A_2AR antagonist IDPU facilitates the restoration of mitochondrial dynamics in 6-OHDA-induced primary mid-brain neuronal cells, via regulating p38MAPK/Parkin/DJ-1/DRP1 axis

Abstract

Parkinson’s disease (PD) is marked by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Adenosine receptors (A_2ARs) modulate the striatopallidal non-dopaminergic pathway to alleviate PD symptoms. In the present study, the neuroprotective mechanism of the selective A_2AR antagonist exhibiting a non-purine scaffold, IDPU (K_i = 0.0038 nM), was explored using the primary mid-brain neuronal (PMDN) cells isolated from P₀/P₁ rat pups that differentiated to form dopaminergic neurons, as validated using tyrosine hydroxylase. PMDN cells, when treated with IDPU (0–10 μM) alone, showed insignificant toxicity. However, they exhibited < 60% cell viability when treated with 6-OHDA (150 μM) after 24h. Cell viability improved to > 80%, and dopamine levels were restored in 6-OHDA- (150 μM) induced PMDN cells when 3h post-treated with IDPU (0.7 μM, 1 μM), including the depletion in ROS generation and [Ca²⁺]_i levels observed after 24h. IDPU treatment further impacts the mitochondrial control by attenuating both mitochondrial SOD production and its membrane potential loss in PD-like conditions. To investigate the mechanism of A_2AR intervention on governing the mitochondrial-associated signalling cascades, the proteins were isolated from 6-OHDA (150 μM) induced PMDN cells 3h post-treated with IDPU (0.7 μM, 1 μM) and ZM241385 (1 μM) for western blot analysis. Our results exhibited that the phosphorylation of both DRP1 (Ser616) (78 kDa) and p38MAPK (Tyr182) (41 kDa) proteins was enhanced when exposed to 6-OHDA; however, the protein levels reduced post-treatment with both A_2AR antagonists. In contrast, 6-OHDA toxicity alleviated the levels of both Parkin (58 kDa) and DJ-1(23 kDa), while exposure to A_2AR antagonists (IDPU and ZM241385) improved their protein levels. This suggests the possible involvement of A_2AR blockade in regulating mitochondrial dynamics, thus promoting survival. These findings present the first evidence that IDPU demonstrates neuroprotection in PDlike conditions via the p38MAPK/DRP1/Parkin signalling, offering a potential therapeutic mechanism for targeting mitochondrial dynamics through A_2AR antagonism.

Introduction

Degeneration of the dopaminergic neurons within the substantia nigra pars compacta (SNpc) marks the Parkinson’s Disease (PD) pathology [1]. Established PD models use the selective catecholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), to replicate the key pathogenic processes of the disease, including reactive oxygen species (ROS) production [2], neuroinflammation [3], dysregulation of calcium homeostasis [4, 5] and mitochondrial function [6–8]. Mitochondrial homeostasis sustains the balance between mitochondrial fission, which segregates the damaged mitochondria [9], and mitophagy, which eliminates them [10–12]. Restoring mitochondrial homeostasis has been recognized as a promising strategy for dopaminergic neuroprotection in PD [13].

6-OHDA-induced oxidative stress and intracellular calcium [Ca²⁺]_i overload are known to activate p38 mitogen-activated protein kinase (p38 MAPK) [14, 15]. This MAPK family enzyme is activated via dual phosphorylation of tyrosine (Tyr) and threonine (Thr) residues [16]. Moreover, Ca²⁺/CaMK/p38MAPK signalling downstream enhances the activity of PGC-1α, directing the mitochondrial control [17]. p38MAPK activation also inhibits Parkin (ubiquitin E3 ligase) mediated mitophagy in PD [18]. These findings emphasize the significance of p38MAPK in maintaining mitochondrial homeostasis. Furthermore, Parkin reportedly interacts with a GTPase, dynamin-related protein (DRP1), through the proteasome-dependent pathway [19, 20]. It is recruited from the cytosol to the mitochondrial outer membrane to induce fission [21]. However, excessive stimulation of DRP-1 results in cell death [22, 23]. Notably, DRP1 phosphorylation at the serine 616 residue is also mediated through p38MAPK activation, leading to neuronal apoptosis in PD [24].

Collectively, p38MAPK activation via elevated ROS and [Ca²⁺]_i levels inhibits the Parkin-mediated mitophagy and DRP1 degradation, consequently accumulating the dysfunctional mitochondria, leading to caspase-dependent apoptosis [18, 24, 25]. As a result, therapies being developed for PD have shifted their approach towards maintaining mitochondrial function [13].

Adenosine A_2A receptors (A_2ARs) have emerged as an effective target for PD therapeutics [26], highly expressed in the basal ganglia, striato-pallidal pathway, GABAergic medium spiny neurons [26], and glutamatergic and dopaminergic nerve terminals [27]. Nevertheless, evidence suggests that A_2ARs mitigate glutamatergic plasticity in cortico-striatal pathways during the presymptomatic stages of PD [28]. In this context, Nunes et al. added by validating their efficacy in improving the early synaptic dysfunction rather than reversing the later events of neurodegeneration [29], elucidating its functional role as an early therapeutic target.

Studies report that A_2AR rescues the staurosporine-induced neurons by preserving the mitochondrial integrity, eventually inhibiting the intrinsic apoptosis [30]. Furthermore, A53T α-synuclein-induced neurotoxicity was attenuated through A_2AR deficiency by restoring autophagic flux in an in-vivo PD model [31]. Additionally, A_2AR-mediated suppression of p38MAPK prevented the β-amyloid-induced synaptotoxicity [32]. Expanding on this view, Ren et al. validated that A_2AR interference prevented p38MAPK activation and apoptosis of hippocampal cells [33]. Collectively, these insights underline the probable impact of A_2ARs on neuronal apoptosis via p38MAPK regulation, eventually restoring the mitochondrial dysfunction in neurodegenerative conditions. Therefore, our study investigates the mechanistic role of A_2AR in maintaining the mitochondrial dynamics under PD-like conditions, using the 6-OHDA-induced primary midbrain neurons treated with the A_2AR antagonist IDPU.

