Protection Against Cellular Toxicity from Rotenone Treatment by the Neuroprotective, Novel Multifunctional Antiparkinsonian Drug D-512

TL;DR

This study shows that D-512 protects brain cells from rotenone toxicity, a pesticide linked to Parkinson's disease.

Contribution

The novel drug D-512 is shown to have multifunctional neuroprotective effects against rotenone-induced toxicity in neuronal cells.

Findings

D-512 protected PC12 and MN9D cells from rotenone-induced toxicity in a dose-dependent manner.

D-512 reversed mitochondrial dysfunction and reduced oxidative stress caused by rotenone.

D-512 inhibited rotenone-induced apoptosis and restored dopamine-related signaling.

Abstract

Objective: Exposure to rotenone, a naturally occurring pesticide, has been linked to an increased risk of developing Parkinson’s disease (PD). Rotenone strongly inhibits complex I of the mitochondrial respiratory chain, inducing oxidative stress both in vitro and in vivo, ultimately leading to cell death. The objective of this study was to evaluate the cytoprotective effects of the multifunctional agonist D-512 against rotenone-induced toxicity in neuronal PC12 and dopaminergic MN9D cell lines. Methods: Various cell-based assays, including cell viability, antioxidant activity, caspase-mediated apoptosis, and other related assays, were performed. Results: Rotenone was found to be toxic to both dopaminergic MN9D cells and neuronal PC12 cells. However, treatment with D-512 protected both cell types from rotenone-induced toxicity in a dose-dependent manner. Rotenone-induced impairment of mitochondrial membrane potential and increased production of reactive oxygen species were reversed by D-512 treatment. Furthermore, rotenone-induced caspase-mediated apoptotic signaling in MN9D cells was inhibited by D-512. In addition, D-512 restored levels of phosphorylated tyrosine hydroxylase in rotenone-exposed cells across various doses, indicating protection of the dopaminergic system. Finally, rotenone-induced activation of phosphorylated ERK was reversed by D-512 treatment, further supporting its neuroprotective potential. Conclusions: This study demonstrates the ability of D-512 to reverse the toxic effects of rotenone across multiple experimental models. The data presented here are consistent with previously reported neuroprotective properties of D-512. The multifunctional nature of D-512, which combines potent dopamine agonist activity with neuroprotective and other beneficial properties, may address therapeutic needs in PD beyond symptomatic relief and could have potential application across PD subgroups as part of a personalized therapeutic approach.

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1. Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder whose incidence increases with age. Clinically, PD is characterized by resting tremor, muscle rigidity, and bradykinesia early in the disease process, whereas postural instability and freezing appear later. Pathologically, PD is characterized by the dopaminergic neuronal loss in the substantia nigra pars compacta and subsequent depletion of dopamine (DA) in the striatum [1]. The presence of intracellular inclusions called Lewy bodies and Lewy neurites is the hallmark of PD pathology [2]. These inclusions are primarily characterized by the presence of α-synuclein along with ubiquitin, neurofilament protein, and other proteins [3,4,5].

Current therapeutic strategies for treating PD mainly offer symptomatic relief by restoring the levels of DA by the administration of levodopa (L-DOPA), a direct precursor of the neurotransmitter DA. However, long-term use of L-dopa gives rise to motor fluctuations with dyskinesias characterized by decreased duration of response to a given L-dopa dose [6,7] and development of “on” and “off” episodes. DA agonists have a long history of therapeutic use in PD, either as a monotherapy or as an adjunct to L-dopa, particularly in the early stages of PD and in younger patients to mitigate L-dopa-induced side effects [8,9,10]. However, there is a significant unmet need in the development of symptomatic and disease-modifying agents for the treatment of PD as the disease progression continues in the current treatment regimen.

