5D, E). These results show that Cur protected against apoptosis in MPP⁺-induced PC12 cells.

Cur Mitigates MPP⁺-Induced Cuproptosis in PC12 Cells

Excess copper has been shown to induce apoptosis through multiple pathways, and it may also trigger cuproptosis [33]. Based on our previous in vivo study, which demonstrated that Cur regulates cuproptosis in DA neurons of PD mice, the present study investigated whether Cur’s anti-apoptotic effects in PD cell models are associated with cuproptosis. As shown in Fig. 5D and E, co-treatment with Cur and ES showed an evidently higher ratio of apoptotic cells than the Cur group.

PC12 cells subjected to various treatments were examined by TEM to evaluate mitochondrial structural integrity. The results (Fig. 6A) show that MPP⁺ exposure resulted in severe mitochondrial ultrastructural damage, manifesting as mitochondrial shrinkage, fragmented cristae, and membrane breakdown, which are consistent with the morphological features of cuproptosis. However, Cur pretreatment markedly alleviated this damage, an effect that was subsequently abrogated by ES (Fig. 6A). Meanwhile, the expression of FDX1 was assessed using immunofluorescence. The results showed that the fluorescence intensity of FDX1 in the MPP⁺ group was lower than that of the control group (Fig. 6B, C). In contrast, the Cur-treated group exhibited increased FDX1 fluorescence intensity compared with the MPP⁺ group (Fig. 6B, C), while co-treatment with Cur and ES decreased FDX1 fluorescence intensity compared with the Cur-treated group (Fig. 6B, C). WB was performed to assess the DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70 protein expression in PC12 cells to further validate these findings. The results showed the significant downregulation of DLAT and FDX1 protein and the upregulation of SLC31A1, HSP70 (Fig. 6D, F), and DLAT oligomerization (Fig. 6E) in the MPP⁺ group compared with the control group. The Cur-treated group displayed an increase in DLAT and FDX1 protein expression and a significant decrease in SLC31A1, HSP70 (Fig. 6D, F), and DLAT oligomerization (Fig. 6E, F) compared with the MPP⁺ group. However, co-treatment with Cur and ES downregulated DLAT and FDX1 and upregulated SLC31A1, HSP70 (Fig. 6D, F), and DLAT oligomerization (Fig. 6E, F). These findings collectively indicate that Cur inhibited MPP⁺-induced cuproptosis in PC12 cells, potentially contributing to its neuroprotective effects.

Fig. 6.

Cur inhibited MPP⁺-induced cuproptosis. PC12 cells were divided into 4 groups: control, MPP⁺ group, Cur group (MPP⁺ + Cur), and ES group (MPP⁺ + Cur + ES). The control group received DMSO (<0.1%). The MPP⁺ group were exposed to 1 mM MPP⁺. In the Cur group, the cells were pretreated with 10 μM Cur for 1 h, followed by exposure to 1 mM MPP⁺ for 24 h. In the ES group, the cells received pretreatment with Cur (10 μM) combined with ES (200 nM) followed by MPP⁺ treatment. A PC12 cells subjected to various treatments were examined by TEM to evaluate mitochondrial structural integrity. Scale bar = 2 μm (L), 500 nm (R). B and C The expression level of FDX1 was detected by IF. D and E The DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70 protein expression was measured by western blot. F Western blot analysis of DLAT/DLAT oligomerization, FDX1, SLC31A1, and HSP70. n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Cur Activates Autophagy in the MPP⁺-Induced PD Cell Model