In our previous work, we synthesized and validated IDPU as a potent and selective A_2AR antagonist (Fig. 1) with a K_i value of 0.0038 nM, approximately 737-fold binding selectivity versus A₁R [34]. This impressive selectivity profile and a non-purine scaffold (Fig. 1) offer advantages in drug development. These results position IDPU as a promising candidate with high functional potency and receptor selectivity for targeting A_2AR signalling in PD.

Fig. 1.

IDPU as a potent and selective A_2AR antagonist

Our earlier reports showcase that A_₂AR influences [Ca²⁺]_i levels via a PKA/IP₃–dependent pathway in HEK 293-A_2AR transfected cells. Subsequently, Sophronea et al. validated that A_2AR antagonism reversed the calcium-dependent cell damage in a 6-OHDA-induced PD model [35, 36]. In addition, we documented that A_2AR blockade mediated via IDPU treatment attenuated haloperidol and NECA-induced PD-like symptoms by exhibiting an upsurge in antioxidant markers [37, 38]. Henceforth, establishing a direct link between A_₂AR and calcium/oxidative stress cascades activates neuronal survival pathways.

Considering the above findings, this study aims to investigate whether the A_2AR antagonist-mediated modulation of [Ca²⁺]_i and ROS levels might potentially regulate p38MAPK phosphorylation, which can further influence mitochondrial dynamics, including its membrane integrity, oxidative stress, and levels of fission (DRP1) and mitophagy (Parkin) associated proteins, to validate its possible mechanism for neuroprotection. This work establishes the neuroprotective role of IDPU in reversing 6-OHDA-induced toxicity, with comparison against an established selective A_2AR antagonist, ZM241385. This is the first report that provides a mechanistic link between A₂_AR signalling and mitochondrial dynamics in a physiologically relevant 6-OHDA-induced primary mid-brain neuronal model, highlighting the feasibility of A_2AR-mediated therapies in PD.

Materials and methodology

Chemicals and reagents

6-OHDA (MCE; Cat No. HY-B1081) and ZM241385 (MCE; Cat No. HY-19532) were purchased while IDPU was synthesized in our laboratory [34, 39]. Neurobasal medium (Gibco; Cat No. 21103049) supplemented with B-27 (Gibco; Cat No. 17504044), fetal bovine serum (FBS) (Gibco; Cat No. RM112), L-glutamine (Invitrogen; Cat No. 25030081), poly-D-lysine (Invitrogen; Cat No. A3890401), antibiotic antimycotic solution (Cell Clone; Cat No. CC403.010L), MitoSOX™ Red (Invitrogen; Cat No. M36008), rhodamine 123 dye (MCE; Cat No. HY-D0816), Annexin V-APC/PI apoptosis detection kit (Elabscience; Cat No. E-CK-A217), H2DCFDA (MCE; Cat No. HY-D0940) ROS fluorescent probe, Fluo-4, AM (Invitrogen; Cat No. F14201), MTT (SRL; Cat No. 298-93−1), dopamine ELISA kit (Elabscience; Cat No. E-EL-R0343), and ECL (Cyanagen; Cat No. XLS142,0250) were procured for this study.

Approval from the institutional animal ethical committee

This project was approved by the Departmental Animal Ethics Committee on 21 st August 2023 for both in vitro (isolation of primary midbrain neuronal cells from P₀/P1 pups of SD rats) and in vivo studies under proposal no. IAEC/ACBR/August 2023/PML/02.

Primary neuronal culture

P₀-P₁ Sprague Dawley (SD) rat pups were used for primary midbrain neuronal (PMDN) cultures according to the method standardized in our laboratory [40]. After dissection, the mid-brain tissue was trypsinized and incubated for 30 min at 37 °C. The dissociated cells were plated at densities of 2 × 10⁶ cells per 90 mm culture petri dish, 1 × 10⁵ cells per 6-well plate, or 2 × 10⁴ cells per 96-well plate in pre-coated poly-D-lysine culture plates and grown in Neurobasal medium with 0.5 mM L-glutamine, 2% B27 supplement serum, 2% heat-inactivated fetal bovine serum, and 1% antibiotic antimycotic solution [41, 42]. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2. PMDN cultures attained morphological uniformity and neurite coverage to become confluent within 7–10 days. The media was replaced every third day until cells reached 80–100% confluency.

Immunocytochemistry

Primary mid-brain neuronal cells (PMDN) 1 × 10⁵ were seeded on poly-D-lysine-coated coverslips in the 6-well plate in Neurobasal media, supplemented with 2% B-27, 2% FBS, 0.5 mM L-glutamine, and 1% antibiotic-antimycotic solution. After 7–10 days, the cells reached 70–90% confluency on coverslips, then were permeabilized with 0.5% Triton X-100 in PBS for 10 min, fixed with 4% paraformaldehyde for 10 min, and followed by washing with phosphate-buffered saline (PBS). The cells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h and then incubated overnight at 4 °C with primary antibody tyrosine hydroxylase (TH) (ABclonal, Cat No. A12756). The fluorescence-tagged secondary antibody (FITC) was incubated at 4 °C for 3 h in the dark [41]. The fluorescence was visualized under a fluorescence microscope (Nikon Ti2 Eclipse).

Cell viability assay

To determine the cell viability, 2 × 10⁴ cells were seeded in a 96-well microplate and cultured until the cells reached 80% confluency. PMDN cells were given the following treatments: (i) incubation with different concentrations of 6-OHDA (0–250 µM) for 24 h; 6-OHDA was dissolved in saline with 0.02% (w/v) ascorbic acid. (ii) Incubation with different concentrations of IDPU (0.01–10 µM) for 24 h. (iii) Pre-incubation of 6-OHDA (150 μM) for 3 h, 6 h, 12 h, and 24 h, followed by treatment with the A_2AR antagonist, IDPU (0.7 µM and 1 μM) and ZM241385 (1 µM) for 24 h. MTT assay was carried out to measure the cell viability. After 24 h, the cells taken from each treatment were incubated with 20 µl of MTT solution (5 mg/ml in PBS) for 4 h. The obtained formazan crystals were dissolved in DMSO [36, 41]. The absorbance was measured at a wavelength of 570 nm in a microplate reader (Tecan Infinite M200). 6-OHDA-induced PMDN cells 3 h post-treated with IDPU (0.7 µM and 1 μM) and ZM241385 (1 µM), observed after 24 h, were used for further experiments.

