Among age-related neurodegenerative disorders, Parkinson’s disease (PD) stands out as the second-highest occurring disorder. The distinctive clinicopathological features of PD involve the gradual and specific loss of dopaminergic (DA-ergic) neurons in the midbrain and the substantia nigra pars compacta (SNpc) regions of the brain. Additionally, a notable characteristic is the pathological buildup of $\alpha$-synuclein ($\alpha$-syn) aggregates [1]. Consequently, the dorsal striatum and other target areas have a dopamine deficiency, which causes chief motor dysregulations such as bradykinesia, stiffness, and tremors. Furthermore, non-motor manifestations, like depression and sleep disturbances in PD patients, are also evident [1].

Numerous studies have revealed that the brains of PD patients and animal models with synucleinopathies contain an abnormal accumulation of P-Ser¹²⁹-$\alpha$-syn, a pathologically active phosphorylated form of $\alpha$-syn [2, 3, 4, 5]. In healthy brains, $\alpha$-syn undergoes minimal phosphorylation, while there are dramatically elevated P-Ser¹²⁹-$\alpha$-syn levels during PD pathology in brains afflicted with Lewy pathology [2, 6]. This suggests a potential correlation between this post-translational modification and heightened deposition of $\alpha$-syn, coinciding with Lewy body formation and the beginning of the neurodegenerative process. Current strategies, including treatments inhibiting $\alpha$-syn’s aberrant aggregation, have generated significant interest as potential interventions that could potentially slow or arrest the advancement of the pathological condition.

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model is widely used due to its ability to replicate several key features of PD, including DA-ergic neuron degeneration and motor deficits. This model is well-characterized and allows for the study of neurodegenerative processes and potential therapeutic interventions. While specific $\alpha$-synucleinopathy models, such as transgenic mice expressing human $\alpha$-syn, provide insights into the role of $\alpha$-syn aggregation in PD, the MPTP model remains valuable for its simplicity, reproducibility, and relevance to DA-ergic system degeneration [7, 8]. We selected the MPTP model for its established utility in investigating neurodegeneration and evaluating neuroprotective strategies, which are critical aspects of PD research.

Oxidative stress mediates vital contributions in PD pathogenesis while activating glycogen synthase kinase-3 beta (GSK-3$\beta$). Increased GSK-3$\beta$ leads to $\alpha$-syn toxicity and the dysregulation of the Wnt/$\beta$-catenin signaling mechanism. The canonical Wnt/$\beta$-catenin signaling process, essential for embryonic developmental and tissue homeostasis processes, also controls neuronal function in the central nervous system (CNS) [9]. This pathway is an intriguing candidate that appears to be dysregulated in PD and has been linked to several neuropathologies [10]. Research suggests activating this pathway could restore neurogenesis and enhance brain self-repair capacity [11], making it a promising therapeutic candidate for countering neurodegenerative conditions. Furthermore, the pathogenesis of PD is associated with the activity of GSK-3$\beta$, a key regulator of the Wnt/$\beta$-catenin signaling pathway. GSK-3$\beta$ restricts $\beta$-catenin’s cytoplasmic stabilization and, hence, nuclear migration, effectively preventing the conventional Wnt pathway activation [12]. GSK-3$\beta$ inhibition has been shown to promote $\beta$-catenin’s stabilization, thus promoting Wnt signaling-mediated neuroprotective benefits in PD models [13]. Elevated expression of GSK-3$\beta$ in the nigral neurons of post-mortem PD brains and its aberrant phosphorylation at the Tyrosine 216 (Tyr216) is also evident in the striatum of individuals with PD [14, 15]. GSK-3$\beta$ activity is strictly controlled through the negative regulation occurring via GSK-3$\beta$ Serine-9 phosphorylation at the N-terminal region [16]. Pharmacological agents inhibiting GSK-3$\beta$activation may mitigate neurodegeneration in PD [17].