LC3 expression, a key marker of autophagy [34], was evaluated to ascertain the regulatory influence of Cur on autophagy in the PD cell model. To confirm whether Cur enhances autophagic flux, cells were treated with Baf-A1. WB analysis revealed that curcumin indeed promoted autophagic flux (Fig. 7A, B) rather than simply blocking it. Meanwhile, the immunofluorescence results displayed a decrease in the average in MPP⁺ treatment compared with the control (Fig. 7C, D). Conversely, Cur treatment increased the fluorescence intensity of LC3 compared with the MPP⁺ treatment (Fig. 7C, D). Furthermore, co-treatment with 3-MA significantly decreased LC3 fluorescence compared with Cur treatment alone (Fig. 7C, D). Furthermore, autophagosome formation was assessed using MDC staining. The number of autophagosomes was significantly reduced in the MPP⁺ treatment compared with the control, indicating reduced autophagosome formation (Fig. 7E, F). The Cur-treated group showed evidently higher autophagosome fluorescence intensity than the MPP⁺ treatment, indicating autophagosome accumulation (Fig. 7E, F). Additionally, co-treatment with 3-MA resulted in significantly lower autophagosome fluorescence intensity than Cur treatment alone (Fig. 7E, F). WB was used to evaluate LC3 II/I and P62. The expression of LC3 II/I was significantly downregulated in the MPP⁺ group, while the expression of P62 was evidently upregulated compared with the control group, indicating impaired autophagic flux (Fig. 7G, H). LC3 II/I expression was significantly increased compared with the MPP⁺ group, while P62 expression was significantly decreased in the Cur-treated group (Fig. 7G, H). However, co-treatment with 3-MA decreased LC3 II/I protein expression and increased P62 protein expression compared with Cur treatment alone (Fig. 7G, H). These findings collectively confirm that Cur activated autophagy in the MPP⁺-induced PD cell model.

Fig. 7.

Cur reduces MPP⁺-induced PC12 cell cuproptosis via autophagy. PC12 cells were divided into control, Baf-A1, Cur, Baf-A1 + Cur and control, MPP⁺ group, Cur group (MPP⁺ + Cur), and 3-MA group (MPP⁺ + Cur + 3MA). The control group received DMSO (<0.1%). The MPP⁺ group were exposed to 1 mM MPP⁺. In the Cur group, the cells were pretreated with 10 μM Cur for 1 h, followed by exposure to 1 mM MPP⁺ for 24 h. In the 3-MA group, the cells received pretreatment with Cur (10 μM) combined with 3-MA (2 mM) followed by MPP⁺ treatment. A and B The expression level of LC3 and P62 was detected by WB. C and D The expression level of LC3 was detected by IF. E and F The expression of autophagosomes was detected by MDC. G and H The LC3 and P62 protein expression was measured by WB. I and J The DLAT, FDX1, SLC31A1, and HSP70 protein expression was measured by western blot. n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Cur Attenuates MPP⁺-Induced Cuproptosis in PC12 Cells via Autophagy Modulation

WB was performed to assess cuproptosis-related protein expression, including DLAT, FDX1, SLC31A1, and HSP70 in PC12 cells co-treated with Cur and 3-MA to further investigate whether Cur-mediated autophagy activation regulates cuproptosis. DLAT and FDX1 expression was significantly reduced, while SLC31A1 and HSP70 expression was significantly increased in the MPP⁺ group compared with the control group (Fig. 7I, J). Compared with the MPP⁺ group, DLAT and FDX1 protein expression was slightly increased, while SLC31A1 and HSP70 expression was evidently reduced in the Cur group (Fig. 7I, J). However, co-treatment with 3-MA significantly decreased DLAT and FDX1 expression and increased SLC31A1 and HSP70 expression levels compared with Cur treatment alone (Fig. 7I, J). These findings suggest that Cur attenuated MPP⁺-induced cuproptosis in PC12 cells via the modulation of autophagy.

Cur Alleviates Cuproptosis via Autophagy Modulation Through AKT/mTOR/P70S6K Signaling Inhibition in PD Models

To determine whether Cur activates autophagy via AKT/mTOR/P70S6K-signal path to regulate cuproptosis in MPTP/MPP⁺-induced PD models, four experimental groups were established: control, MPTP/MPP⁺, MPTP/MPP⁺ + Cur, and MPTP/MPP⁺ + Cur + SC79 (an AKT activator) groups. Immunohistochemical analysis showed a decrease in the number of TH-positive neurons in SC79 group compared with the Cur group (Fig. 8A, B). WB was conducted to evaluate the expression of key proteins associated with the AKT/mTOR/P70S6K pathway, including P-AKT/AKT, P-mTOR/mTOR, and P-P70S6K/P70S6K. The results revealed a significant rise in the levels of P-AKT/AKT, P-mTOR/mTOR, and P-P70S6K/P70S6K in the MPTP/MPP⁺ treatment group compared with the control (Fig. 8C–F). Cur treatment decreased the levels of P-AKT/AKT, P-mTOR/mTOR, and P-P70S6K/P70S6K proteins compared with the MPTP/MPP⁺ group, indicating pathway inhibition. Co-treatment with SC79 significantly increased the levels of P-AKT/AKT, P-mTOR/mTOR, and P-P70S6K/P70S6K proteins compared with Cur treatment alone. The findings suggest that Cur activated autophagy by inhibiting the AKT/mTOR/P70S6K signaling pathway, thereby mitigating cuproptosis and providing neuroprotection in MPTP/MPP⁺-induced PD models.