Annexin V-APC-PI detection of apoptosis

According to the manufacturer’s protocol, apoptotic PMDN cells were detected with the Annexin V-APC/PI apoptosis detection kit. After the treatment, the PMDN (2 × 10⁵ PMDN) cells were trypsinized and suspended in 500 μl (1×) binding buffer, incubated with 5 μl APC-conjugated Annexin V and 5 μl of PI for 15 min in the dark. The stained cells were analyzed immediately using a FACS Calibur flow cytometer (BD Biosciences), and a minimum of 10,000 events were acquired per sample [36]. Quadrant gating was performed using CellQuest Pro software to distinguish between live (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and necrotic (Annexin V⁻/PI⁺) cell populations. The number of events in each quadrant was recorded and converted into percentages to quantify the extent of apoptosis. This analysis enabled a comparative evaluation of treatment-induced cytotoxicity and the potential neuroprotective effects of the IDPU in 6-OHDA-induced PMDN cells.

Measurement of intracellular reactive oxygen species (ROS)

ROS generation was analyzed using an oxidation-sensitive probe, H2DCFDA. After the treatment, the 2 × 10⁵ PMDN cells were washed with PBS and incubated with a 5–10 μM probe for 30–45 min at 37 °C in the dark. Post incubation, cells were washed thrice with PBS, and DCFDA fluorescence was analyzed using Tecan Infinite M200 at an excitation and emission wavelength of 485 nm and 535 nm [36].

Live-cell imaging of Ca2 + transients

PMDN cells (2 × 10⁶) were seeded in a 90-mm culture petri dish; maintained with Neurobasal medium supplemented with 2% B-27, 2% FBS, 0.5 mM L-glutamine, and 1% antibiotic antimycotic solution; and were given the desired treatment. After the treatment, the media was discarded and washed twice with 200 μl Krebs-HEPES buffer, followed by incubation with 4 μM Fluo-4 AM dye in the Krebs-HEPES buffer for 30 min at RT in the dark. The buffer was discarded to remove the dye and then washed twice with the same. The fluorescence intensity was measured at excitation λ 494 nm and emission λ 506 nm, and its fluorescence intensity kinetics was carried out for 800 s with an interval of 5 s using Tecan Infinite M200. Data are presented as F/F₀, where F₀, fluorescence intensity at 0 s; F, fluorescence intensity at the endpoint, normalized to control [35].

Measurement of mitochondrial superoxide and mitochondrial membrane potential

Mitochondrial superoxide production was measured using a Mito-superoxide anion fluorescent probe, MitoSOX™ Red. After the treatment, 2 × 10⁵ PMDN cells were incubated with the fluorescent probe (5 μM) for 30 min at 37 °C. ROS production gives red fluorescence intensity, measured at excitation λ 385 nm and emission λ 405 nm by fluorescence microscope (Nikon Ti2 Eclipse) [36]. Mitochondrial membrane potential was measured using a cell-permeant, green fluorescent cationic dye, Rhodamine 123, which is readily sequestered by active mitochondria without cytotoxic effects. After the treatment, the PMDN cells were stained with 10 μM of Rhodamine 123 for 15 min at 37 °C in the dark. The fluorescence intensity was measured at excitation λ 488 nm and emission λ 529 nm using flow cytometry (FACS Calibur, BD Bioscience) as per the manufacturer’s protocol [36].

Measurement of dopamine levels by ELISA

To estimate dopamine levels, 2 × 10⁵ PMDN cells were seeded and treated according to experimental conditions, followed by analysis using a dopamine ELISA kit (Elabsciences) [41]. After treatment, cells were lysed using lysis buffer, and the supernatant was collected after centrifugation. Protein concentration was determined using BSA as a standard [36]. For the ELISA, 50 μL each of standards, blank, and samples (in duplicate) were added to the wells, followed by 50 μL of biotinylated detection antibody. The plate was sealed, incubated at 37 °C for 45 min, and then washed. Next, 100 μL of HRP conjugate was added, incubated at 37 °C for 30 min, and washed again. Subsequently, 90 μL of substrate reagent was added and incubated for 15 min in the dark at 37 °C. The reaction was stopped with 50 μL of Stop Solution, and absorbance was read at 450 nm using a microplate reader.

Western blot analysis

PMDN cells (2 × 10⁵) were incubated with RIPA lysis buffer, followed by centrifugation at 14,000 g for 20 min; the supernatant obtained was used for protein estimation by Bradford assay using BSA as a standard [36]. Proteins of equal concentration (60 μg/lane) were separated on 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% BSA for 1 h and incubated overnight at 4 °C with the following primary antibodies (dilution 1:1000): TH (Cat No. A12756), DJ-1 (Cat No. SC32874), Parkin (Cat No. SC-136989), p-p38MAPK (Tyr182) (Cat No. AF3455), p38-MAPK (Cat No. A14401), DRP1 (Cat No. A2586), p-DRP1 (ser 616) (Cat No. AF8470), cleaved caspase-3 (E-AB-3004), and β-actin (3598R-100). After washing thrice with PBS-T, the blots were incubated with horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse secondary antibodies (Cat No. E-AB-1003 and E-AB-1001) at a dilution of 1:1000 for 4 h at 4 °C [41]. Specific bands were detected using an enhanced chemiluminescence (ECL) kit. The relative band intensity was measured using ImageJ Software and expressed as a value normalized by the intensity of the β-actin signal [41].

Data collection and statistical analysis

The data is represented as mean ± SEM. All statistical calculations were made with the GraphPad Prism 10.5.0 and ImageJ 1.53e; Java 1.8.0_172 [64-bit] software. The statistical significance was tested by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparison tests [41]. p-values ≤ 0.05 were considered significant.

