Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system that impairs motor skills and cognitive function. To date, the disease has no effective therapies. The identification of new drugs that provide benefit in arresting the decline seen in PD patients is the focus of much recent study. However, the lengthy time frame for the progression of neurodegeneration in PD increases both the time and cost of examining potential therapeutic compounds in mammalian models. An alternative is to first evaluate the efficacy of compounds in Caenorhabditis elegans models, which reduces examination time from months to days. n-Butylidenephthalide is the naturally-occurring component derived from the chloroform extract of Angelica sinensis. It has been shown to have anti-tumor and anti-inflammatory properties, but no reports have yet described the effects of n-butylidenephthalide on PD. The aim of this study was to assess the potential for n-butylidenephthalide to improve PD in C. elegans models.
In the current study, we employed a pharmacological strain that expresses green fluorescent protein specifically in dopaminergic neurons (BZ555) and a transgenic strain that expresses human α-synuclein in muscle cells (OW13) to investigate the antiparkinsonian activities of n-butylidenephthalide. Our results demonstrate that in PD animal models, n-butylidenephthalide significantly attenuates dopaminergic neuron degeneration induced by 6-hydroxydopamine; reduces α-synuclein accumulation; recovers lipid content, food-sensing behavior, and dopamine levels; and prolongs life-span of 6-hydroxydopamine treatment, thus revealing its potential as a possible antiparkinsonian drug. n-Butylidenephthalide may exert its effects by blocking egl-1 expression to inhibit apoptosis pathways and by raising rpn-6 expression to enhance the activity of proteasomes.
Citation: Fu R-H, Harn H-J, Liu S-P, Chen C-S, Chang W-L, Chen Y-M, et al. (2014) n-Butylidenephthalide Protects against Dopaminergic Neuron Degeneration and α-Synuclein Accumulation in Caenorhabditis elegans Models of Parkinson's Disease. PLoS ONE 9(1): e85305. doi:10.1371/journal.pone.0085305
Editor: Anne C. Hart, Brown University/Harvard, United States of America
Received: April 24, 2013; Accepted: November 25, 2013; Published: January 8, 2014
Copyright: © 2014 Fu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Science Council in Taiwan (101-2314-B-039-010-MY2), the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004) and China Medical University (DMR-102-054). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Parkinson's disease (PD) is the second most common disorder of the central nervous system, the incidence of which quickly augments in the people over the age of 60 years. The apparent symptoms of PD are movement disorders, including muscle rigidity, bradykinesia, and tremors. Nevertheless, behavioral and cognitive problems, including dementia, depression, anxiety, and sleep disturbances, are also involved in the later stages of the disease . Two clinical-pathological findings describe both familial and sporadic PD: the development of Lewy bodies (aggregation of α-synuclein) in the brain tissue and the selective loss of midbrain dopaminergic (DA) neurons in the substantia nigra . The reason of PD remains unclear, but it most likely results from complex interactions between a number of genetic and environmental factors .
PD is associated with ageing and its progression, and a raise in free radicals has been suggested as the fundamental factor. 6-hydroxydopamine (6-OHDA) is a neurotoxin that is thought to enter neurons via dopamine reuptake transporters and is used to selectively destroy DA neurons and then reduce dopamine levels in vivo treatment. The main use for 6-OHDA in medical research is to induce experimental parkinsonism in laboratory animals to establish and test new medicines for treating PD in humans . The α-synuclein protein is encoded by the SNCA gene (PARK1) and is abundant in the human brain. In PD, the accumulation of α-synuclein is a critical challenge to DA neurons, especially as they aggregate in inclusions and destroy the cellular machinery required for their degradation. These influences may be compounded by PD-related dysregulation of chaperones , a continually accelerating loss in general cellular homeostasis , . Some reports also have implied that α-synuclein associates with the negatively charged surface of phospholipids and participates membrane composition and turnover , . The interacting of α-synuclein with lipid membranes changes the phospholipid bilayer structure, and leads to the formation of small vesicles. Moreover, the toxic property of these protein aggregates tends to promote lipid peroxidation in the tissues via a raise in the reactive oxygen species load .