Fig. 8.

Cur reduces microglial activation through the AKT/mTOR/P70S6K-signaling pathway in MPTP/MPP⁺-induced PD models. The mice were randomly assigned included control, MPTP group, Cur group (MPTP + Cur), and SC79 group (MPTP + Cur + SC79). The MPTP group received daily intraperitoneal injections of MPTP (25 mg/kg/day). The Cur group received daily intraperitoneal injections of Cur (80 mg/kg/day) 1 h before MPTP administration. The SC79 group was administered daily intraperitoneal injections of SC79 (10 mg/kg/day) and Cur (80 mg/kg/day) 1 h before MPTP administration. The control mice received intraperitoneal injections of normal saline (25 mL/kg). A TH-positive cells were stained by IHC and observed through a fluorescence microscope. The scale bar represents 200 μm (top) and 50 μm (bottom). B Number of TH-positive cells. C and D The P-AKT/AKT, P-mTOR/P-mTOR, and P-p70S6K/p70S6K protein expressions in MPTP-induced PD mice model were measured by western blot. PC12 cells were divided into 4 groups: control, MPP⁺ group, Cur group (MPP⁺ + Cur), and SC79 group (MPP⁺ + Cur + SC79). The control group received DMSO (<0.1%). The MPP⁺ group were exposed to 1 mM MPP⁺. In the Cur group, the cells were pretreated with 10 μM Cur for 1 h, followed by exposure to 1 mM MPP⁺ for 24 h. In the SC79 group, the cells received pretreatment with Cur (10 μM) combined with SC79 (10 μM) followed by MPP⁺ treatment. E and F The P-AKT/AKT, P-mTOR/P-mTOR, and P-p70S6K/p70S6K protein expressions in MPP⁺-induced PD cell model were measured by western blot. n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. The data are expressed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test

Discussion

In this work, MPTP was used to establish a PD mouse model. Pole-climbing, rope suspension, and NOR tests were used to evaluate motor function. The results showed that Cur-treated mice demonstrated significantly shortened pole-climbing and rope suspension times and increased NOR index values compared with the MPTP group (Fig. 1). Cur has been suggested to ameliorate the motor, learning, and memory dysfunction in PD animal models. Meanwhile, in vitro experiments showed Cur significantly attenuated the MPP⁺-induced reduction in cell viability (Fig. 5). Our study confirmed that Cur has neuroprotective effects; however, the mechanism of Cur in the treatment of PD is not entirely clear.

Cuproptosis has been implicated in the etiopathogenesis of neurodegenerative disorders [35]. Nevertheless, its role in PD and the potential effects of Cur on this process remain elusive. Our results showed that MPTP/MPP⁺ induced cuproptosis and Cur prevented cuproptosis in PD models (Figs. 3, 6). Cur treatment reduced copper ion concentrations in vitro and inverted FDX1, SLC31A1, and HSP70 expression levels induced by MPTP/MPP⁺ (Figs. 3, 6). Moreover, the protective benefits of Cur were partially reversed by the known cuproptosis inducer ES (Figs. 3, 6). However, we observed a marked decrease in the total copper content in the SN of MPTP-treated mice (Fig. 3) (this observation contrasts with the results obtained in cellular experiments). This reduction is likely attributable to the extensive loss of dopaminergic neuronal cell bodies in this region, as Cur treatment—which mitigated neuronal loss and preserved tissue integrity—significantly restored total copper levels. Converging experimental evidence robustly establishes MPTP-induced disruption of brain copper homeostasis, characterized by marked copper depletion in the striatum and midbrain [36], spatially resolved loss within the periventricular region and dentate gyrus [37], and corroborated by independent reports documenting a ~71% downregulation of Cu, Zn-SOD expression in the substantia nigra [38]. These findings are consistent with our results. It is worth noting that our observation of reduced copper in the SNpc appears to contrast with the recent report by Zhang and Wang [39] showing copper accumulation in the striatum. However, this divergence reflects the spatiotemporal heterogeneity of copper homeostasis in PD models rather than a contradiction. First, regionally, our data reflect SNpc neuronal loss, whereas Zhang and Wang focused on the striatum. Second, our data capture the early neuronal copper deficit in the SNpc, complementing Zhang and Wang’s finding of late striatal copper overload, together mapping the full pathological landscape of cuproptosis in PD. In summary, all data converge on the conclusion that cuproptosis plays an important role in the etiopathogenesis of PD, and it emphasizes the inhibitory effect of Cur on cuproptosis.