Results

Establishment of 6-OHDA-induced primary mid-brain neuronal (PMDN) cells toxicity model

In our study, primary midbrain neuronal (PMDN) cells isolated from P₀/P₁ rat pups (Fig. 2a–c) were differentiated into dopaminergic neurons. They were characterized using the expression of the dopaminergic neuronal marker, tyrosine hydroxylase (TH) (Fig. 2d–f), an enzyme involved in dopamine biosynthesis. The identification of TH-positive cells assured the successful dopaminergic differentiation of the cultured PMDN cells. These dopaminergic neurons, when exposed to 6-hydroxydopamine (6-OHDA), exhibit characteristic features of PD pathogenesis, including oxidative stress, calcium overload, mitochondrial dysfunction, and dopamine depletion, thereby validating this system as a suitable in vitro model for studying Parkinson’s disease [8, 40]. To establish the optimal concentration for modelling PD-associated neurotoxicity, the IC₅₀ value of 6-OHDA in PMDN cells was determined. The cells were treated with 6-OHDA at various concentrations (10–200 μM) for 24 h. We recorded a decrease in cell viability (85%–25%) in a concentration-dependent manner. The IC₅₀ value was obtained at 150 μM; 51.15 ± 1.893% (p < 0.001) for 6-OHDA against PMDN cells.

Fig. 2.

Primary midbrain neuronal (PMDN) cells isolated from P₀/P₁ rat pups

A_2AR antagonist, IDPU-induced reversal of 6-OHDA-induced PMDN cell toxicity

PMDN cells treated with IDPU alone at 0–10 μM showed > 95% cell viability (Fig. 3a), indicating that IDPU is non-toxic and safe for PMDN cells. The standardization of IDPU at various concentrations (0.1–3 µM) was carried out against 6-OHDA- (150 μM) induced PMDN cells. Since IDPU did not show a toxic effect on the PMDN cells, low concentrations (0.1–3 µM) were selected for standardization as reported for other standard A_2AR antagonists [36].

Fig. 3.

PMDN cells treated with IDPU alone at 0–10 μM

PMDN cells pre-incubated with 6-OHDA (150 μM), 3 h post-treated with IDPU, depicted increased cell viability (65–86%) after 24 h. However, IDPU-induced insignificant changes in cell viability at lower concentrations (0.1–0.5 µM). At the same time, significant (p < 0.001) improvement was measured from 0.7 to 1 µM in PMDN cells (Fig. 3b). Further, all experimental studies with IDPU were carried out at 0.7 µM and 1 µM.

To elucidate the optimum exposure time of IDPU at 0.7 µM and 1 µM concentrations, PMDN cells incubated with 6-OHDA were post-treated with IDPU for 3 h, 6 h, 12 h, and 24 h. The IDPU-induced cell viability (%) monitored after 24 h was found to be 80.39 ± 0.9156 (0.7 µM, 3 h), 85.36 ± 1.842 (1 µM, 3 h); 64.65 ± 2.670 (0.7 µM, 6 h), 68.26 ± 1.797 (1 µM, 6 h); 58.92 ± 2.537 (0.7 µM, 12 h), 61.12 ± 2.752 (1 µM,12 h); and 50.88 ± 2.321 (0.7 µM, 24 h), 50.59 ± 2.128 (1 µM, 24 h) respectively (Fig. 3c–f). We identified that the optimum exposure time of IDPU was in parallel with the standard A_2AR antagonist ZM241385(1 µM), demonstrating the % cell viability to be 82.96 ± 1.045 (3 h), 64.33 ± 2.693 (6 h), 58.2 ± 1.351 (12 h), and 50.75 ± 2.6 (24 h), respectively, in 6-OHDA-induced PMDN cells Fig. 3c−f). The concentrations of IDPU and ZM241385 were selected based on their non-toxic profiles (Fig. 3), optimal rescue of cell viability post-insult, and supportive evidence from existing literature on A_2AR antagonists. Previous studies in our laboratory [34, 37–39] further validate dose relevance in both in vitro and in vivo models.

Nevertheless, 6-OHDA-induced PMDN cells 3 h post-treated with IDPU at 0.7 µM and 1 µM significantly increased the cell viability to 80.39 ± 0.9156% (p < 0.001) and 85.36 ± 1.842% (p < 0.001), respectively, after 24 h; hence, this designated treatment was used for further experiments.

Neuroprotective effect of A_2AR antagonist, IDPU, on 6-OHDA-mediated toxicity in PMDN cells reduces ROS generation, intracellular calcium overload, apoptosis, and restores dopamine levels

Previously, it has been reported that 6-OHDA induces intracellular calcium overload, leading to ROS generation, thus activating the cleaved caspase-3, subsequently triggering the dopaminergic neuronal cell death and reducing the dopamine levels in PD [42–45]. PMDN cells were pre-incubated with 6-OHDA (150 μM) for 3 h followed by IDPU (0.7 µM and 1 µM) and ZM241385 (1 µM) treatment for 24 h. Cellular morphological changes like shrinkage and fragmentation were identified post-treatment under phase contrast microscopy, as shown in Fig. 4a–g, across all groups. These treated cells were then processed for ROS, intracellular calcium overload, and apoptosis analysis. Proteins isolated from these cells were used to estimate the restored dopamine levels.

Fig. 4.

Phase contrast microscopy

To validate the neuroprotective effect of A_2AR antagonist IDPU, ROS generation was measured in 6-OHDA- (150 μM) induced PMDN cells 3 h post-treated with IDPU (0.7 µM and 1 µM) and ZM241385 (1 μM) using H₂DCFDA as a fluorescent probe after 24 h. 6-OHDA-treated PMDN cells showed a six-fold increase in ROS production with respect to control (6.174 ± 0.5013 fold; p < 0.001); however, 3 h post-treatment with IDPU (0.7 μM; 4.373 ± 0.4503 fold; p < 0.05 and 1 μM; 3.540 ± 0.1952 fold; p < 0.01) and ZM241385 (1 μM, 3.210 ± 0.5498 fold; p < 0.001) significantly depleted the ROS levels as detected after 24 h (Fig. 4h).

Additionally, the 6-OHDA-induced [Ca²⁺]_i overload in PMDN cells (1.988 ± 0.02856 fold; p < 0.001) with respect to control was markedly decreased upon treatment with 0.7 μM IDPU (1.455 ± 0.02874 fold; p < 0.001), 1 μM IDPU (1.342 ± 0.03971 fold; p < 0.001), and 1 μM ZM241385 (1.291 ± 0.1127 fold; p < 0.001). (Fig. 4i, j).

6-OHDA-induced PMDN cells entered apoptosis (47.31% ± 0.8626; p < 0.001); however, when 3 h post-treated with IDPU 0.7 µM (29.42% ± 1.404; p < 0.001), 1 µM (26.75% ± 2.616; p < 0.001), and ZM241385 1 µM (20.73% ± 1.948; p < 0.001), cells were found to be rescued from apoptosis (Fig. 4k, l).