Due to the complex pathogenesis of PD, it is recognized that a multifunctional drug addressing the underlying disease processes will be more effective than a drug targeting a single pathological feature of the disease. In this regard, we have been focusing on the development of multifunctional dopamine agonists for symptomatic and neuroprotective treatment for PD [11,12,13]. One of our lead compounds, D-512, a novel, highly potent D2/D3 receptor agonist, has been developed as a novel symptomatic and neuroprotective treatment agent for Parkinson’s disease [14,15,16,17].

Previous work has shown that D-512 (Figure 1) significantly attenuated 6-OHDA- and MPP^+^-induced toxicity in dopaminergic MN9D cells in a dose-dependent manner, as well as in neuronal PC12 cells against the toxicity of 6-OHDA [11,18]. Furthermore, in a well-known MPTP neuroprotection animal model, D-512 has shown significant efficacy in protecting dopaminergic neurons from the toxicity of MPTP [16]. Inhibition of caspase 3/7 activity and reduction in lipid peroxidation with restoration of tyrosine hydroxylase levels in 6-OHDA-treated cells may partially explain the mechanism of action of D-512 [11]. D-512 has the potential to inhibit reactive oxygen species (ROS) produced by 6-OHDA. This may be due to its ability to scavenge free radical species, or by enhancing the endogenous cellular antioxidant defense system, or a combination of both processes [18].

Rotenone is a natural plant compound extracted from certain tropical plant species and has been extensively used as an insecticide and a pesticide [19]. Rotenone has been shown to be specifically toxic to the brain dopaminergic system, leading to loss of DA neurons and development of motor dysfunction like bradykinesia and rigidity [20,21,22]. Administration of low doses of rotenone to rats has been known to recapitulate most of the symptoms of PD pathogenesis, including dopaminergic neuronal loss and the appearance of Lewy body-like inclusions, which are immunopositive for both ubiquitin and α-synuclein [23]. Thus, the rotenone model is considered a useful model to explore the pathology and the molecular mechanisms of PD.

In this report, we sought to study the neuroprotective effects of D-512 in a rotenone-induced PD model using dopaminergic MN9D and neuronal PC12 cells. We have shown that D-512 is capable of reversing the toxic effects caused by rotenone. D-512 leads to an increase in cell viability, decreased ROS, increased mitochondrial membrane potential, and reduced caspase 3 activation caused by rotenone. Furthermore, we have also shown that dose-dependent treatment with D-512 could reverse the loss of phospho-tyrosine hydroxylase levels and decrease the activation of ERK.

2. Methods

2.1. Materials

Synthesis of D-512 [(6S)-N6-(2-(4-(4-chloro-1H-indol-5-yl) piperazin-1-yl)ethyl)-N6-propyl-3a, 4,5,6,7,7a-hexahydrobenzo[d]thiazole-2,6-diamine] was carried out in house [14,17]. Rat adrenal pheochromocytoma PC12 cell lines (ATCC, Manassas, VA, USA, CRL1721.1™) cells were purchased from ATCC. RPMI 1640, heat-inactivated horse serum, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from GIBCO (Grand Island, NY, USA) Rotenone, dimethyl sulfoxide, methyl thiazolyl blue tetrazolium bromide (MTT), thiobarbituric acid, 2′,7′-dichloro-fluorescein diacetate (DCF-DA), Dulbecco’s phosphate-buffered saline, and Triton-x-100 were purchased from Sigma-Aldrich (Burlington, MA, USA). BCA protein assay reagents were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). RIPA lysis buffer and anti-GAPDH antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Ten percent mini-protean TGX gels were obtained from Bio-Rad laboratories (Hercules, CA, USA).

2.2. Cell Culture

The dopaminergic MN9D cells were cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 μg/mL) at 37 °C 95% air/5% CO_2_. PC12 cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 95% air/5% CO_2_. Stock solutions of D-512 and rotenone were prepared in DMSO and stored at −20 °C. Different drug concentrations were made by diluting the stock solution with the appropriate medium volume. Controls were made with media containing 0.01% DMSO. We used both the cell lines from early passage numbers.