Previous studies suggest that cuproptosis may be an autophagy-dependent cell death modality [40, 41]; however, the interplay between these processes in PD is not fully understood. This study demonstrates that curcumin suppresses cuproptosis through autophagy activation. 3-MA (an autophagy inhibitor) co-treatment reversed the effects of Cur, supporting the role of autophagy in cuproptosis regulation (Figs. 4, 7). However, the precise molecular mechanism by which Cur regulates this cuproptosis-autophagy axis warrants further investigation.

The AKT/mTOR/p70S6K signaling pathway is an established regulator of autophagy, with its activation known to inhibit autophagic processes [42, 43]. Nevertheless, the precise role of the AKT/mTOR/p70S6K signaling pathway has not been fully clarified in PD models. In this study, MPTP/MPP⁺ treatment significantly upregulated phosphorylated AKT, mTOR, and P70S6K levels, indicating pathway activation and the suppression of autophagy. In contrast, Cur treatment decreased the phosphorylation of these proteins (Fig. 8), suggesting pathway inhibition. Furthermore, pretreatment with the AKT activator SC79 attenuated the autophagy-promoting and cuproptosis-inhibiting effects of Cur (Fig. 8). These findings suggest that Cur activates autophagy by inhibiting the AKT/mTOR/p70S6K signaling pathway. This pathway-mediated autophagy activation exerts important effects on regulating cuproptosis and contributes to the neuroprotective effects of Cur in PD (Fig. 9). However, the precise molecular mechanisms by which Cur regulates autophagic flux remain elusive, underscoring the need for further investigation.

Fig. 9.

Cur inhibits DA neuron cuproptosis in MPTP/MPP⁺ PD models via activating autophagy through inhibition of the AKT/mTOR/p70S6K signaling pathway

This study has several limitations. First, although we established that Cur modulates cuproptosis through autophagy, the specific mechanistic interface between these processes in PD warrants further study. Notably, while Cur was shown to regulate FDX1 expression in an autophagy-dependent fashion, we did not confirm whether FDX1 is a direct substrate for autophagic degradation (e.g., via cycloheximide chase or lysosomal inhibition). Elucidating the post-translational modifications controlling FDX1 stability remains a key direction for future work. Second, the MPTP-induced mouse model, although widely used, does not fully recapitulate the multifactorial and progressive nature of human PD. Third, despite its potential to induce autophagy, Cur faces challenges as a therapeutic candidate due to its suboptimal pharmacokinetics and limited blood-brain barrier accessibility. Future studies could explore combining novel nanoformulations or structural modifications to enhance its brain-targeting efficacy, thereby providing a stronger foundation for its clinical translation.

Conclusions

In summary, our findings demonstrate that Cur alleviates DA neuronal damage in MPTP/MPP⁺-induced PD models, involving the suppression of the AKT/mTOR/p70S6K pathway and the subsequent modulation of autophagy and cuproptosis. These results provide novel insight into the functional interplay between autophagy and cuproptosis in the context of PD. Furthermore, this study provides a theoretical basis for understanding the role of cuproptosis in PD pathology. It highlights the AKT/mTOR/autophagy/cuproptosis signaling cascade as a potential therapeutic target and identifies Cur as a promising candidate for PD management.

Author Contribution

Z.J. and S.H. made equal contribution in roles/writing—original methodology and draft. R.F. did behavioral tests. S.Y.P. made data curation. Z.C. did formal analysis. W.M. designed the methodology. Z.K. data analysis.

Funding

Natural Science Foundation of Hubei Province of China, No. 2024AFB818; the Cultivating Project for Young Scholar at Hubei University of Medicine No. 2020QDJZR013.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.