At present there is no effective cure for PD. Obtainable drugs, including levodopa, coenzyme Q10, creatine, dopamine agonists (ropinirole and pramipexole), and monoamine oxidase B inhibitors (rasagiline and selegiline), only delay and relieve the symptoms . The most hopeful therapeutic route for PD involves the finding of small molecules that can improve the disease as well as prevent its symptoms. Many herbs in China have been found to be valuable neuroprotective agents. Angelica sinensis, commonly called "dong quai", is a biennial and perennial herb that is distributed in scattered locations across China and Japan. The radix of Angelica sinensis is officially recorded in the Chinese Pharmacopoeia . It has been available for treatments such as anemia, female irregular menstruation, cardiovascular disease, amenorrhoea, hypertension, chronic bronchitis, asthma, and rheumatism . n-Butylidenephthalide (Figure 1) is the key bioactive component derived from the chloroform extract of Angelica sinensis herbs, has been confirmed to have a variety of potential pharmacological activities, such as anti-cancer –, anti-angiogenesis , anti-inflammatory , anti-platelet , vasorelaxant –, anti-anginal , , and anti-atherosclerotic  effects. Here, we study that n-butylidenephthalide could be examined as a prophylactic as well as an adjuvant agent for its advantageous effects on PD.
Drug development for PD is a main challenge. Testing the therapeutic value of candidate compounds in vertebrate disease models requires time-consuming and costly experimental studies. The establishment of rapid, simple and inexpensive in vivo assays to assess the small molecule is therefore of foremost importance. Utilizing the simple well-studied nematode Caenorhabditis elegans as an animal model system affords several advantages in the study of PD. (1) This animal is reasonably small, has a short life cycle, and is inexpensive to grow in liquid culture. Large-scale analysis is possible. (2) It has 8 DA neurons, with completely mapped neuronal networks . (3) It also has PD-related homologous gene, and the pathways involved in the function and metabolism of the DA neurons have been well conserved through evolution –. (4) Specific behavioral responses in this animal are well-known to be connected to DA signaling , . (5) The large number of mutant strains are available and the transgenic/knockdown methods can be easily operated . (6) This animal is wholly transparent; DA neurons can be directly observed through the expression of a fluorescent protein , , and a transgenic strain that expresses human α-synuclein-fluorescent protein fusion proteins can be used to estimate the amount of α-synuclein accumulation –. (7) It is convenient to use neurotoxins, including 6-OHDA and 1-methyl-4-phenyl pyridinium, to induce DA neuron degeneration in this animal, thus producing a useful pharmacological model of PD –. Here, we used the C. elegans animal model system to evaluate the effects of n-butylidenephthalide on PD and to study the potential mechanism of drug action.
Materials and Methods
Strains, culture and synchronization
C. elegans of wild-type Bristol N2, transgenic BZ555 (Pdat-1:GFP; GFP expressed specifically in dopaminergic neurons) and transgenic OW13 (Punc-54:α-synuclein:YFP+unc-119; human α-synuclein protein fused to YFP expressed specifically in body wall muscles) were provided by the Caenorhabditis Genetics Center (University of Minnesota). On the basis of previous standard protocols , we cultured the animals on nematode growth medium (NGM) plates seeded with the Escherichia coli strain OP50 or HB101 as food sources (OP50 for compound efficacy analyses and HB101 for food clearance tests) at 22°C. Fertilized eggs (embryos) were isolated from gravid adults by hypochlorite treatment (2% sodium hypochlorite and 0.5 M NaOH). After 20 h incubation at 22°C in M9 buffer to collect synchronized L1 larvae, the animals were transferred to OP50/NGM plates and then incubated for 24 h at 22°C to obtain L3 larvae.
Food clearance test
Synthesized n-butylidenephthalide (mol. wt. 188.23, 95% purity) was purchased from Lancaster Synthesis Ltd. (Newgate Morecambe, UK), dissolved in dimethyl sulfoxide (DMSO) to 1 M, and stored at −20°C as a master stock solution. A food clearance test was used to determine the impact of n-butylidenephthalide on C. elegans physiology , . A culture of E. coli was grown overnight and then resuspended at a final optical density (OD) of 6.6 in nematode S-medium . n-Butylidenephthalide was diluted into the E. coli suspension to the desired concentrations. The final concentration of DMSO in all n-butylidenephthalide-treated cultures was 2% (v/v). Fifty microliters of the final mixture was loaded per well in a 96-well plate. Approximately 20–30 synchronized L1 animals in 10 µl of S-medium were added to an E. coli suspension containing a series of concentrations of n-butylidenephthalide and incubated in a 96-well microtiter plate at 25°C. The absorbance (OD 595 nm) of the culture was determined every day for 6 days using a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
6-OHDA and n-butylidenephthalide treatment
Animals were treated with 6-OHDA (Sigma, St. Louis, MI) in order to induce selective degeneration of DA neurons . In brief, 50 mM 6-OHDA and 10 mM ascorbic acid were added to OP50/S-medium mix with n-butylidenephthalide. The final concentration of DMSO in all treated cultures was 1% (v/v). Synchronized L3 larvae were then transferred onto the treated cultures, incubated for 1 h at 22°C and mixed gently every 10 min. After 1 h of treatment, animals were washed three times with M9 buffer and then incubated in OP50/NGM plates with n-butylidenephthalide. After 24 h, animals were transferred to OP50/NGM plates containing n-butylidenephthalide and 0.04 mg/mL 5-fluoro-2′-deoxyuridine (FUDR, Sigma, St. Louis, MI) to reduce the production of progeny. Animals were scored with various assays 72 h after treatment.