These results affirmed that post-treatment of A_2AR antagonists (IDPU and ZM241385) in 6-OHDA-induced PMDN cells decreased ROS generation, intracellular calcium overload, and apoptosis.

We also measured that 6-OHDA-induced PMDN cells 3 h post-treated with IDPU at 0.7 µM, 1 µM, and ZM241385 at 1 µM suppressed cleaved caspase-3 protein levels (IDPU at 0.7 µM; 1.225 ± 0.02678; p < 0.001); (IDPU at 1 µM; 1.192 ± 0.07506; p < 0.001); and (ZM241385 at 1 µM; 1.011 ± 0.05470; p < 0.001) as compared to PMDN cells incubated with 6-OHDA (150 µM; 1.999 ± 0.04633; p < 0.001) alone (Fig. 4m, n).

Furthermore, the dopamine levels decreased to 2.903 ± 0.2694 (p < 0.001) in PMDN cells incubated with 6-OHDA compared to the control 8.676 ± 0.3257. However, 3 h post-treatment of 6-OHDA-induced PMDN cells with A_2AR antagonists (IDPU at 0.7 μM; 4.820 ± 0.5836); (IDPU at 1 µM; 5.843 ± 0.2416; p < 0.01); and (ZM241385 at 1 µM; 6.116 ± 0.5485; p < 0.01) (Fig. 4o) significantly restored the dopamine levels.

Our results presented that IDPU arrests apoptosis and restores dopamine levels mediated through the attenuation of ROS and intracellular calcium levels, protecting 6-OHDA-induced PMDN cells.

A_2AR antagonist–induced alleviation of mitochondrial stress via reducing the mitochondrial superoxide generation and rescuing the mitochondrial membrane potential (Δψ) in 6-OHDA-induced PMDN cells

Mitochondrial dysfunction is a marked pathogenic feature of PD. Maintaining mitochondrial homeostasis is crucial to protect the cells from cellular stress and, eventually, cellular demise [44, 45]. Therefore, the mitochondrial oxidative stress (superoxide) was measured using a mito-targeted reporter, MitoSOX Red, to validate the effect of IDPU on the improvement of mitochondrial stress, subsequently causing the neuroprotective effect. Our results revealed that 6-OHDA-induced PMDN cells generated approximately sevenfold (7.342 ± 0.2390; p < 0.001) mitochondrial superoxide compared to the control after 24 h. Significant attenuation of mitochondrial superoxide was visualized in 6-OHDA-induced PMDN cells 3 h post-treated with IDPU (0.7 µM; 4.648 ± 0.5878; p < 0.01 and 1 µM; 3.193 ± 0.3662; p < 0.001) and ZM241385 (1 µM; 4.680 ± 0.4092; p < 0.01) (Fig. 5a–h).

Fig. 5.

Significant attenuation of mitochondrial superoxide

Rhodamine 123 fluorescence dye detects mitochondrial membrane potential (Δψ) to determine membrane integrity. The 6-OHDA treatment diminished the mitochondrial membrane potential (Δψ) to 0.3332 ± 0.06279; p < 0.001 compared to the control cells. However, 6-OHDA-induced PMDN cells 3 h post-treated with A_2AR antagonists rescued the mitochondrial potential (Δψ) from further damage (ZM241385 at 1 μM; 0.6443 ± 0.03166; p < 0.001), (IDPU at 0.7 μM; 0.6126 ± 0.03238; p < 0.001), (IDPU at 1 μM; 0.6572 ± 0.02179; p < 0.001) (Fig. 5i–p).

These results illustrate that the A_2AR antagonist treatment significantly attenuated the mitochondrial ROS generation and restored the mitochondrial membrane integrity in 6-OHDA-induced PMDN cells.

A_2AR antagonist attenuates mitochondrial dynamics via regulating p38MAPK/DRP1/Parkin signalling in 6-OHDA-induced PMDN cells

Meanwhile, IDPU treatment attenuated the 6-OHDA-induced oxidative stress, calcium overload, mitochondrial potential, and apoptosis in PMDN cells. We further explored the effect of IDPU on mitochondrial dynamics by evaluating the protein levels of DJ-1, which acts as a sensor for oxidative stress and helps neutralize ROS, thus possibly maintaining mitochondrial health and protecting cells from apoptosis.

We observed that DJ-1 protein levels were significantly alleviated in 6-OHDA-induced PMDN cells (0.6611 ± 0.01968; p < 0.001), suggesting that depleted DJ-1 levels in PMDN cells upon 6-OHDA induction likely contributed to mitochondrial dysfunction. However, 3 h post-treatment with IDPU (0.7 μM; 0.8964 ± 0.04192; p < 0.01; 1 μM; 0.9550 ± 0.06952; p < 0.01) and ZM241385 (1 μM; 0.8604 ± 0.02985; p < 0.05) elevated the DJ-1 (Fig. 6a, d) protein expression. The results further assert that IDPU-induced increase in mitochondrial-associated oxidative stress markers promoted 6-OHDA-induced PMDN cell survival.

Fig. 6.

IDPU treatment attenuated the mitophagy and fission-associated proteins to improve the mitochondrial dynamics: (a) The protein extracted from PMDN cells treated with 6-OHDA (150 μM) for 3h, followed by treatment of A_2AR antagonists (IDPU- 0.7, 1 μM and ZM241385- 1 μM) for 24 hours, was immunoblotted for p-p38MAPK, p38MAPK, p-DRP1, DRP1, DJ-1 and Parkin proteins. The blots were re-probed with β-Actin antibody (b-e), and quantification of the western blot was carried out using Image J and GraphPad Prism software. Data are represented as the mean ± SEM of three independent experiments (n=3). *p < 0.05; **p< 0.01; ***p < 0.001 vs 6-OHDA group and p < 0.001 vs control group; one-way ANOVA with Tukey’s post hoc test.

