Current therapeutics for Parkinson’s disease (PD) lack neuroprotective properties and are only effective in providing symptom relief [1
]. To overcome the limitations of PD drugs, researchers have focused on early pathological changes in PD [1
], with the goal of developing strategies for early interventions, prior to the onset of severe motor symptoms, such as bradykinesia, rigidity and resting tremors, in patients with preclinical or prodromal stage PD [4
Oxidative stress on dopaminergic neurons causes neurodegeneration and induces behavioral symptoms of PD. More than 90% of intracellular reactive oxygen species (ROS) are produced by aberrant electron transfer during mitochondrial respiration [5
]. There is some evidence to suggest that mitochondrial alterations lead to PD-like pathologies. For example, genetic mutations in the PD-related genes, Parkin, DJ-1
or PTEN-induced kinase 1 (PINK1)
, cause mitochondrial dysfunction in offspring of familial-type PD patients, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, which are known to be PD-inducing toxins, inhibit mitochondrial complex I [6
]. These two neurotoxins are suitable to show the effects of auraptene (AUR) in PD models, which results from mitochondrial dysfunction because both toxins lead to PD by inducing oxidative stress. The accumulation of α-synuclein, which has neurotoxic effects prior to the onset of PD symptoms, can also cause mitochondrial alterations and ROS production [7
]. Therefore, modulating mitochondrial function during the pathogenesis of PD could be an effective preventive therapeutic strategy in prodromal stage PD.
Auraptene (AUR) is a 7-geranyloxylated coumarin isolated from citrus fruit [8
]. Natural compounds such as AUR might generally be expected to offer advantages of safety and minimal adverse effects [9
]; notably, AUR is able to cross the blood-brain barrier [10
]. We previously showed that AUR inhibits progression of renal cell carcinoma by altering mitochondrial metabolism [11
]. In addition to its anticancer effects, AUR has been used in conjunction with various toxins, including N-methyl-D-aspartate, lipopolysaccharide (LPS) and scopolamine, to study the neuroprotective effects of AUR against various neurotoxic defects (e.g., cerebral ischemia and neurodegenerative diseases), focusing on movement disorders and memory impairments [12
]. Although AUR treatment inhibits microglial activation and prevents dopaminergic neuronal loss in an LPS mouse model [14
], the molecular and cellular mechanisms for the protective effects of AUR in PD models are not yet clear, and the effects of AUR on motor function in PD have not yet been investigated.
In the context of cancer, biosynthetic substrates and energy supplied by mitochondria support cancer cell proliferation and metastasis. Because AUR treatment suppresses mitochondrial function, it leads to inhibition of cancer proliferation. However, in the context of neurodegeneration, maintenance or protection of neurotoxin-induced reduction in mitochondrial respiration increases neuronal activity and survival. In order to clarify the antioxidative effect by treatment with AUR, we investigated the alteration of cell viability, antioxidant enzyme expression and ROS generation by using rotenone, MPP+ in SN4741 cell line. We demonstrated that pretreatment with AUR improves movement deficits in association with an increase in the number of dopamine neurons in the substantia nigra (SN) of MPTP-induced PD mouse models which inhibits the mitochondrial complex I. On the basis of these findings, we suggest that AUR pretreatment acts through protection of a decrease in mitochondrial respiration by neurotoxins and down-regulation of ROS of dopaminergic neurons to produce its beneficial PD-related neurobiological changes.
The complexity of PD and the variety of causative factors that contribute to its development create difficulties in identifying specific targets for effective treatments that might achieve complete disease remission. In the present study, we focused on modulation of mitochondrial energy metabolism and inhibition of ROS production by damaged mitochondria using the natural compound AUR. We postulate a dual preventive mechanism of AUR: (1) Induction of expression of genes encoding antioxidant enzymes, which protect against ROS, and (2) reduction of mitochondrial respiration by neurotoxins.