Quantitative assay of dopaminergic neurodegeneration
Assay of dopaminergic neurodegeneration was performed in animals treated with 6-OHDA or n-butylidenephthalide/6-OHDA, as described previously. After 72 h of treatment at 22°C, BZ555 animals were washed three times with M9 buffer, and then mounted onto a 2% agar pad on a glass slide using 100 mM sodium azide (Sigma, St. Louis, MI) and enclosed with a coverslip. Imaging of immobilized animals was carried out with an Axio Observer inverted fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen Germany). Fluorescence intensity was estimated using AxioVision software (Carl Zeiss, Göttingen, Germany).
Quantitative assay of α-synuclein accumulation
Accumulation of α-synuclein protein was assayed in control and n-butylidenephthalide-treated OW13 animals. Synchronized OW13 L3 larvae were cultured on OP50/NGM plates containing 0.04 mg/mL FUDR (Sigma, St. Louis, MI) and n-butylidenephthalide for 72 h at 22°C, then washed three times using M9 buffer and transferred to 2% agarose pads on glass slides, mounted with 100 mM sodium azide and enclosed with a coverslip. Immobilized animals were imaged on an Axio Observer inverted fluorescence microscope (Carl Zeiss, Germany) to monitor the accumulation of α-synuclein protein, and accumulation was estimated using AxioVision software by measuring fluorescence intensity.
Quantitative assay of lipid deposits
Nile red (Invitrogen, Carlsbad, CA) is a fluorescent stain specific for to detect intracellular lipid droplets. The stock solution was prepared by dissolving 0.5 mg Nile red dye in 1 mL of acetone, which was then mixed with E. coli OP50 suspension in the ratio 1∶250 as described previously . Synchronized OW13 L3 larvae were cultured on Nile red/OP50/NGM plates containing 0.04 mg/mL FUDR and n-butylidenephthalide for 72 h at 22°C. Animals were washed and mounted onto 2% agarose pads using 100 mM sodium azide and enclosed with a coverslip. Immobilized animals were imaged on an Axio Observer inverted fluorescence microscope (Carl Zeiss, Germany) to monitor the lipid deposits. Fat staining was estimated using AxioVision software by measuring fluorescence intensity.
Food-sensing behavior test
The food-sensing behavior test was carried out by evaluating the function of C. elegans DA neurons , . Briefly, test plates were prepared by spreading E. coli overnight at 37°C in a ring with an inner diameter of 1 cm and an outer diameter of 8 cm on 9-cm diameter NGM agar plates to avoid the animals reaching the edge of the plate during the test. Well fed 6-OHDA-treated or n-butylidenephthalide/6-OHDA-treated adult animals were washed with M9 buffer and then transferred to the center of an assay plate with or without bacterial lawn in a drop of M9 buffer. Five minutes after transfer, the locomotory rate of each animal was measured in 20-s intervals. The slowing rate was estimated as the percentage of the locomotory rate in the bacteria lawn compared with that in the no bacteria lawn. The average slowing rate among 10 animals was defined as the result of each analysis. In all analyses, plates were numbered so that the experimenter was blind to the treatment of the animal.
Dopamine content analysis
The dopamine content in animals treated with 6-OHDA or n-butylidenephthalide/6-OHDA was evaluated by high-performance liquid chromatography (HPLC) analysis combined with a chemiluminescence reaction assay as described previously . Animals were washed with M9 buffer, and were then temporarily frozen in liquid nitrogen. For the preparation of the extract, frozen animal pellets were resuspended in buffer A (2 mM EDTA, 200 mM perchloric acid, 2 mM sodium metabisulfite), sonicated and centrifuged at 10,000 g for 5 min. The supernatants were analyzed by HPLC.