DJ-1interacts with mitophagy-linked proteins (PINK1, Parkin) in PD. Since A_2AR inhibition elevated the DJ-1 protein levels in PD-like conditions (Fig. 6a, d), we further investigated the effect of A_2AR inhibition on Parkin in 6-OHDA-induced PMDN cells. Our findings revealed significant attenuation of Parkin protein levels when treated with IDPU at 0.7 μM; 0.7479 ± 0.084; IDPU at 1 μM; 0.9794 ± 0.01678; p < 0.001, and ZM241385 at 1 μM; 0.8763 ± 0.08768; p < 0.01 when compared to PMDN cells incubated with 6-OHDA (150 μM; 0.4269 ± 0.1430; p < 0.001) alone, thus implicating the activation of mitophagy-associated cascades, essential for neuronal survival (Fig. 6a, e).

We have determined that A_2AR tunes mitophagy-related mechanisms by enhancing Parkin protein levels in 6-OHDA-induced PMDN cells, and evidence indicates that Parkin degrades DRP1 to reduce mitochondrial fragmentation [24]. Therefore, we further investigated A_2AR-mediated impact on fission-associated protein, DRP1, in 6-OHDA-induced PMDN cells 3 h post-treated with IDPU and ZM241385.

Our results displayed that phosphorylation of DRP1 at Ser 616 was enhanced (2.499 ± 0.1181; p < 0.001) in 6-OHDA-induced PMDN cells (Fig. 6a, c). However, the phosphorylation of DRP1 at Ser 616 was mitigated post A_2AR antagonist treatment (IDPU at 0.7 μM; 1.543 ± 0.2662; p < 0.01; IDPU at 1 μM; 1.298 ± 0.1415; p < 0.01 and ZM241385 at 1 μM; 1.279 ± 0.1102; p < 0.01), resulting in the reduced expression of mitochondrial fission-associated cascades (Fig. 6a, c).

IDPU (A_2AR antagonist) treatment attenuated the 6-OHDA-induced ROS generation, calcium overload, mitophagy, and fission-associated proteins, which could be plausibly mediated through p38MAPK activation to reduce apoptosis. To validate that A_2AR downregulation leads to neuroprotection via reducing the p38MAPK activation, the phosphorylated p38MAPK levels were analyzed using western blot. We noted that phosphorylation of p38MAPK at Tyr182 was elevated with exposure to 6-OHDA (3.329 ± 0.3148; p < 0.001) in PMDN cells (Fig. 6a, b), consequently activating p38MAPK, upon increased ROS generation and calcium overload, leading to apoptosis. However, the 3 h post-treatment of cells with A_2AR antagonists (IDPU at 0.7 μM; 2.679 ± 0.1010; p < 0.05; IDPU at 1 μM; 2.224 ± 0.01953; p < 0.01, and ZM241385 at 1 μM; 2.413 ± 0.06537; p < 0.01) (Fig. 6a, b) led to a decrease in the phosphorylation of p38MAPK at Tyr 182, thereby resulting in inhibition of apoptosis. (Fig. 4k, l).

According to our results, the proteins linked to the stress cascade and mitochondrial fission were activated in 6-OHDA-induced PMDN cells, as evidenced by enhanced levels of phosphorylated DRP1 at Ser616 and p38MAPK at Tyr182. The phosphorylation levels were reduced post-A_2AR antagonist treatment, indicating that A_2ARs potentially influence pathways triggered by mitochondrial stress. Conversely, 6-OHDA treatment significantly reduced the protein levels of Parkin and DJ-1, which were found to be attenuated upon exposure to A_2AR antagonists (IDPU and ZM241385). Therefore, A_2AR modulation is important in rescuing mitophagy-associated proteins and antioxidant defense mechanisms in conditions similar to Parkinson’s disease.

These findings add confidence to the idea of employing A_2AR antagonism as a probable therapeutic tool for directing mitochondrial dynamics in Parkinson’s disease.

Discussion

Oxidative stress and mitochondrial dysfunction both add to the pathology of PD, leading to the de-innervation of dopaminergic neurons in the SNpc [45]. Adenosine A_2AR acting through the striatopallidal pathway has emerged in the therapeutics of PD [26, 34]. Recent reports suggest that ROS generation activates the p38MAPK phosphorylation, parkin translocation to mitochondria, and DRP1-mediated mitochondrial fission to attenuate the PD symptoms in the striatonigral pathway [25, 46–48]. However, our interest was to explore the mechanism of A_2AR-mediated neuroprotection via the striatopallidal pathway for PD therapeutics.

Nevertheless, most in vitro studies have been conducted on immortalized and modified cell lines such as SH-SY5Y, PC12, LUHMES, and MN9D [49–51]. Highly differentiated primary mid-brain neuronal cells (PMDN) isolated from rat pups P₀/P₁, characterized as mature dopaminergic/TH-positive neurons, were employed (Fig. 2) for this study. These TH⁺ neurons, when incubated with 6-hydroxydopamine (6-OHDA), mimicked PD pathogenesis, including oxidative stress, calcium overload, mitochondrial dysfunction, and dopamine depletion [6–8, 36], thereby increasing the significance of this in vitro model for exploring the mechanistic action of PD therapeutics.

The pharmacological intervention of A_2AR involved in the study was achieved through IDPU, a novel A_2AR antagonist (K_i = 61 nM) [34]. To validate the specificity and compare the endpoints of all experiments, a well-established A_2AR antagonist, ZM241385, was employed as the positive control for this study [35]. The concentration of IDPU used in this report was selected based on preliminary in vitro evaluations and established safety profiles. Our studies revealed that PMDN cells, when treated with IDPU (0.01–10 μM) alone, displayed no toxicity (> 90% viability) (Fig. 3a). However, we noted that IDPU treatment post 3 h, 6-OHDA exposure in PMDN cells up to 24 h, increased the viability to > 80% (Fig. 3b). Similarly, our previous findings standardized the post-treatment protocol for ZM241385 in a similar in vitro PD model [36]. Henceforth, 6-OHDA-induced PMDN cells 3 h post-treated with IDPU (0.7 µM and 1 µM) and ZM241385 (1 µM), observed after 24 h, were used for this investigation. These concentrations were effective in restoring cell viability without inducing toxicity. For translational relevance, the chosen range aligns with concentrations that have shown behavioral and neurochemical improvements in rodent models of Parkinsonism [37, 38]. This supports the feasibility of extending IDPU’s use into in vivo studies, which are currently being streamlined as part of our future experimental workflow.