2.3. Evaluation of Neuroprotective Effect of D-512 in a Rotenone-Induced Toxicity Model

MN9D cells were plated at a 10,000 cells/well density in a 96-well plate. After 48 h of plating, cells were treated with D-512 at different concentrations for 1 h, following which rotenone at a concentration of 1 μM was added to the cells, and the cells were incubated for 24 h. In another treatment group, cells were treated with 1 μM rotenone alone. The MTT assay was used to determine the cell viability as described previously [11].

In a similar way, PC12 cells were seeded at 17,000 cells/well density in a 96-well plate. After 24 h of plating, cells were treated with D-512 at different concentrations for 1 h, followed by the addition of 1 μM rotenone for another 24 h. Cells were also treated with 1 μM rotenone alone for 24 h. The MTT assay was used to determine the cell viability as described by us previously [11,16]. After 24 h incubation, 5 mg/mL MTT solution (prepared in 1×PBS) was added to the cells (to a final concentration of 0.5 mg/mL) and was incubated at 37 °C in 95% air/5% CO_2_ atmosphere for 3 h to produce dark blue formazan crystals. Afterwards, the plate was centrifuged at 450× g for 10 min, and the supernatants were carefully removed. Formazan crystals were dissolved by adding 100 μL of methanol:DMSO (1:1) mixture to each well and shaking at 25 °C for 30 min. Absorbance values were measured on a microplate reader (Biotek Epoch, Winooski, VT, USA) at 570 nm with background correction performed at 690 nm. Data from at least three experiments were analyzed using Graphpad software (Version 10, San Diego, CA, USA). Cell viability was defined as the percentage reduction in absorbance compared to untreated controls.

2.4. Study of Mitochondrial Membrane Potential by JC1 Staining

The probe 5,5′,6,6′–tetrachloro-1,1′,3,3′ tetraethylbenzymidazolyl carbocianyne iodine (JC-1) forms multimeric aggregates upon interaction with intact mitochondria with high membrane potential and emits in the high orange wavelength of 590 nm. In mitochondria with low membrane potential, JC-1 forms monomers that emit in the green wavelength (525 to 530 nm) when excited at 488 nm. This study is used as a measure for mitochondrial health. We followed our previously published procedure to carry out this assay [12].

MN9D cells were plated at a 10,000 cells/well density in 100 μL media in 96-well plates for 48 h. PC12 cells were also plated at the same density for 24 h, following which cells were treated with different concentrations of D-512 (0.1 μM to 30 μM) for 1 h, followed by a co-treatment with rotenone (1 μM) for 24 h [24]. A JC1 solution at a dilution of 2 μg/mL was prepared in serum-free media. The media containing D-512 or rotenone was removed, and 100 µL of the JC1-containing media was added to the cells and incubated for 2 h in the incubator. After incubation, cells were washed with PBS 2 times, and 100 μL of serum-free media was added. Fluorescence was read using the Synergy Hybrid H1 fluorescence microplate reader (BioTek, VT, USA). JC-1 J-aggregates display fluorescence with excitation and emission at 550 nm and 600 nm, while JC-1 monomers exhibit excitation and emission at 485 nm and 535 nm, respectively. The ratio of fluorescence intensities of J-aggregates to monomers (red/green) was used as an indicator for mitochondrial health. Data from 3 different experiments were analyzed using Graphpad software (Version 10, San Diego, CA, USA).

2.5. Determination of Intracellular ROS Levels by ′7′-Dichlorofluorescein Diacetate (DCF-DA) Assay

Briefly, PC12 cells were seeded at 10,000 cells/well density in 100 μL media in 96-well plates for 24 h. After incubation, the media were removed from the 96-well plates, and 100 μL of 20 μM DCF-DA solution was added to each well. The cells were incubated for 30 min. After incubation, the DCF-DA solution was removed and washed with PBS. Different concentrations of D-512 were added and co-treated with 1 mM rotenone, and the fluorescence was measured immediately for baseline. After the incubation period of 24 h, fluorescence at 495 nm excitation and 529 nm emission was measured [25].

