Discovery of L-dopa was a major step forward in the treatment of PD, but its major drawback was the generation of various side effects. In addition, many combinational drugs were reported to reduce the side effects, but the success rate was very low . Besides, the researchers also focused on other molecular targets as well as the alternative treatment options to improve the lifestyle of PD patients. PD attributed by mitochondrial dysfunctions, impairment of protein degradation, oxidative stress and death of DAergic neurons in SN, but the continuous progression of molecular events leading to cell death remains unclear . Prior to designing any preventive intervention, in-depth knowledge of the molecular mechanisms underlying neurodegeneration in PD is required.
ROT, is a well-known pesticide acting selectively on mitochondrial complex I, causing its inhibition like MPP+. A growing body of proofs suggest that the insecticide model offers more advantages over different experimental models as it will effectively mimic the behavioral and neuropathological symptoms of the illness through the selective degeneration of DAergic neurons [46,47].
PD have various motor (tremor, slowness of movement, rigidity, falls and dizziness, freezing, postural imbalance)  and non-motor symptoms (pain, low blood pressure, fatigue, restless legs, swallowing and saliva control, sleep, skin and sweating, bowel and bladder problems) . Various studies reported that non motor symptoms generally develop at the initial stage of PD progression so that it is not able to differentiate PD patients from others. Zahra et al. (2020) reported the characteristic motor impairment in ROT-intoxicated Parkinsonian mice . To check the behavioral deficit in mice, we performed various behavioral tests such as catalepsy, rotarod, pole, and footprint, which highlighted severe motor abnormalities in ROT-intoxicated PD mouse model [42–45] (Figure 1). Indistinguishable co-ordinates and deterioration of motor skills were also observed in our result due to ROT toxicity, whereas the behavioral tests demonstrates that Tc improves the neurobehavioral shortfall as compared with previous studies .
Various medicinal herbs are used for therapeutic functions since ancient times in India, China, and other Asian countries. Medicinal plants are used to treat various neurodegerative diseases  and showed very promising results with minimal side effects. Herbs like Bacopa monnieri, Withania somnifera, Mucuna pruriens, Tc, are tested for its therapeutic potential against neurodegenerative diseases via redox balancing and immunomodulation activities [18,52,53]. In our previous study, we reported that Tc has neuroprotective role via suppressing oxidative stress as well as neuroinflammation .
With the application of proteomic profiling analysis, the quantitative and qualitative analysis of thousands proteins from the tangled mixtures along with the demonstrations of their PTMs could be efficiently performed. Based on our findings, we have identified various new molecules and pathways as alternate treatment approach. Here, we used comparative proteome profiling through LFQ/LC-MS/MS to investigate DEP in nigrostriatal region of mice brain. Out of total 800 DEPs, 101 were upregulated and 76 were downregulated in ROT-intoxicated mice compared to control; whereas, out of total 133 DEPs in Tc-treated mice, 13 were upregulated and 11 were downregulated as compared to ROT. Some proteins which are significantly expressed in PD group were reversed by Tc treatment. On the basis of expression and co-relation with PD marker proteins, we selected 21 proteins (Table 2A, Table 2B). Further in-silico analysis suggested that some of DEPs (Smad7, Tfam, Grin2a, Gpr37, and Tiam7) play a key role in PD pathogenesis via regulating different signaling pathways (Table 3) and could be a new therapeutic avenue for neuroprotection.
Smad7 is an inactive record factor in pathogenesis of PD, which is constitutively upregulated in triggered microglia . It leads DAergic neurons towards apoptosis by means of p53 and TGF-β cascade. Interestingly, Pal et al. (2016) indicated that activators of either NF-κB (IL-1β, TNF-α) or STAT-1 (IFN-γ) signaling pathways enhances the expression of Smad7 [54,55]. In the present study, we also found an upregulated expression of Smad7 in PD mice which is in agreement with the results of Uberham et al. (2006) whereas, its expression was downregulated after Tc treatment. Upregulation of Smad7 in triggered microglial cell has possessed threat to the neuroprotective TGF-β1 signaling [55,56]. TGF-β signaling favors cell survival by inactivating Bad (pro-apoptotic protein of the Bcl-2 family) thereby enhancing its phosphorylation through activation of the ERK/MAP kinase pathway. TGF-β1 signaling also suppresses the secondary neuronal damage caused by neuroinflammation [57–59]. Thus, TGF-β/Smad signaling pathway plays a crucial role in neuroprotection. Our results also suggests that Tc potentially suppresses the death of DAergic neurons via downregulation of Smad7  .