It has been reported that 6-OHDA and LPS treatment decreased the antioxidants and dopamine levels in animal models, which were counteracted by treatment with A_2AR antagonists, SCH412348, istradefylline, caffeine, etc. [36, 52, 53]. Additionally, 6-OHDA and LPS-induced inflammation triggered the neurodegeneration in primary rat striatal neurons, SHSY5Y cells, and PC12 cells [54]. In alignment with other studies, we found that IDPU and ZM24185-mediated blockage of A_2AR reduced ROS production and restored dopamine levels in 6-OHDA-induced PMDN cells (Fig. 4).

Our previous reports confirmed that A_2AR altered the [Ca²⁺]_i levels in HEK-A_2AR transfected cells through the cAMP/PKA/IP₃ pathway. Furthermore, we detected the attenuation of the 6-OHDA-induced [Ca²⁺]_i overload post A_2AR antagonist treatment [35, 36]. Collectively, these findings propose an interplay between A_2AR/[Ca²⁺]_i signalling, influencing downstream cascades of neuronal survival, including pathways of mitochondrial homeostasis [55].

In alignment with the above findings, we observed that [Ca²⁺]_i overload was depleted in 6-OHDA-induced PMDN cells when incubated with IDPU and ZM241385 (Fig. 4) compared to 6-OHDA alone. However, this study did not include the direct measurement of cAMP levels, but our prior results presented that IDPU and ZM241385 inhibited cAMP accumulation in A_2AR-transfected CHO (Chinese Hamster Ovary) cell lines [34]. Hence, we infer that the alleviation of [Ca²⁺]_i and ROS levels in our study might involve the opposition of the cAMP/PKA/IP3 axis downstream of A_2AR. However, this mechanistic link remains speculative and requires further experimental validation.

Furthermore, 6-OHDA-induced oxidative stress compromises mitochondrial function by enhancing ROS generation, disrupting polarization, and causing depletion of mitochondrial antioxidant proteins such as DJ-1, ultimately leading to neurodegeneration [8, 56–58]. However, Fang et al. and Castro et al. show that A_2AR downregulation relieves mitochondrial dysfunction by improving mitochondrial oxidative stress, membrane potential loss, and altering ATP production in retinal degeneration and osteoarthritis–chondrocytes model, respectively [59, 60]. In line with these investigations, our results provided evidence that IDPU and ZM241385 treatment lead to the attenuation of DJ-1 protein levels (Fig. 6a, d), mitochondrial superoxide production (Fig. 5a–h) and membrane potential loss (Fig. 5i–p), exhibiting the improvement of mitochondrial oxidative stress.

Identifying downstream effectors is significant given an established understanding of intricate crosstalk among oxidative stress, disrupted calcium homeostasis, and apoptosis [4–8]. In this context, p38MAPK is reported to be a sensor for ROS and calcium overload, further activating caspase-induced cell death. Studies in PD patient brains and animal models of PD also presented a positive correlation of activated caspase-1, caspase-3, caspase-8, and caspase-9 in dying dopaminergic neurons [21, 61, 62]. Nevertheless, Silva et al. recorded the protection of staurosporine-induced apoptotic neurons via A_2AR blockade [30]. Another study on hippocampal neurons elucidated that A_2AR suppression inhibits p38MAPK to prevent β-amyloid-induced synaptotoxicity [31]. Furthermore, Ren et al. confirmed that A_2AR antagonism prevented p38MAPK activation and apoptosis of hippocampal cells in the chronic hypoxic–hypercapnia model [33]. These insights strongly support the possibility of targeting A_2ARs to govern the p38MAPK-mediated survival in neurodegenerative conditions.

Similar observations were made; 6-OHDA-exposed PMDN cells exhibiting amplified ROS generation and calcium overload (Fig. 4) were found to be apoptotic (47.31%) (Fig. 4k, l), thus validating the increased protein levels of cleaved caspase-3 and phosphorylated p38MAPK (Fig. 4m, n) (Fig. 6a, b). However, post-treatment of IDPU and ZM241385 implicated that only 15.50% and 12.35% of cells were apoptotic (Fig. 4k, l), respectively, due to a reduction in protein levels of phosphorylated p38MAPK (Fig. 6a, b) and cleaved caspase-3 (Fig. 4m, n), leading to significant improvement in the 6-OHDA-induced PMDN cell death.

Evidence supports that 6-OHDA-induced oxidative stress and calcium overload triggered p38MAPK activation, which, in turn, can compromise mitochondrial homeostasis, leading to disturbed mitophagy by negatively tuning Parkin protein [17, 18, 25]. Additionally, Parkin cross-talking with DRP1 protein to persuade mitochondrial fission-related processes [19, 20].

In correlation, we observed that 6-OHDA-induced ROS production (Fig. 4) led to p38MAPK activation (Fig. 6a, b) that negatively regulated the protein levels of Parkin (Fig. 6a, e) and positively regulated the protein levels of DRP1 (Fig. 6a, c), causing activation of fission-related pathways, while suppressing the mitophagy-linked cascades promoting cell death. These findings lend credence to p38MAPK-mediated regulation of mitochondrial stress, recorded in our study (Fig. 5).

Furthermore, the alteration of PINK1/Parkin and DRP1 depletes α-synuclein aggregation in PD [63, 64]. Additionally, an investigation by Hu et al. employing KW6002 (A_2AR antagonist) ensured protection against A53T mutant α-synuclein-induced apoptosis via restoring the autophagic flux in the PD mouse model [31]. In support of this view, Castro et al. demonstrated enhanced mitophagy in chondrocytes post A_2AR counteraction [60], thereby highlighting the possible role of A_2AR/p38MAPK-mediated fission and mitophagy signalling in PD. Consistently, we found that IDPU and ZM241385 treatment in 6-OHDA-induced PMDN cells reduced the phosphorylation of DRP1 at serine 616 (Fig. 6a, c) compared to 6-OHDA alone. At the same time, Parkin levels were elevated post-treatment, depleted on 6-OHDA exposure (Fig. 6a, e), accompanied by a parallel decrease in p38MAPK phosphorylation (Fig. 6a, b), as discussed above. This indicates that A_2AR antagonism potentially contributes to the attenuation of mitochondrial dynamics via this cascade.