2.6. Western Blot Analysis

MN9D cells were seeded at a cell density of 1.5 × 10^5^ cells/well in a 6-well plate and allowed to attach for a period of 48 h. Cells were treated with 1 μM rotenone at varying time points (3 h, 6 h, 9 h, 12 h, and 24 h) to determine the time points at which a significant decrease in the levels of phospho-tyrosine hydroxylase could be observed. In order to observe whether D-512 has any protective effects in restoring the levels of phospho-tyrosine hydroxylase, MN9D cells were pretreated with D-512 at different concentrations (5 μM, 10 μM, and 20 μM) of D-512 for 1 h, followed by co-treatment with 1 μM rotenone for 24 h. At the end, the media was removed, and the cells were washed with PBS two times, followed by the addition of lysis reagent (RIPA buffer), protease inhibitor cocktail, and phosphatase inhibitor cocktail. The cells were scraped and transferred to a 1.5 mL Eppendorf tube and kept on ice for 30 min with intermittent mixing. The lysate was centrifuged for 10 min at 15,000× g at 4 °C. The protein concentration was determined by the Bicinchoninic acid (BCA) assay. A total of 40 μg of the cell lysates were loaded on a 10% SDS-PAGE, and the blots were blocked with 5% nonfat dry milk in TBST, followed by incubation with anti-phospho-tyrosine antibody (cell signaling Technology, Beverly, MA, USA, # 2791, 1:1000 dilution) and total tyrosine hydroxylase antibody (cell signaling Technology # 2792, 1:1000 dilution). Afterward, the membrane was incubated with appropriate HRP-conjugated secondary antibody (anti-rabbit, Cell Signaling, Catalog # 7074S) (1:1000 dilution). GAPDH was used as a loading control. GAPDH was detected after stripping the membrane in a stripping buffer, Mili-pore Sigma, St. Louis, MO, USA, Re-Blot plus Mild solution (10X) (LOT:3885644).

For the analysis of caspase-3 levels, cells were seeded in a similar way as mentioned above. Cells were treated with 5 μM, 10 μM, and 20 μM of D-512 for 1 h, followed by co-treatment with 1 μM rotenone for 24 h. A total of 40 ug of the cell lysates were resolved on a 10% SDS-PAGE, and the blots were developed using Caspase-3 Antibody (cell signaling Technology # 9665, 1:1000 dilution), and a-tubulin (catalog # 2144) was used as the loading control.

In order to evaluate the activation of phopsho- and total ERK upon treatment with rotenone and to observe the effect of D-512 in this process, the following immuno experiment was carried out. Briefly, PC12 cells at a density of 5 × 10^5^ cells/well in a 6-well plate were allowed to adhere for a period of 24 h. Cells were treated with 1 μM rotenone at varying time points—30 min, 1 h, 2 h, 4 h, and 6 h—to determine the optimum treatment time for maximum activation of ERK. In order to evaluate the effect of pretreatment of D-512 on rotenone-induced activation of phospho-ERK, the cells were first treated with different concentrations of D-512 (5 μM and 10 μM) for 24 h, followed by co-treatment with 1 μM rotenone for 30 min. A total of 40 ug of the cell lysates was resolved on a 10% SDS PAGE. The blots were probed with phospho-Erk1/2 (cell signaling Technology # 4370, 1:1000 dilution) and total Erk1/2 Abs (Cell signaling Technology # 9107, 1:1000 dilution). The membranes were incubated with appropriate HRP-conjugated secondary antibody (anti-mouse or anti-rabbit). Beta Actin (Cell Signaling Technology, Cat no. 4970S) was detected as a loading control after stripping the membrane in a stripping buffer, Mili-pore Sigma Re-Blot plus Mild solution (10X) (LOT:3885644). The images were visualized using ECL-Plus reagent (Perkin-Elmer, Waltham, MA, USA) and Image Quant LAS 4000 imager (GE Healthcare Biosciences, Pittsburgh, PA, USA). Densitometric analysis was performed using ImageJ 1.54g software. For quantification, target band intensities were normalized to the corresponding loading control (GAPDH, α-tubulin, or β-actin). For phosphorylation-dependent readouts, phospho-protein levels were expressed relative to total protein (e.g., phospho-TH/total TH and p-ERK/total ERK) and normalized to control values. All experiments were performed using independent biological replicates (n ≥ 3).

