Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that manifests as selective death of dopaminergic neurons in the substantia nigra [10,11,15]. As an excitatory neurotransmitter, glutamate is thought to act through ionotropic N-methyl- D-aspartic acid (NMDA) receptors, kainate, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and G-protein-coupled metabotropic receptor subtypes [1,28,35]. The progression and maintenance of PD are associated with increased glutamatergic neurotransmission associated with hyperphosphorylation of NMDA receptor subunits. It is believed that NMDA receptors primarily contain the subunits NR1 (NMDA receptor type 1), NR2A (NMDA receptor type 2A), and NR2B (NMDA receptor type 2B). It has been reported that NR2 subunits are unevenly distributed throughout the central nervous system, causing calcium overloads and dopaminergic neuron death in the basal ganglia and striatum [28,41].
Abnormal excitation of neurons in the nigrostriatal pathway is likely to be mediated by NR2B, which directly activates neurons in the globus pallidus and substantia nigra in PD [24,45]. Previous studies conducted on monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have demonstrated the antiparkinsonian effects of two different NR2B-selective NMDA antagonists [6,43]. However, inhibiting NMDA receptors throughout the central nervous system can result in undesirable cognitive and psychiatric effects, so targeted inhibition of NMDA receptors has been proposed as a treatment [2,14,17].
Multiple neurodegenerative disorders, including Alzheimer’s disease and PD, have been observed to impair the autophagy process, which degrades proteins and organelles [3,31]. It has been shown in numerous studies that PD reduces microtubule-associated protein 1 light chain 3B (LC3-I) to LC3-II transitions, as well as increasing SQSTM1/p62 levels, indicating that autophagy is suppressed [19,21,38]. The hypothesized involvement of autophagy in PD arises from the delayed inhibition of autophagic flux caused by NMDA receptor activation [25]. It remains unclear, however, whether autophagy contributes to excitotoxicity in PD or increased autophagy functions as a protective response.
In this study, we examined whether ifenprodil, a NR2B antagonist, intracerebrally injected can reduce excitotoxic injury caused by excessive autophagy and whether this effect is related to autophagy stimulation and inhibition.
Material and methods
Chemicals
Antibodies for LC3B/A (83506), SQSTM1/p62 (23214), beclin-1 (3495), BAX (5023), BCL-2 (15071), BNIP3L/Nix (12396), PARP (9532), and TH (58844) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for NR2B (ab28373), NR2B phosphor-S1303 (ab81271) and phosphor-T1472 (ab3856), NMDAR1 (ab109182), NNMDAR1 phosphor-S890 (ab52184), iba-1 (ab178846), PSD95 (ab18258), CaMK (ab227108), and phosphor-T287 (ab182647) were obtained from Abcam (Cambridge, UK). -Ca (PA5-114937) and -Ca (PA5-82589) were acquired from Thermo Fisher Scientific (Carlsbad, CA).
Drugs
Ifenprodil was obtained from MedChemExpress (MCE, Qingdao, China) and dissolved in sterile saline followed by filtration through sterile filters (Millipore Millex) for animal injection.
Animals and experimental arrangement
Male Sprague-Dawley rats (SPF) (200 ±20 g) were obtained from the Pengyue Experimental Animal Center (Jinan, China) (age: 6 weeks) under the laboratory animal license SCXK (Lu) 20190003. Rats were acclimated to the animal facility for 1 week in an animal room maintained at 23°C with a 12 : 12-hour light-dark cycle. Ethical approval for this study was obtained from the Experimental Animal Care and Use Research Committee of Yantai University (APPROVAL NUMBER: YT-YX-2019-0097). Figure 1 shows the allocation of rats to the saline group, model group, I.P. group (intraperitoneal, 6.25 mg/kg, twice/day), CPU group (100 nm, speed: 0.25 ml/min, twice/day), and SNr group (100 nm, speed: 0.25 ml/min, twice/day) for a 28-day experiment.