While neuronal autophagy is widely recognized as a defensive and beneficial mechanism for the physiological functioning of the nervous system, its paradoxical involvement in neuronal cell death has become increasingly evident. Numerous studies employing environmental toxins, hereditary PD-risk genes, and postmortem PD brain samples have illuminated the contributions of autophagy in instigating PD-associated DA-ergic neuronal loss [18, 19]. Particularly noteworthy is the identification of autophagic-regulated loss of nigral neurons in PD patients [20]. Oxidative stress induced by MPTP or dopamine toxicity has been demonstrated to amplify the formation of autophagic vacuoles, intensifying autophagic activity and subsequently leading to neuronal cell death [21, 22]. This dual role of autophagy, serving both neuroprotection and contributing to neurodegeneration, underscores its multifaceted involvement within the intricate framework of neuronal processes.

The drug targeting strategies in PD treatment have limited efficacies and are often associated with side effects. Phytochemicals present alternative, more efficacious, and safer treatment strategies against PD pathology. Owing to its diverse neuropharmacological characteristics, ranging from anti-oxidative to anti-inflammatory, the phenolic aldehyde vanillin exhibits promise against the pathophysiology of PD. The aim of this study was to determine if vanillin inactivates GSK-3$\beta$, thus potentiating the Wnt/$\beta$-catenin signaling in MPTP-induced nigrostriatal degeneration while protecting the DA-ergic system. By examining critical pathways implicated in PD pathogenesis, such as increased $\alpha$-syn expression, autophagic-neuronal loss, and Wnt/$\beta$-catenin/GSK-3$\beta$ activity, we seek to understand the underlying mechanism of therapeutic potential of Vanillin in PD.

In the present study, we have determined the potential neuroprotective effects of Vanillin in PD by assessing its effect on motor balance and coordination in the MPTP-induced PD mice model. Combining results from all three behavioral tests—the FST, wire hanging, and cylindrical tests allows for a more comprehensive assessment of the animal’s motor function. In coherence with previous studies, we found that MPTP administration induces depressive behavior and impaired motor activity [28]. However, vanillin effectively reversed these behavioral and motor dysfunctions. A similar motor deficit alleviating effect of vanillin has been previously reported in a rotenone-induced rat model of PD [29]. PD, which involves the aberrant accumulation of $\alpha$-syn inside the SNpc, which causes the selective and gradual death of neurons [30]. The post-translational modification of $\alpha$-syn is a crucial factor in its aggregation [31]. Phosphorylated $\alpha$-syn, particularly P-Ser¹²⁹-$\alpha$-syn, is a key hallmark of PD and related synucleinopathies [31, 32]. Research has demonstrated that the administration of MPTP leads to elevated levels of P-Ser¹²⁹-$\alpha$-syn in mice SNpc [33]. Consistent with these, our immunofluorescence and western blot results also showed a significant increase in P-Ser¹²⁹-$\alpha$-syn expression in MPTP-intoxicated mice. In contrast, Vanillin treatment significantly ameliorated the P-Ser¹²⁹-$\alpha$-syn expression in MPTP-induced mice.