2.7. Statistics

Statistical analyses were carried out by using GraphPad Prism 10.0. Comparisons between groups were performed using one-way ANOVA analysis followed by Tukey’s post hoc test. The differences were considered to be significant if the p values were less than 0.05. All the experiments were performed at least in triplicate unless otherwise specified.

3. Results

3.1. Neuroprotective Effect of D-512 in a Rotenone Induced Toxicity Model

We carried out dose-dependent toxicity of rotenone to MN9D cells (see Supplemental Materials section, Figure S1) and selected 1 μM for our study. Treatment with 1 μM rotenone led to a decrease in the viability by 31.4% in MN9D cells. D-512 showed a neuroprotective effect against the toxicity induced by 1 μM rotenone, as seen by an increase in the viability when compared to rotenone alone. The increase in the viability shown by D-512 was most significant at doses of 5 μM and 10 μM, with an increase in viability by 17.5% and 12.2% when compared to 1 μM rotenone alone (Figure 2A).

We carried out dose-dependent toxicity of rotenone to PC12 cells (see Supplemental Materials section, Figure S1) and selected 1 μM for our study. PC12 cells, when treated with 1 μM rotenone for a 24 h period, showed a reduction in viability by 22.2% when compared to the control. When PC12 cells were pre-treated with D-512 for a period of 1 h, followed by treatment with 1 μM rotenone for 24 h, they showed a protective effect. The increase in viability shown by treatment of D-512 at doses of 0.1 μM, 1 μM, 5 μM, and 10 μM D-512 were 3.7%, 5.8%, 16%, and 10.6%, respectively, when compared to 1 μM rotenone alone (Figure 2B).

3.2. Measurement of Mitochondrial Membrane Potential by JC1 Staining

Rotenone treatment has been shown to lead to a decrease in the mitochondrial membrane potential (Δψ_m_).

In MN9D cells, 1 μM rotenone treatment led to a decrease in the mitochondrial membrane potential (MMP) by 18% when compared to the control. Pretreatment with D-512 at a concentration of 1 μM, 5 μM, and 10 μM followed by treatment with rotenone, reversed the decrease in MMP and led to an increase in the mitochondrial membrane potential by 26%, 78%, and 88% when compared to rotenone alone, with the most significant effect shown by 10 μM D-512 (Figure 3A).

In PC12 cells, 1 μM rotenone treatment led to a decrease in the mitochondrial membrane potential by 40% when compared to the control. Similar to MN9D cells, pretreatment with 5 μM and 10 μM D-512 followed by treatment with rotenone reversed the rotenone-induced decrease in mitochondrial membrane potential by increasing 16% and 14% when compared to rotenone alone (Figure 3B).

3.3. D-512 Attenuates ROS Generated by Rotenone in PC12 Cells

ROS was measured by the DCFDA assay. When PC12 cells were treated with 1 μM rotenone, it led to an increase in ROS by 48.4%. Pretreatment with D-512 at a concentration of 5 μM and 10 μM led to a decrease in the ROS by 64.8% and 62.8%, respectively (Figure 4).

3.4. Attenuation of Rotenone-Induced Phospho-Tyrosine Hydroxylase Levels by D-512 in Dopaminergic MN9D Cells

Tyrosine Hydroxylase (TH) is a highly specific, tetrahydrobiopterin-dependent non-hemeprotein and is considered a marker of the viability of dopaminergic cells. MN9D cells, when treated with 1 μM rotenone, led to a decrease in the phospho-tyrosine hydroxylase levels in a time-dependent manner. The highest decrease was seen at 24 h by 25% when compared to the control (Figure 5A,B). D-512 pre-treatment at a concentration of 5 μM, 10 μM, and 20 μM led to the recovery of the p-TH levels by 35%, 30%, and 27%, respectively (Figure 5C,D).