6-OHDA-lesioned rats
Rats were pretreated with desipramine hydrochloride (25 mg/kg, I.P.) 30 min prior to the injection of 6-OHDA to protect noradrenergic fibers. The 6-OHDA- lesioned rat model was created as described in previous studies [9,34]. For anesthesia, 1% phenobarbital (0.1 ml/100 g, I.P.) was administered. The nigrostriatal pathway was unilaterally lesioned by stereotaxic injection of 6-OHDA (4 µl with 0.5 µl/min, 4 mg/ml, dissolved in 1 mg/ml ascorbate) over a period of 8 min at the following coordinates: anteroposterior (AP), –4.0 mm; lateral (LA), –1.65 mm; dorsoventral (DV), –8.0 mm from the bregma. The microinjector was left in place for 8 min to allow for diffusion after injection. The group that received saline only was injected with the same amount of saline. Three weeks after injection, the 6-OHDA-lesioned rats were given apomorphine (0.5 mg/kg). Rats that exhibited more than 50 contralateral turns per 10 minutes were selected for further investigation.
Microinjection
Microinjection was performed as described previously [47]. The procedure involved inserting a cannula into the brain and anchoring it to the skull using screws and dental cement. Two cannulas were inserted, one at the striatum (bregma: AP, –0.26 mm; LA, ±4.0 mm; DV, 4.0 mm) and the other at the midbrain (bregma: AP, –4.5 mm; LA, 1.65 mm; DV, 8.0 mm). Injection cannulas, attached to a 10 µl Hamilton syringe, were then inserted into guide cannulas for drug infusion. After injection, the cannulas were left in place for 8 minutes.
Behavior test
Open field test
As previously reported, the experiment involved placing rats in an open field chamber measuring 50 × 50 × 70 cm (W × D × H) and allowing them to move freely for 10 minutes [15]. To measure locomotor activity, the rats were videotaped, and their total movement distance was calculated using SMART 3.0, a video tracking software system developed by Panlab in the United States.
Rotarod test
The rotarod unit comprised a revolving spindle with a diameter of 7.3 cm and four separate compartments, as previously described [16]. The rats were placed on a rotarod apparatus that accelerated from 0 to 40 rpm in 10 minutes. The retention time on the rod was recorded to evaluate the rats’ motor coordination.
Perfusion fixation
The rats were sedated with pentobarbital (65 mg/kg, I.P.) and then transcranially perfused with ice-cold 0.9% NaCl [12]. After decapitation, the brain was re- moved.
Immunofluorescence staining
Briefly, 2.5 µm paraffin slices were deparaffinized in xylene and rehydrated using graded alcohols, as previously described [48]. The slices were washed three times with PBS for 5 minutes each time. The homologous serum of the secondary antibody was blocked for 30 minutes. The primary antibodies TH (1 : 50) were incubated overnight at 4°C. The slices were washed three times with PBS for 5 minutes each time and then incubated with fluorescent secondary antibody (1 : 250) for 1 hour at room temperature. The nuclei were visualized by counterstaining the sections with DAP and observing them using a digital pathology scanner (OLYMPUS VS120-S6-W).
Transmission electron microscopy
Transmission electron microscopy was used to observe autophagosomes in the midbrain of mice. The midbrain specimens were fixed using a solution consisting of 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer. Subsequently, the specimens were incubated in a mixture of 1% osmium tetroxide and 0.1 M sodium cacodylate buffer, followed by dehydration using a gradient of alcohol and acetone. The specimens were then embedded, and ultrathin sections were obtained. These sections were stained with uranyl acetate and lead citrate before being examined under electron microscopy to evaluate the presence of autophagosomes.
Western blot analysis
The Western blot analysis was performed following the methods described in previous studies [17,26]. Total protein extracts were obtained using RIPA buffer. The protein concentration of the supernatant was determined using an enhanced BCA protein assay kit (Beyotime). Subsequently, 50 µg protein aliquots from the supernatant of the brain tissue were subjected to SDS-PAGE (10%) and transferred onto polyvinylidene difluoride (PVDF) membranes (Whatman Westran PVDF membrane, Sigma). The membranes were blocked with nonfat dry mivvlk at room temperature. Primary antibodies were applied overnight at 4°C, followed by treatment with secondary antibodies for 2 hours at room temperature the next day. Antibody-reacting bands were detected using enhanced chemiluminescence (ECL) luminous fluid (Solarbio, Beijing, China).