A Life-span examine was carried out by transferring control, 6-OHDA-treated and n-butylidenephthalide/6-OHDA-treated adult animals every 3 days to a fresh control or treated plate. A total of 0.04 mg/mL of FUDR was added to each plate to diminish the production of progeny. The number of live, dead and missing animals was counted each day until the last animal was dead. Experiments were performed by three different experimenters. Survival curves were plotted by the product-limit method of Kaplan and Meier; statistical analyses were carried out by SPSS software.
Quantitative real-time PCR
Total RNA was isolated from synchronized control or experimental adult animals using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was synthesized using the SuperScript One-Step RT-PCR system (Invitrogen, Carlsbad, CA). SYBR Green real-time qPCR assays were carried out with a 1∶20 dilution of cDNA using an ABI StepOnePlus system (Applied Biosystems). Data were calculated with the comparative 2ΔΔCt method using the geometric mean of cdc-42, pmp-3 and Y45F10D.4 as the endogenous control . Table S1 shows details of the primers used in the current study .
26S proteasome activity analysis
In vitro 26S proteasome activity analyses were carried out as previously described . Briefly, using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), animals were lysed using proteasome activity assay buffer containing 50 mM Tris-HCl (Ph 7.5), 250 mM sucrose, 5 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol and 0.5 mM EDTA. The lysate was centrifuged at 10,000 g for 15 min at 4°C. For each assay, 25 µg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. For determining the chymotrypsin-like activity of the proteasome, Z-Gly-Gly-Leu-AMC (Enzo Life Sciences, Farmingdale, NY) was used as a substrate. After incubation for 1 h at 25°C, fluorescence (an excitation wavelength 380 nm and an emission wavelength 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA).
Statistical analyses are shown as mean ± standard deviation (SD) from independent experiments. Three replicates were done of each experiment. The differences among groups were determined by One-way ANOVA analysis followed by a Newman-Keuls post hoc test. Values of p<0.05 were determined to be statistically significant.
Determining the n-butylidenephthalide concentration range for treatment of C. elegans by food clearance test
To assess the effects of n-butylidenephthalide on DA neuron degeneration and α-synuclein accumulation, we first determined the optimal concentrations of n-butylidenephthalide to evaluate in our C. elegans PD models by food clearance test. Given the advantage of the ability of C. elegans to grow in a liquid culture of E. coli and the short life cycle, n-butylidenephthalide was tested at the rate at which the food source (E. coli suspension) was consumed. Each adult animal can generate hundreds of offspring that rapidly consume the limited E. coli supply. Therefore, the OD of the wells without n-butylidenephthalide significantly reduced in 3 days in N2, BZ555 and OW13 strains (Figure 2A). The DMSO solvent had no influence on the food consumption rate. The addition of 2 mM or 5 mM n-butylidenephthalide to the cultures containing N2, BZ555 or OW13 strains showed no effect on food clearance compared to that in control animals, whereas animals treated to 10 mM or 20 mM n-butylidenephthalide had significantly delayed food clearance (Figure 2A). Optical observation revealed that animals treated with 10 mM or 20 mM n-butylidenephthalide were smaller than untreated animals, whereas animals treated with 2 mM or 5 mM n-butylidenephthalide were unaffected (Figure 2B). In addition, animals treated to 20 mM n-butylidenephthalide did not generate offspring over the time course of the experiment, which was related with the lack of clearance of the E. coli source. This simple assay for determining the concentration range of n-butylidenephthalide to test food clearance in C. elegans uses a small amount of n-butylidenephthalide. In the experiments following this test, animals were exposed with n-butylidenephthalide at concentrations of up to 5 mM.
Between 20 and 30 newly hatched L1 synchronized animals of N2, BZ555 or OW13 were incubated in E. coli (OD A595 = 0.6) in a 96-well plate at 25°C containing different n-butylidenephthalide concentrations to a total volume of 60 µL. The OD of the plate was measured daily for 6 days. Note the lower OD (∼0.6) in a well due to the reduced path length of the 60 µL final suspension compared to the 1 cm path length in a spectrophotometer. (A) The OD of E. coli recorded daily for each concentration of n-butylidenephthalide. (B) Animals treated with no n-butylidenephthalide and the indicated n-butylidenephthalide concentrations. Animals treated with 10 mM n-butylidenephthalide were alive but concentrations over 20 mM caused death. Scale bar is 100 µm.