3.5. Attenuation of Rotenone-Induced Apoptosis Through Caspase 3 by D-512 in MN9D Cells

As rotenone is a mitochondrial complex 1 inhibitor, it destabilizes the membrane, causing leakage of Cytochrome-C. Cytochrome-C binds to Apoptotic Protease Activating Factor-1 (APAF-1) and Caspase–9, forming an apoptosome, which leads to activation of Caspase 3 from procaspase-3. Caspase3 levels as a measure of apoptosis were measured after treatment with 1 μM rotenone in MN9D cells. Rotenone treatment led to an increase in the cleaved caspase-3 levels by 25%, and these levels were attenuated significantly by pre-treatment with D-512 at concentrations of 5 μM, 10 μM, and 20 μM, bringing them back to the level of the control (Figure 6).

3.6. Attenuation of Rotenone-Induced ERK Activation by D-512 in PC12 Cells

Treatment with rotenone at a concentration of 1 μM led to an increase in the phospho-ERK levels in PC12 cells. The maximum activation is seen at 30 min with a 3-fold increase in phospho-ERK/total ERK when compared to the control (Figure 7A,C). Pre-treatment with D-512 mitigates this effect. D-512 at a concentration of 5 μM and 10 μM showed a significant decrease in the phosphorylation of ERK by 1.4-fold (Figure 7B,D).

4. Discussion

Parkinson’s disease is a complex neurodegenerative disorder that involves multiple pathogenic factors. The current therapies available provide only symptomatic relief but do not stop the progression of the disease. We are focused on developing multi-functional dopamine D2/D3 agonists to stop or slow the progression of the disease while providing symptomatic relief [11,12,16].

D-512 is one of the lead compounds developed in our lab, which provides protection of dopaminergic neurons from the toxicity of 6-OHDA in cells and from MPTP toxicity in mice [14,16]. Its mechanism of neuroprotection might be due to inhibition of oxidative stress, lipid peroxidation, and DNA fragmentation; protection of glutathione levels; and tyrosine hydroxylase levels in the 6-OHDA toxicity model [11,16,18]. In the present study, as we have done earlier [11,16], we utilized both PC12 and MN9D cells as in vitro PD cell models to study the effect of D 512 on rotenone-induced cell death, oxidative stress, and apoptosis. MN9D cells express a high level of tyrosine hydroxylase with high dopamine content and exhibit other similarities to DA neurons [26]. These cell lines are frequently used as suitable in vitro PD models. Thus, the justification for using two different cell lines in our study is to characterize the drug fully in different study endpoints.

Rotenone has been known to recapitulate the pathological characteristics of PD in in vitro and in vivo models by increasing oxidative stress, eventually leading to degeneration of dopaminergic neurons, which is implicated in PD initiation and progression. Rotenone, a mitochondrial complex I inhibitor, has been known to induce cell death in dopaminergic neurons [27], neuroblastoma cells [28], and retinal ganglion cells [29]. Therefore, drugs that reduce the rotenone-induced oxidative stress have been proposed to have potential therapeutic benefits for PD by increasing the survival of dopaminergic neurons [30,31]. Our results demonstrated that treatment with rotenone led to a decrease in cell viability in both PC12 and dopaminergic MN9D cells, although MN9D cells were adversely affected compared to PC12 (Figure 2). However, pretreatment with D-512 effectively protected these cells from the toxicity of rotenone (Figure 2A,B).