Collectively, our results suggested that post-treatment of A_2AR antagonists with IDPU and ZM241385 in 6-OHDA-induced primary midbrain neuronal cells is associated with attenuated cell viability, depleted oxidative stress and intracellular calcium overload, restoration of mitochondrial membrane potential loss, and SOD activity. At the molecular level, A_2AR modulation (via IDPU and ZM241385) alleviated the phosphorylation of p38MAPK (Tyr182) and DRP1 (Ser616), which were elevated under 6-OHDA exposure. Conversely, protein levels of Parkin and DJ-1 were depleted when incubated with 6-OHDA alone; however, both protein levels were enhanced when treated with both A_2AR antagonists. These findings imply that A_2AR blockade possibly attenuates the mitochondrial dynamics in 6-OHDA-induced PMDN cells, promoting survival via the p38MAPK/Parkin/DJ-1/DRP1 axis (Fig. 7).

Fig. 7.

Schematic Illustration of the potential neuroprotective mechanism of the A_2AR antagonist (IDPU) in 6-OHDA-induced PMDN cells. This figure illustrates that pharmacological blockade of A_2A receptors by IDPU and ZM241385 which attenuates 6-OHDA-induced intracellular calcium overload and enhanced reactive oxygen species (ROS) production in primary neurons. These upstream effects suppress the aberrant activation of the p38MAPK signalling cascade, thereby potentially preventing DRP1-mediated mitochondrial fission and preserving mitochondrial integrity. Concomitantly, IDPU restored Parkin-dependent mitophagy, maintaining mitochondrial membrane potential (MMP) and redox balance. Collectively, A_2AR antagonism mitigated oxidative stress, calcium dysregulation, apoptosis, and mitochondrial dysfunction through the coordinated modulation of the p38MAPK/DRP1/Parkin axis, thereby establishing its neuroprotective effect. (Red arrows depict the potential effect of A_2AR antagonistic intervention in 6-OHDA induced cells.)

However, we need further confirmation, especially in in vivo models, as direct measurements of mitochondrial fission, mitophagy, or oxidative phosphorylation, targeting these proteins, were not carried out to confirm its long-term neuroprotective effects. This step is already part of the future work plan. Moreover, the current investigation explores the short-term effects, while chronic exposure or repeated dosing for long-term gains needs further investigation. Additionally, the involvement of PGC-1α or AMPK pathways, maintaining mitochondrial homeostasis, cannot be ruled out and needs further investigation [17, 36]. Future studies incorporating co-culture systems with astrocytes or microglia may offer valuable insights into the broader neuroimmune context of IDPU-mediated neuroprotection [65].

These findings strongly support IDPU as a strong candidate for PD therapeutics, especially from a translational perspective. Prior in vivo data from our laboratory had recorded the neuroprotective effects of IDPU and other A_2AR antagonists in haloperidol- and NECA-induced Parkinsonism, including restoration of dopamine levels and antioxidant status [37, 38]. With current in vitro findings, IDPU exhibits dual functionality, marking its ability to influence mitochondrial integrity and redox homeostasis, eventually promising not only as a symptomatic treatment but also as a possible disease-modifying agent in Parkinson’s disease. However, these results need further validation in the context of preclinical studies to evaluate IDPU’s pharmacokinetics, blood-brain barrier permeability, and behavioral efficacy in PD models. Such repositioning of A_2AR antagonists expands their therapeutic value in addressing the metabolic and oxidative underpinnings of neurodegeneration.

Conclusion

This is the first report to demonstrate that A_2AR antagonist-induced attenuation of intracellular calcium and ROS levels potentially regulated the p38MAPK phosphorylation, fission-associated DRP1 phosphorylation, and mitophagy-associated Parkin protein, which perhaps contributed to the improved mitochondrial potential and oxidative stress in 6-OHDA-induced primary midbrain dopaminergic neurons. This work established the functional role of IDPU in reversing 6-OHDA-induced neurotoxicity, validated against ZM241385, a selective A_2AR antagonist. The exposure of 6-OHDA to TH-positive primary midbrain dopaminergic neurons used in this study recapitulated PD pathogenic features, increasing the translational relevance of our findings. This study mechanistically links A_2AR signalling with mitochondrial dynamics and dopaminergic recovery, offering a promising model for neuroprotection in Parkinson’s disease.

Abbreviations

A_2AR

Adenosine receptor

IDPU

1-(7-Imino-3-propyl-2,3-dihydrothiazolo[4,5-d]pyrimidin-6(7H)-yl)urea)

ROS

Reactive oxygen species

SD

Sprague Dawley (SD)

[Ca²⁺]_i

Intracellular calcium

TH

Tyrosine hydroxylase

PMDN

Primary mid-brain neuronal

PD

Parkinson’s disease

SNpc

Substantia nigra pars compacta

6-OHDA

6-Hydroxydopamine

GTPase

Guanosine triphosphatase

DRP1

Dynamin-related protein

MAPK

Mitogen-activated protein kinase

p38MAPK

P38mitogen-activated protein kinase

cAMP

Cyclic adenosine monophosphate

PKA

Protein kinase A

IP₃

Inositol triphosphate

NECA

Adenosine-5′-N-ethyluronamide

CaMK

Calmodulin-dependent protein kinase

CHO

Chinese hamster ovary

GPCRs

G-protein-coupled receptors

Author contribution

Vaishali Walecha (V.W) was involved in the study’s conceptualization, investigation, project administration and methodology. Jyoti Mishra (J.M.) and Tuithung Sophronea (T.S.) contributed to the methodology, and Namrata Kumari (N.K.) established the initial work on IDPU in the laboratory. Prof. Pratibha Mehta Luthra (PML) supervised, coordinated, and funded the study. Vaishali Walecha (V.W.) compiled the whole manuscript, including writing, editing, data curation, analysis, validation, and visualisation. Prof. Pratibha Mehta Luthra (P.M.L) reviewed the final draft of the manuscript.

Funding

Pratibha Mehta Luthra received research funds from the IoE, University of Delhi, needed to perform this study.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This project was approved by the Departmental Animal Ethics Committee on 21 st August 2023 for both in vitro (isolation of primary midbrain neuronal cells from P₀/P₁ pups of SD rats) and in vivo studies under proposal no. IAEC/ACBR/August 2023/PML/02.

Conflict of interests

The authors declare no competing interests.