The Wnt/$\beta$-catenin signaling is implicated in the pathogenesis of PD, regulating ventral midbrain precursor development, proliferation, and differentiation [34, 35, 36]. Dysregulated Wnt/$\beta$-catenin signaling is reported to be involved in the development of PD [37, 38, 39]. This signaling is altered by GSK-3$\beta$, an inhibitor of this crucial pathway, which phosphorylates and ubiquitinates $\beta$-catenin. The inactivation of GSK-3$\beta$ increases $\beta$-catenin levels, potentially offering neuroprotective effects [40]. Reports suggest that activating the Wnt/$\beta$-catenin may facilitate the differentiation and regeneration of DA-ergic neurons [41, 42]. This highlights the potential of targeting Wnt/$\beta$-catenin signaling components, as well as GSK-3$\beta$, which may be an effective therapeutic intervention for PD [34]. Blocking GSK-3$\beta$ reduced $\alpha$-syn protein expression and, consequently, prevented cell death in PD disease models [14]. Neuroinflammation, involving activated astrocytes and microglia, contributes to $\alpha$-syn fibril formation [43]. GSK-3$\beta$, an enzyme implicated in this process, exacerbates neuroinflammation when overactive. A recent study shows that inhibiting GSK-3$\beta$ via Ser⁹ phosphorylation reduces astroglial and microglial reactivity, lowering pro-inflammatory cytokine levels and ROS, thereby offering neuroprotection [44]. These findings suggest that GSK-3$\beta$ inhibitors could be a promising therapeutic approach to mitigate neuroinflammation and slow the progression of neurodegenerative diseases. Notably, in vivo, studies have also revealed that human GSK-3$\beta$^(S9A) mutated form exhibit elevated P-Ser¹²⁹-$\alpha$-syn and p-Tau in TH-positive DA-ergic neurons in mice as they age [17, 45]. These findings underscore the intricate interplay between GSK-3$\beta$, $\alpha$-syn phosphorylation, and the subsequent neurodegenerative processes, shedding light on potential targets for therapeutic intervention. Phosphorylation regulates the activity GSK-3$\beta$ activity. The enzymatic GSK-3$\beta$ activity gets reduced following the phosphorylation at serine-9 [46]. The results from both our immunofluorescence and western blot analysis showed a decrease in the protein levels of P-Ser⁹-GSK-3$\beta$ in MPTP-intoxicated mice. The MPTP-induced aberrant protein levels of P-Ser⁹-GSK-3$\beta$ was markedly alleviated following the Vanillin treatment. Next, we assessed the protein levels of Wnt-3a, $\beta$-catenin in the SNpc region of mice. Our results revealed that MPTP-lesioned mice had significantly reduced protein expression of Wnt-3a. Vanillin was significantly able to rescue this decreased Wnt-3a levels. $\beta$-catenin protein levels were also considerably downregulated in MPTP-intoxicated mice. These reduced levels of $\beta$-catenin upregulated following Vanillin treatment in MPTP-exposed mice. These findings imply that the neuroprotective properties imparted by Vanillin in vivo may be largely mediated by the activation of the Wnt/$\beta$-catenin signaling, mainly by suppressing the GSK-3$\beta$ activity.

Although neuronal autophagy is primarily recognized as a defensive mechanism, it can paradoxically contribute to neuronal cell death. Numerous studies have revealed the involvement of dysregulated autophagy in DA-ergic neuronal degeneration [47, 48]. Notably, PD patients showed autophagy in nigral neurons [49]. Studies have observed that MPTP administration elevates LC3-II expression and decreases p62 expression, indicating the activation of the autophagy-lysosomal pathway [50, 51]. Furthermore, a recent study indicates that induced autophagy can cause cell death in MPP⁺-exposed human neuroblastoma cells [52]. Our immunofluorescence findings demonstrate that increased LC3 immunoreactivity in the MPTP-induced mouse model is consistent with these observations. Vanillin treatment significantly lowered the LC3 immunoreactivity in the MPTP-administrated group. Reduced p62 expression and an elevated LC3-II/LC3-I ratio indicate increased autophagic activity. Our results also show that the expression of LC3$\mathrm{\Pi }$/I protein was elevated and Vanillin treatment significantly reduced the ratio of LC3$\mathrm{\Pi }$/I protein expression in MPTP-intoxicated mice. Additionally, p62 levels were decreased in the MPTP-administrated rats, which further shows the increased autophagic-cell death in MPTP-induced mice, and Vanillin administration markedly increased the p62 expressions. The effect of autophagy and autophagic cell death in dopaminergic neurodegeneration appears to be contingent upon the particular cellular setting and the initial causal cause. Improving the comprehension of autophagy stress [53] and the mechanisms governing autophagic-cell death holds promise for developing therapeutics to restore DA-ergic neuronal homeostasis in PD. Furthermore, additional physiologically relevant knowledge on the autophagic pathway’s activity status in PD patients is required to assess whether promoting or suppressing autophagy would be more beneficial in reducing the symptoms of the disease and delaying its progression. Future studies aimed to addressing the present dispute will be critical in translating these discoveries into viable therapies for PD.