Statistical analysis
The data are presented as mean ± standard deviation (x ±SD). Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA). One-way or two-way ANOVA (two-sided) was used. A p-value of less than 0.05 was considered statistically significant.
Results
Ifenprodil ameliorated behavioral deficits in PD animals
Figure 2 shows that the administration of ifenprodil consistently improved the duration that 6-OHDA-lesioned rats remained on the rod across all drug delivery methods. The CPU group exhibited a statistically significant divergence starting on the 14th day (p < 0.05) compared to the model group, while the SNr group showed a significant difference on both the 14th and 17th days (p < 0.05). Compared to the I.P. group, the CPU group showed a significant difference on the 14th (p < 0.05) and 28th days (p < 0.01). Furthermore, when analyzing two distinct brain injection regions, only the CPU group and SNr group exhibited a significant difference on the 14th day (p < 0.01). Therefore, it is impossible to independently assess the impacts of the two brain injection regions. Figure 2B, C presents the findings of the open-field test, indicating that the ifenprodil-treated groups exhibited a higher frequency of behavior compared to the model group. Moreover, the CPU group showed a statistically significant difference from the model group (p < 0.05). Additionally, the I.P. group and SNr group showed a tendency towards improvement.
Ifenprodil provided a protective effect on neurons
The findings from projection electron microscopy are presented in Figure 3A, B. In comparison to the control group, the model group exhibited a reduction in the quantity of autophagy bodies, whereas ifenprodil treatment (CPU group) ameliorated the inhibition of autophagy in the model, resulting in an increase in the number of autophagy bodies and the promotion of autophagy (p < 0.001). The presence of TH neurons in the striatum and substantia nigra was identified through immunofluorescence analysis. As depicted in Figure 3C-F, a reduction in the positive area of TH neurons was observed in the model group compared to the normal group, accompanied by significant neuronal synapses loss in the striatum and evident vacuolation. However, ifenprodil exhibited a protective effect on the neuronal synapses and neurons, particularly in the CPU group (p < 0.001).
Ifenprodil protects synaptic functional plasticity
The objective of this study was to examine the effects of 6-OHDA-induced lesions and ifenprodil treatment on the expression of NMDA receptor subunits and their involvement in the PSD95/NMDAR2B-CaMKII signaling pathways. Figure 4 demonstrates that there were no significant differences in the overall concentration of NR2B protein among the experimental groups. However, the model group showed an increase in the phosphorylated forms at positions S1303 and T1472. There were no significant differences in the expression levels of CaMKII and -Ca protein among the experimental groups. However, the model group showed a significant increase in the levels of phosphorylated CaMKII and -Ca. Ifenprodil was effective in all treated groups, resulting in a significant reduction in PSD95, S1303 phosphorylation, T1472 phosphorylation, phosphorylated-CaMKII, and -Ca, especially in the CPU group (p < 0.001). The decrease in CaMKII phosphorylation was particularly noteworthy, especially in the CPU group (p < 0.001).
Involvement of the autophagy pathway in the neuroprotective effect of ifenprodil
To assess autophagic activity, we examined alterations in autophagic and apoptotic proteins in the striatum and midbrain, as shown in Figure 5. The findings suggest that striatal lesions induced by 6-OHDA significantly decreased the levels of LC3b, beclin-1, and bcl-2, while increasing the levels of p62, BAX, and PARP. The treatments with ifenprodil showed a tendency to increase the expression of LC3b, beclin-1, and bcl-2, while simultaneously reducing the levels of p62, BAX, and PARP, especially in the CPU group (p < 0.001). Additionally, the receptor for mitochondrial autophagy BNIP3L/Nix, which binds to LC3 during cellular development and pathological conditions, increased in response to the treatments, especially in the CPU group (p < 0.001).