The lack of available treatment options for preventing or slowing the progression of PD has driven increased efforts to delay the occurrence of PD symptoms—the primary concept in current drug development strategies [36
]. One disease-modifying agent, vitamin E, counteracts oxidative stress, and its intake is inversely correlated with PD occurrence [37
]. In addition, the green tea polyphenol, (–)-epigallocatechine-3-gallate [38
], and two Mediterranean plant-based extracts, Padina pavonica
(EPP) and Opuntia ficus-indica
(EOFI), ameliorate neurodegeneration in PD [39
]. However, the mechanisms by which these treatments affect PD pathogenesis have not been identified. Unlike these latter studies, we focused specifically on mitochondrial respiration—considered the first target of environmental causative factors such as paraquat—and ROS overproduction by damaged mitochondria [36
]. We assessed the protective effect of AUR by measuring mitochondrial oxygen consumption rate (OCR) and antioxidant enzyme expression levels in a neuronal cell line model of mitochondrial toxicity. We found that the overall changes in cellular metabolism induced by AUR are just a slight change in mitochondrial respiration. In the AUR-pretreated and MPP+
-treated groups, basal OCR was higher than that of the control. However, there was no significant difference in behavioral tests such as the open-field test and the vertical grid test between control and AUR-treated groups (Figure 5
). These results suggest that AUR increases OCR of dopaminergic neurons in the presence of MPP+
and it is consequently sufficient to improve MPTP-induced PD-like behavior to a normal level. But, additive beneficial effects on behavior or hypermobility were not found. Therefore, AUR could be used for prevention purposes by reducing adverse effects. Thus, our findings suggest that AUR, a coumarin from a source as simple and natural as citrus peel oil, could assist in preventing PD.
In general, enhancing mitochondrial respiration is expected to increase ROS generation, because the mitochondrial respiratory chain is a major source of intracellular ROS production and many enzymes that convert molecular oxygen to ROS are present in mitochondria [40
]. Impairment of mitochondrial respiration plays a major role in the pathogenesis of PD, and increased ROS levels are known to be among the important causes of PD [40
]. The key strength of AUR is its dual function described above, which enables AUR to protect a decrease in mitochondrial respiration caused by neurotoxins without increasing cellular ROS, although how these two effects are linked is not yet clear.
In a previous study, we reported that AUR suppresses mitochondrial respiration in the renal cell carcinoma cell line, RCC4 [11
]. It has also been reported that AUR acts as a mitochondrial poison in the T-47D human breast cancer cell line [8
]. However, our study suggests that AUR increases mitochondrial function in PD-like conditions. Although these two observations are seemingly at odds, they might actually be compatible, given that cancer cells possess exceptional cellular pathways compared with normal cells. Activation of NRF2 has been reported in several types of cancer cells [41
]. NRF2, which is responsive to oxidative stress, is constitutively expressed in normal cells, but its protein level is low because of KEAP1-mediated ubiquitination and degradation [42
]. Considering that AUR acts, at least in part, through induction of NRF2, its actions on cellular pathways could be different in cancer cells and normal cells. It is also worth noting that the AUR concentration range was significantly different between these two studies. In the cancer cell study, cellular metabolism was targeted by inhibiting translation of the HIF-1α transcription factor using an AUR concentration of 100 μM. At a high concentration, AUR reduced basal OCR to 67% of that in untreated cancer cells, which show immature mitochondrial function. In the current study, we tested AUR at a concentration of 1 μM, and found that it increased basal OCR in dopaminergic neuron-like cells in the presence of neurotoxins. Notable in this context, some antioxidants, including EGCG, have been reported to show neuroprotective activity at low concentrations, but pro-oxidant activity at high concentrations [38
We also suggest the potential of AUR in trials of combined therapy with levodopa. Levodopa is one of the main drugs used for relief of PD symptoms, but it should be used with caution in younger patients with early PD [36
]. If there were a drug that could prevent progression of the disease, it should be used starting as early as possible. Although drugs currently used in combination with levodopa, such as benserazide and carbidopa, reduce the peripheral effects of levodopa and increase levodopa concentrations in the brain [36
], combination therapy with AUR would provide additional neuronal protective effects through a different pathway. If an early diagnosis of pre-symptomatic PD patients is possible in the near future, AUR could be beneficial to delay the loss of dopaminergic neurons and PD-behavior symptoms. Combining these drugs in a single therapeutic regimen would seek to relieve symptoms while delaying disease progression.
4. Materials and Methods
4.1. Cell Culture
SN4741 mouse embryonic substantial nigra dopaminergic neuronal cell line was cultured in RF media containing Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Korea), 10% FBS (Hyclone, MA, USA), 1% penicillin and streptomycin (Hyclone, MA, USA), 0.6% D-glucose and 0.7% 200 mM·L-glutamine at 33 °C under 5% CO2 and 21% O2 condition.