Tfam (Mitochondrial Transcription Factor A) is a 25 kDa DNA-binding protein contained in mtDNA. Tfam protects mtDNA from ROS, and mtDNA protects Tfam from Lon protease degradation [60, 61]. Loss of Tfam expression was found to be linked with degeneration of the DAergic neurons , whereas its enhanced expression improves mitochondrial ailment phenotypes via accelerating mtDNA copy variety and delays neurodegeneration . Consequently, Tfam is a good indicator of defects in mitochondrial integrity. In this study, we observed that ROT-intoxication upregulates Tfam expression whereas, its expression was downregulated by Tc pretreatment. Lu et al. (2018) also demonstrated a downregulated Tfam expression in ROT-intoxicated cultured SH-SY5Y [64,65]. Thus, it is suggested that by downregulating Tfam expression, Tc improved the mitochondrial function, which is a major pathogenic hallmark of PD.
Gpr37 is a substrate of the E3 ligase Parkin, and henceforth is also called Parkin-related endothelin-like receptor (Pael-R) . Missense type of mutation in PARKIN causes aggregation of Gpr37 in brain of PD patients . Besides, overexpression of Gpr37 prompts its amassing in totals causes ER stress, and neuronal death [66, 68]. On other hand, it has been proposed that native Gpr37 shows neuroprotective property by binding prosaptide and prosaposin . Interestingly, Lundius et al. (2013) reported that overexpression of Gpr37 was robustly protected against ROT, MPP+ or 6-OHDA-induced cytotoxicity in N2a cells . Furthermore, it has been suggested that Gpr37 regulates oligodendrocyte separation and myelination by means of ERK signaling . However, Tc-treatment to the PD mice suppresses the expression of Gpr37 in ROT-intoxicated mice model. Here, we present that Gpr37 plays a broader role in neuroprotective and glioprotective activity of Tc.
Tiam1 (T lymphoma invasion and metastasis 1) is a Rac-specific GEF (Dbl family member), potentially involved in neurodegenerative diseases (AD and PD). It is a Ras effector molecule, stimulated by the Ca2+-dependent activation of the NMDA receptor (NMDAR) . Tiam1 controls the activation of Rho GTPase and plays an important role in PD by regulating oxidative stress and neuroinflammation . Tiam1 controls neurite extension and DAergic neurons differentiation [74,75] . After exposing hippocampal neurons to amyloid-β peptide, Tiam1 is activated and mobilized to the membrane, which may affect the pathology of AD . In the present study, expression of Tiam1 is downregulated by Tc extract treatment against ROT-intoxication. Interestingly, Smith et al. (2017) also reported that, an elevated expression of Tiam1 contributes to neuronal damage . In contrast, Cajanek et al. (2013) showed, Tiam1 as a positive regulator of DAergic neuron differentiation . Together, these studies indicate that ROT-induced neurotoxic effects are linked with an upregulated expression of Tiam1 and Tc reverts the ROT-induced neurotoxicity via downregulating Tiam1 expression.