In this study, we primarily focused on assessing the activity of GSK-3$\beta$ and the activation of the Wnt/$\beta$-catenin pathway in the context of MPTP-induced nigrostriatal degeneration, as these pathways are known to be involved in neuroprotection. While our study aimed to investigate the protective effect of Vanillin on the dopaminergic system by targeting GSK-3$\beta$ activity and the Wnt/$\beta$-catenin pathway, demonstrating the co-expression of related signaling molecules with dopaminergic neuron-specific markers would have provided stronger evidence. Thus, future studies should incorporate techniques such as immunofluorescence co-staining with specific markers for dopaminergic neurons to further validate our findings and enhance the understanding of Vanillin’s potential therapeutic effects on PD. Additionally, our study focused specifically on the SNpc region, and the mechanisms directly related to the Wnt/$\beta$-catenin signaling pathway. However, we did not investigate this pathway in the striatum region, which indeed plays a crucial role in the dopaminergic system and the pathophysiology of PD in the striatum. Future studies should consider examining this pathway in the striatum to provide a more comprehensive understanding of PD and to identify additional therapeutic targets against PD.

In conclusion, our study highlights the intricate mechanisms involved in PD etiology and the potential therapeutic role of Vanillin. The aberrant accumulation of $\alpha$-syn in the SNpc contributes to neurodegeneration in PD. Vanillin treatment effectively mitigates the elevated levels of phosphorylated P-Ser¹²⁹-$\alpha$-syn. Moreover, our study of Wnt/$\beta$-catenin signaling highlights the critical role it plays in PD pathogenesis and highlights the possibility of using GSK-3$\beta$ as a target for neuroprotection and neurorestoration. GSK-3$\beta$, implicated in both $\alpha$-syn phosphorylation and $\beta$-catenin degradation, emerges as a critical player in PD pathophysiology. Vanillin treatment not only mitigates GSK-3$\beta$ activity but also positively modulates Wnt/$\beta$-catenin signaling, suggesting its neuroprotective effects. The dual role of autophagy in neuroprotection and neurodegeneration, revealing its involvement in DA-ergic neuronal degeneration in PD. Treatment with vanillin significantly reduces abnormal autophagic-cell death, suggesting a possible function for vanillin in reestablishing DA-ergic neuronal homeostasis. Thus, by inhibiting GSK-3$\beta$ activity and reducing autophagic cell death in DA-ergic neurons, vanillin may stimulate the Wnt/$\beta$-catenin signaling pathway, which in turn may explain its neuroprotective effect in our study Fig. 6. This study enhances our understanding of the complex interplay of molecular pathways in PD and underscores Vanillin’s promising therapeutic implications for the disease.

Fig. 6.

Possible mechanisms that mediate the neuroprotective action of Vanillin on MPTP-intoxicated PD mice model. DVL, Dishevelled; CK-1, casein kinase 1; APC, adenomatosis polyposis coli; Lrp, lipoprotein receptor-related protein; TrcP, transducin repeats-containing protein; Ub, ubiquitin; Axin, axis inhibition protein; DA-ergic, dopaminergic.

All the data produced during the study are already included in the manuscript, and no further data is required to reproduce the results.

ACM conceptualized, supervised the study and edited the final manuscript. LR performed the experiments and wrote the manuscript. Both authors read and approved the final manuscript. Both authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All the experiments were carried out on C57BL/6 mice following standard guidelines and regulations of the Institutional Animal Ethics Committee (IAEC), Jawaharlal Nehru University (JNU), New Delhi (10/GO/ReBi/99/CPCSEA/March 10, 1999). All the Standard methods and protocols were followed for animal handling and experiments. All efforts were made to reduce animal suffering.

The authors would like to acknowledge the Central Instrumentation Facility (CIF), School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.

This study was supported by the Department of Biotechnology (DBT), Govt. of India (BT/PR38493/TRM/120/465/2020), and (BT/PR47726/CMD/150/26/2023), Ministry of Science and Technology (Govt. of India).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/j.jin2309175.