4.2. Measurement of Cell Viability
In the sulforhodamine B assay, SN4741 cells (5 × 103 cells per well) were seeded in triplicate in 96-well plates and incubated overnight. Added to each well were media containing Rot (0, 0.5, 1 and 10 uM, Sigma-Aldrich, MO, USA) for 6 h or MPP+ (0, 1, 4, 8 mM, Sigma-Aldrich, MO, USA) for 24 h in the presence or absence of AUR 1 uM (Sigma-Aldrich, MO, USA). The media were removed and cells were fixed with 10% TSA at 4 °C for 1 h. After washing, the cells were incubated with 0.4% SRB (Sigma-Aldrich, MO, USA) solution at room temperature for 20 min. The wells were washed with 1% acetic acid five times and dried in air. After resolving the proteins with 10 mM unbuffered Tris, absorbance was read at 490 nm using a Multiskan Ascent plate reader.
4.3. Flow Cytometry
For analyzing ROS generation, the fluorescent dye, MitoSOX™ red reagent (Invitrogen, CA, USA) and DCFDA (Invitrogen, CA, USA) were used following the manufacturers’ instructions. SN4741 cells (2–4 × 105 cells in 60 mm dish) were incubated with Rot for 6 h and AUR was pretreated for 1 h. Media was discarded and washed with HBSS and incubated for 30 min in the dark with DCFDA or MitoSOX™ (5 μM final concentration). Cells were washed with PBS and trypsinized, then resuspended in PBS/EDTA. After washed with PBS, cells were collected and kept on ice in the dark for immediate detection with the flow cytometer. Fluorescence was measured on a FACScan (BD Biosciences, NJ, USA) using excitation/emission wavelengths of 485/535 nm, and 510/580 nm for DCFDA and MitoSOX™, respectively. The values were expressed as mean fluorescence of the cell population.
4.4. Measurement of Oxygen Consumption Rate (OCR)
SN4741 cells cultured with rotenone or MPP+ ± treatment with AUR 2 uM were plated 2 × 104 cells at each well. Basal OCR was analyzed by XF24 analyzer (Seahorse, MA, USA). Then, 20 µg/mL of oligomycin A (an ATPase inhibitor, Sigma-Aldrich, MO, USA), 50 µM of carbonyl cyanide 3-chlorophenylhydrazone (CCCP, an uncoupler, Sigma-Aldrich, MO, USA) and 20 µM rotenone (a mitochondrial complex I inhibitor, Sigma-Aldrich, MO, USA) were sequentially added into each well and OCR was measured at 37 °C.
4.5. RNA Isolation and Real Time PCR
Total RNA was isolated using Trizol from SN4741 cells treated with Rot (0, 0.5 or 1 uM) or MPP+ (0, 50, 75 or 100 uM) and AUR for 24 h. cDNA was synthesized from total RNA with 5× RT premix. After mixing cDNA, primers and SYBR mix, mRNA expression was analyzed using a Rotor Gene 6000 system (Corbett Life Science, Venlo, Netherlands) and normalized to 18s rRNA. Primers used in this study: NRF2, 5′-CCAGAAGCCACACTGACAGA-3′ (forward) and 5′-GGAGAGGATGCTGCTGAAAG-3′ (reverse); NQO1, 5′-TTCTCTGGCCGATTCAGAGT-3′ (forward) and 5′-GGCTGCTTGGAGCAAAATAG-3′ (reverse); GPX, 5′- GTCCACCGTGTATGCCTTCT-3′ (forward) and 5′-TCTGCAGATCGTTCATCTCG-3′ (reverse); GST, 5′-GGCATCTGAAGCCTTTTGAG-3′ (forward) and 5′-GAGCCACATAGGCAGAGAGC-3′ (reverse); Gclc, 5′-AGGCTCTCTGCACCATCACT-3′ (forward) and 5′- TGGCACATTGATGACAACCT-3′ (reverse); Gclm, 5′-TGGAGCAGCTGTATCAGTGG -3′ (forward) and 5′-AGAGCAGTTCTTTCGGGTCA-3′ (reverse); GR, 5′-CACGACCATGATTCCAGATG-3′ (forward) and 5′-CAGCATAGACGCCTTTGACA-3′ (reverse); 18s rRNA, 5′-CGACCAAAGGAACCATAACT-3′ (forward) and 5′-CTGGTTGATCCTGCCAGTAG-3′ (reverse).