n-Butylidenephthalide attenuated 6-OHDA-induced degeneration of DA neurons
C. elegans contains accurately eight DA neurons, including one pair of anterior deirid (ADE) neurons and two pairs of cephalic (CEP) neurons in the head position, and one pair of posterior deirid (PDE) neurons in the posterior lateral region. Selective degeneration of these DA neurons was observed through treatment to 6-OHDA. To analyze n-butylidenephthalide efficacy, we assessed neuronal viability by measuring the loss of expression of a GFP reporter gene in DA neurons of 6-OHDA-treated BZ555 animals. We found that CEP and ADE neurons showed a partial GFP loss with slight reduction in GFP expression in PDE neurons after 6-OHDA treatment (Figure 3A). The DMSO solvent had no influence on 6-OHDA-induced DA neuron degeneration. When animals were exposed with n-butylidenephthalide, remarkable protection was noticed in DA neurons with CEP, and ADE neurons presenting an augmented expression of GFP (Figure 3A). We further measured the fluorescence intensity in DA neurons using AxioVision software. In 6-OHDA-treated animals, the mean fluorescence (GFP) intensity reduced by about 57% (p<0.01) compared to that of untreated animals (Figure 3B). n-Butylidenephthalide increased the GFP expression in a dose-dependent manner. At 5 mM n-butylidenephthalide, the fluorescence intensity of GFP expression in DA neurons of 6-OHDA-treated animals raised by about 1.9-fold (p<0.01) compared to that in animals treated only with 6-OHDA (Figure 3B).
(A) GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain BZ555. The left side shows the differential interference contrast (DIC) image. The right side shows the fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of GFP expression pattern in dopaminergic neurons of a transgenic C. elegans strain BZ555 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.01); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
n-butylidenephthalide arrested accumulation of α-synuclein protein
C. elegans does not contain an orthologous gene of α-synuclein. However, the genetic flexibility of the nematode allows transgenic expression of human α-synuclein genes and the investigation of α-synuclein accumulation. OW13 animals of untreated and n-butylidenephthalide-treated groups were measured for their α-synuclein protein accumulation. The DMSO solvent had no influence on α-synuclein accumulation. Treatment of animals with n-butylidenephthalide showed significantly reduced fluorescence intensity of accumulation compared to that of untreated animals (Figure 4A). n-butylidenephthalide decreased the YFP expression of OW13 animals in a dose-dependent manner. At 5 mM n-butylidenephthalide, the fluorescence intensity of YFP expression associated with α-synuclein protein accumulation in OW13 animals lessened by about 48% (p<0.01) compared to that in untreated animals (Figure 4B).
(A) YFP expression pattern in muscles of transgenic C. elegans strain OW13. The left side shows the differential interference contrast (DIC) image. The right side shows fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of YFP expression pattern in muscles of transgenic C. elegans strain OW13 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). An asterisk (*) indicates significant differences between the control samples and the n-butylidenephthalide-treated samples (*p<0.05, **p<0.01).
n-butylidenephthalide restored lipid content in a transgenic C. elegans model overexpressing human α-synuclein
PD is reported to be connected with altered levels of fatty acids and lipid content by α-synuclein expression . We assayed the lipid levels in OW13 animals untreated or treated with n-butylidenephthalide. Nile red staining was employed to fluorescently label the lipids within animals. Animals of the control group N2 revealed an optimum level of lipids. The lipid content in α-synuclein-overexpressing OW13 animals was reduced. The DMSO solvent had no influence on the lipid level (Figure 5A). n-Butylidenephthalide raised the lipid content of OW13 animals in a dose-dependent manner. At 5 mM n-butylidenephthalide treatment, the fluorescence intensity of Nile red in the entire body of OW13 animals augmented by about 1.8-fold (p<0.01) compared to that in untreated OW13 animals (Figure 5B).