Inhibition of mitochondrial complex I by rotenone leads to the formation of ROS, which in turn inhibits electron transfer and reverses electron transport [32,33]. This process leads to the collapse of mitochondrial membrane potential, leading to the release of cytochrome C from mitochondria to the cytoplasm [34]. Cytochrome C then combines with apoptotic protease-activating factor 1, leading to the recruitment of caspase-9 for its activation. Activated caspase-9 then leads to the activation of caspase-3 and initiates apoptosis [35]. Our results confirmed that rotenone treatment led to an increase in the ROS (Figure 4), a decrease in mitochondrial potential (Figure 3), and activation of caspase-3 (Figure 6). However, pre-treatment with D-512 led to a significant decrease in the ROS generation, reduction in the loss of mitochondrial membrane potential, and a decrease in the levels of cleaved caspase-3 (Figure 3, Figure 4 and Figure 6). PC12 cells are used for the detection of ROS, as these cells have a long track record in the DCF-DA assay for producing reliable data. MN9D cells were chosen for the caspase-3 study as we observed more acute toxicity from rotenone treatment with these cell lines compared to PC12 cells.

Rotenone is known to inhibit tyrosine hydroxylase and phospho-tyrosine hydroxylase activities [27,36]. We chose MN9D cells for this study as they express high levels of tyrosine hydroxylase. When MN9D cells were treated with rotenone, it led to a decrease in the levels of phospho-tyrosine hydroxylase, the rate-limiting enzyme that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), and the phosphorylation form of the enzyme is the active one. Pretreatment with D-512 restored the levels of phospho-tyrosine hydroxylase to those of the control (Figure 5). This data indicates the potential of D-512 in restoring the level of phospho-tyrosine hydroxylase, a marker of dopamine neurons, from oxidative stress induced by rotenone.

The effect of stress or stimuli causes the initiation of various cellular signaling cascades, such as cell survival, cell proliferation, cell differentiation, cell migration, cell death, etc. Such activation of the mitogen-activated protein kinase (MAP-kinase) signaling molecule, extracellular signal regulated kinase (ERK1/2), has been shown to facilitate neuronal cell death [37,38]. Thus, the ERK signal has been shown to be pro-apoptotic when induced by rotenone. In our experiment with PC12 cells, exposure to rotenone led to the activation of ERK and phospho-ERK. However, D-512 was able to bring the level of phospho-ERK 1/2 back to that of the control level at two different doses (Figure 7). Thus, the potency of D-512 in inhibiting activation of ERK in the presence of rotenone correlates well with inhibition of caspase signaling.

Motor and non-motor symptoms of Parkinson’s disease (PD) appear in varying ways among individuals, reflecting the heterogeneous nature of the disease [39,40]. It is now evident that distinct subtypes of PD exist within the patient population [40]. To date, personalized medicine has not been fully implemented in PD due to several factors, the most significant being the lack of reliable biomarkers [41]. In addition, depression is one of the major non-motor symptoms in PD, and the dopamine D3 selective anti-Parkinsonian drug pramipexole is also used to treat depression in this disease [42]. Although D-512 has not been evaluated in an antidepressant animal model, its high affinity for dopamine D3 receptors could potentially provide antidepressant activity to a subpopulation of PD patients [10,42]. A multifunctional compound such as D-512, with potential neuroprotective and other beneficial properties, could provide therapeutic benefits beyond the treatment of motor dysfunction alone and may find useful application in subtype-specific Parkinson’s disease patients within future personalized medicine approaches.

In conclusion, the current data corroborate the neuroprotective capability of D-512 in a rotenone-based in vitro model of PD. The results from the current studies are in line with the data from various in vitro and in vivo neuroprotection studies carried out with D-512 [11,16]. All these data underscore some critical inherent properties of D-512, which are associated with its antioxidant and antiapoptotic properties. This is further reinforced in the current study, as the protective effect of D-512 is most likely not mediated via interaction with dopamine receptors, as the cell lines used in the study do not express those receptors. D-512 has been shown to produce superior locomotor activation compared to ropinirole in vivo and to rescue the dopaminergic system from an in vivo MPTP experiment [15,16]. In brief, the current cell-based results alone may not be sufficient to judge the neuroprotective potential of D-512. However, when combined with our earlier in vivo MPTP data and a host of other in vitro findings described above, these results strengthen the case for D-512 as a potential neuroprotective dopamine agonist.