4.6. Animal Experiments
Temperature was maintained to 22 °C and light condition was adjusted to a 12 h light-dark cycle. Animal experiments were approved by the Institutional Animal Care and Use Committee of Chungnam National University. The ethical approval number is CNU-00912 and approval date is March-1-2017. To establish the MPTP-induced PD mouse model, C57BL/6 mice (8-week-old, male) were intraperitoneally injected with MPTP (1-methyl-4phenyl-188.8.131.52-tetrahydropyridine, Sigma-Aldrich, MO, USA, 2 mg/mL in saline, 20 mg/kg for one injection) four times with 2 h intervals in a day. Control mice were injected with saline. Before 24 h and 48 h of MPTP injection, auraptene (25 mg/kg) was injected intraperitoneally.
4.7. Immunofluorescence Staining and Immunohistochemistry
Saline and MPTP injected Mice were perfused and fixed with 4% paraformaldehyde (PFA). The whole brain was dipped in the 4% PFA and then moved to 30% sucrose solution to dehydrate for three days. The samples were frozen and sectioned, 25 μm of each slice. For the immunofluorescence staining, after 15 min of PBS washing, sections were blocked for 1.5 h with 3% donkey serum (Dako, Glostrup, Denmark) and 0.3% triton x-100 with PBS. Then, sections were incubated with anti-TH antibody (Millipore, MA, USA), anti-GFAP (1:1000, Abcam, Cambridge, UK) diluted with blocking solution overnight at 4 °C. Sections were washed with PBS and incubated with anti-mouse Alexa 594 and anti-chicken Alexa 488-conjugated anti-IgG secondary antibodies containing solution for 1 h at room temperature. For immunohistochemistry, brain slices were incubated with anti-TH antibody for overnight at 4 °C and then incubated with a secondary antibody (Dako EnVision+ system-HRP, CA, USA) for 1 h. The slices were reacted with DAB+ substrate buffer. After mounting with mounting medium (Dako North America Inc., CA, USA), the slides were visualized using an IX70 confocal microscope (Olympus, Tokyo, Japan).
4.8. Protein Isolation and Western Blotting
The protein of mice tissues and SN4741 cells, treated with Rot (0, 0.5 or 1 uM) or MPP+ (0, 50, 75 or 100 uM) and pretreated with 10 uM Auraptene or DMSO for 1 h, were extracted using RIPA buffer (1% Nonidet P-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris–HCl pH 7.5 and 0.5% deoxycholate) with 10% of phosphatase inhibitor and protease inhibitor (Roche, Basel, Switzerland). Equal amounts of proteins were loaded on SDS-PAGE gel and run by electrophoresis. After, they were transferred to polyvinylidene fluoride (PVDF) membrane, blocked by 5% skim milk for 1 h. Then, membranes were incubated with primary antibody including anti-NRF2 (Santa Cruz Biotechnology, CA, USA) and anti-α-Tubulin (Santa Cruz Biotechnology, CA, USA) antibody at 4 °C overnight. Anti-IgG horseradish peroxidase antibody (Pierce Biotechnology, MA, USA) correspond with the host of primary antibody was used as secondary antibody. Protein bands were detected by ECL system (Thermo Scientific, MA, USA).
4.9. Behavior Test
Open-field test: Mice were placed in a 40 × 40 × 40-box respectively. Movement was recorded for 1 h and analyzed with EthoVision XT 11.5 software.
Vertical grid test: The vertical grid test was performed following the previous study [35
]. For performing the vertical grid test, mice were habituated to the apparatus. After habituation for 3 days, a mouse was placed inside the apparatus and was allowed to turn and climb down. The movement was recorded.
4.10. Statistical Analysis
All data are represented as mean values ± SEM (error bars). The statistical analysis of data was performed using Prizm version 5 software (Graphpad, CA, USA). Significance of differences between two groups were analyzed by one-tailed student’s t-test. A P value <0.05 was considered statistically significant.