Discussion
Glutamatergic neurons project to the anterior and posterior striatum, as well as the substantia nigra, from various areas of the prefrontal cortex [9,20,27,49]. Compared with normal levels, glutamate levels and gluta- mate receptor density increased by 156% in the rat striatum following a 6-OHDA lesion [9,11]. Based on the reported mechanisms, the significant increase in intracellular Ca2+ caused by glutamate-induced NMDA receptor over-activation plays a crucial role in initiating intracellular events that lead to autophagy dysfunction and ultimately cell death [22]. However, current research suggests that the role of glutamate in motor control, and thus the effects of glutamate antagonists in the brain, may be more complex than previously thought [37]. There is evidence that NR2B receptor antagonists can alleviate dyskinesia in PD models in rodents and primates [8,33]. However, the mechanisms related to autophagy remain unclear, and adverse effects associated with the full course of administration continue to limit their development. Thus, this study aimed to examine the mechanism of ifenprodil involvement in autophagy in 6-OHDA-lesioned rats, meanwhile evaluating the effect under different intracerebral administration (CPU and SNr).
The rotarod test and open field test showed that ifenprodil could improve rats’ exercise capacity, and the CPU group’s effect was better than systemic administration, which may be related to ifenprodil increasing the release of dopamine from rat striatal slices.
Previous studies have reported that both dopamine depletion induced by PD and treatment with L-DOPA result in the redistribution of NMDA receptor subunits. Specifically, in PD patients and levodopa-treated dyskinetic rats and monkeys, there is an increase in the GluN2A subunits and the GluN2A/GluN2B subunit ratio [13,18,42]. The binding of NMDA-sensitive glutamate in the striatum and nucleus accumbens exhibited a significant increase in both experimental models of PD and the tissue samples obtained from patients with PD [5,51]. This heightened binding has the potential to expedite the degenerative progression of the disease. The striatum is suggested as a possible site for abnormal synaptic plasticity in PD, with NMDARs being well known for their important role in inducing synaptic plasticity [7,36]. NR2A and NR2B, the primary components of NMDARs, are present in the striatum, showing unique pharmacological properties. Phosphorylation of the NR2 subunits by the Fyn and Src protein tyrosine kinases from the Src family in PD leads to an excessive increase in channel activity [39,40,50]. In our findings, the administration of 6-OHDA resulted in a reduction in the overall protein expression of NR1, an elevation of the 1472/NR2B ratio, and enhanced phosphorylation of CaMKII. These observations indicate aberrant functioning of the NR2B receptor in calcium channel activation, as well as upregulation of PSD95 expression, suggesting an abnormality in synaptic plasticity. Ifenprodil reduced the expression of NR2B and inhibited phosphorylation, thereby protecting synaptic functional plasticity, with the CPU group showing the best effect.
Beclin-1 and p62 play crucial roles in autophagy regulation [44,46]. Our results indicate increased PARP expression and impaired autophagy in rats treated with 6-OHDA, ultimately resulting in apoptosis [4,23,29,30,32]. The effects of ifenprodil on the striatum, however, have been demonstrated as enhanced autophagy and reduced cell death. The regulation of Ca2+ in autophagy and apoptosis is important in cellular processes. Our study further demonstrated that ifenprodil can alleviate the aberrant Ca2+ signal, aligning with previous research findings.
Conclusions
Our results indicate that in the 6-OHDA rat model, direct administration of ifenprodil to specific brain regions has better therapeutic efficacy compared to systemic administration, especially intra-striatal administration. In addition, ifenprodil can ameliorate autophagy impairment and improve synaptic plasticity to exert anti-PD effects in 6-OHDA-lesioned rats. Therefore, we believe that the improvement of NR2B receptor antagonists and their targeted therapies have the potential to improve their efficacy in PD.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (21877095), and Taishan Scholar Project (Kehao Zhao).
Funding
This research received no external funding.
Disclosures
The study was approved by the Bioethics Committee of the Yantai University (Approval No. YT-YX- 2019-0097).
The authors report no conflict of interest.
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