Akt, a serine/threonine kinase [also known as protein kinase B (PKB)] is an important molecule necessary for neuronal survival as it plays a major role in phosphorylating its substrates, including GSK3, NF-κB, BAD, and forkhead proteins . Downregulation of Akt signaling are seen in PD . Previous studies suggested that PD-inducing neurotoxins including 6-OHDA and MPP+ decreases the expression of pAkt . Durgadoss et al. (2012) demonstrated the Akt signaling impairment in the MPTP-induced mouse model, showing pAkt levels and loss of Akt kinase activity (Thr308 and Ser473). Similarly, we have also reported a downregulated expression of Akt1 in ROT-induced parkinsonian mice. Growing evidence also suggested the importance of mTOR pathway in autophagy and apoptosis which can lead to neuronal death, but later it was found that was the inhibition of the Akt phosphorylation, rather than mTOR activation that eventually led to neuronal loss . Raghu et al. (2009) reported, activation of Akt by G1-4A (a polysaccharide from Tc) . In an attempt to unravel the anti-apoptotic action of Tc against ROT-intoxication, our study revealed an increased expression of Akt1, also supported by Salama et al. (2020) [83,84].
The present study showed that Tc significantly reversed ROT‐induced downregulation of Grin2A expression and exhibits neuroprotective effect against ROT‐induced neurotoxicity. Moreover, downregulation of Grin2A (NR2A, a glutamate ionotropic NR type subunit 2A) expression resulted in Ca2+-mediated neurotoxicity. NRs (N-methyl-D-aspartate or NMDA receptors) play a crucial role in the pathogenesis of neurological disorders . Reduced expression of the NRs was found in the patient’s brain with neurodegenerative diseases [86,87]. NRs require proper complex formation between GRIN1 and GRIN2 (A-D) subunits to permit Ca2+ influx into the cell . GRIN2A, regulates excitatory neurotransmission in the brain and thus plausibly influence the course of PD. Kong et al. (2015) confirmed that Grin2 is downregulated significantly in PD flies against α-synuclein neurotoxicity . Interestingly, in present study we have documented a downregulated expression of Grin2A in ROT-induced parkinsonian mice, whereas, Tc upregulate the Grin2A expression against ROT-intoxication. Similarly, Simon et al. (2017) and Hamza et al. (2011) also reported Grin2A association with the risk of PD [86, 87]. Downregulation of Grin2A showed lack of binding to Grin1 resulting in reduced NMDAR complex formation. By considering all the aforementioned studies the potential of targeting Grin2A for the therapeutics of PD pathogenesis is confirmed. Tc plays an important role in attenuation of NMDAR-dependent Ca2+-mediated signaling via downregulating Grin2A.
Epigenetic dysregulation have emerged as important component in the PD pathogenesis . Kdm3A is a JmjC (Jumonji) domain-containing lysine (K)-specific demethylase 3A. Kdm3A-mediated demethylation of PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) causes inhibition of subsequent mitochondrial biogenesis and oxidative metabolism via reduction in PGC-1α and Nrf1/2 binding and Nrf1/2-dependent Tfam expression . Dnmt 1 [DNA (cytosine-5)-methyltransferase 1] an enzyme catalyzing DNA methylation via methylating CpGs on hemimethylated DNA. Hypermethylation of PGC-1α promoter results in the reduction of PGC-1α protein expression. Downregulated PGC-1α exacerbates age-dependent neuroinflammation by enhancing the release of IL6 .
From the result of qRT-PCR, it is clearly evident that Smad7, Gpr37, Kdm3a, Dnmt1, and Tiam1 are increased in ROT-intoxicated mice, while it is significantly restored in Tc treated group. On the other hand, the mRNA level of Tfam, Akt1, and Grin2a were downregulated in ROT group while it was restored in Tc treated group. This demonstrates that Tc exhibits neuroprotection of DA neurons in ROT mice via regulating different signaling pathways at mRNA level.
Overall, epigenetic methylation of PGC−1α positively regulates initiation and progression of PD. Downregulation of PGC−1α via inhibition of Dnmts-mediated hypermethylation, or Kdm3A-mediated demethylation, play central role in PD pathogenesis by improving mitochondrial dysfunction, oxidative and inflammatory stress. In this study, results have shown the downregulation of Dnmts and Kdm3A gene after Tc treatment compared to ROT-induced parkinsonian mice. Hence, Tc may be a potential therapeutic agent for epigenetically modified targets in PD pathogenesis (Figure 8).