(A) Nile red staining pattern in transgenic C. elegans strain OW13. The left side shows the differential interference contrast (DIC) image. The right side shows fluorescence images. Scale bar, 50 µm. (B) Graphical representation for fluorescence intensity of the Nile red pattern in transgenic C. elegans strain OW13 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between N2 and OW13 animals (p<0.01); an asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated samples (*p<0.05, **p<0.01).
n-Butylidenephthalide recovered food-sensing behavior of 6-OHDA-exposed C. elegans
It has been confirmed that 6-OHDA-exposed animals show a phenotype of DA neuron degeneration that should affect dopamine synthesis and food-sensing behavior , . C. elegans move themselves by bending their bodies for transportation, and the rate of movement is estimated by the bending frequency. Once animals come across food, they lessen the bending frequency to feed themselves more effectively. 6-OHDA-treated animals, however, lose to display a decremental bending frequency in response to food sensing. Accordingly, the function of dopamine neurotransmission in C. elegans is linked with this food-sensing behavior. We examined whether 6-OHDA treatment in C. elegans generates a defect in this function in 3-day-old animals that were synchronized for age. Wild-type N2 animals revealed a 45% lessening in bending frequency upon contact with bacteria (Figure 6). In contrast, 6-OHDA-treated animals showed a significant decreasing in this decremental response compared with wild-type N2 animals (22%, p<0.01). The DMSO solvent had no influence on the 6-OHDA-induced lessening in the decremental response. n-butylidenephthalide recovered the decremental response of 6-OHDA-treated animals in a dose-dependent manner. At 5 mM n-butylidenephthalide treatment, 6-OHDA-treated animals displayed reduced bending movement upon contact with bacteria by about 1.8-fold (p<0.01) compared with that in untreated animals (Figure 6).
The locomotory rate (frequency of bending) of 6-OHDA-untreated animals, 6-OHDA-treated animals, or n-butylidenephthalide/6-OHDA-treated animals with or without bacteria lawns was assayed. Shown are the slowing rates calculated as the percentage decrease of the locomotory rate in the bacteria lawn as compared with that in no bacteria lawn. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.001); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
n-butylidenephthalide restored the level of dopamine of 6-OHDA-treated C. elegans
We next measured the level of dopamine in 3-day-old wild-type N2, 6-OHDA-treated, or n-butylidenephthalide/6-OHDA-treated animals that were synchronized for age. N2 animals comprised ∼6 ng of dopamine per gram of animals. In 6-OHDA-treated animals, the level of dopamine reduced by about 64% (p<0.01) compared to that of untreated animals (Figure 7). The DMSO solvent had no influence on 6-OHDA-induced lessening in the level of dopamine. n-butylidenephthalide augmented the level of dopamine in a dose-dependent manner. At 5 mM n-butylidenephthalide, the level of dopamine of 6-OHDA-treated animals raised by about 2.2-fold (p<0.01) compared to that in animals treated only with 6-OHDA (Figure 7).
Shown is quantitation of DA levels (ng/g wet weight of animals) by HPLC-chemiluminescence detection in 6-OHDA-untreated animals, 6-OHDA-treated animals, or n-butylidenephthalide/6-OHDA-treated animals. Three-day-old animals synchronized for age were used. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.001); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (*p<0.05, **p<0.01).
n-butylidenephthalide prolonged the life span of 6-OHDA-treated animals
The effect of n-butylidenephthalide on the longevity of 6-OHDA-treated animals was observed. 6-OHDA-exposed animals have a shorter life span compared to wild-type N2 animals (Figure 8). The DMSO solvent had no influence on the longevity of 6-OHDA-treated animals. n-butylidenephthalide increased the life span in a dose-dependent manner. We noted that 5 mM of n-butylidenephthalide appreciably improved the life span of 6-OHDA-treated animals. Figure 8 represents the cumulative survival patterns, as calculated by Kaplan–Meier survival analysis of each group. The mean survival for the n-butylidenephthalide/6- OHDA (5 mM) group was 21.82±2.11 days vs. 13.00±2.43 days for the 6-OHDA condition (p<0.01).
n-butylidenephthalide lessened aopotosis modulator egl-1 expression in 6-OHDA-exposed C. elegans
We hypothesized that a key aspect of the apoptosis pathway might be regulated in the 6-OHDA-treated animals by n-butylidenephthalide. To evaluate whether the observed attenuation in DA neuron degeneration of C. elegans was the result of lessened apoptosis activity, after treating the animals with n-butylidenephthalide, we employed a real-time PCR technique to determine the mRNA levels of egl-1, ced-3, ced-4 and ced-9, which are associated with apoptosis of C. elegans. As represented in Figure 9, the expression level of egl-1, ced-3, ced-4 and ced-9 was not raised in 6-OHDA-treated animals compared to that in untreated animals. The DMSO solvent had no influence on the expression level of egl-1, ced-3, ced-4 and ced-9 in 6-OHDA-treated animals. At 5 mM n-butylidenephthalide, the expression level of egl-1 in 6-OHDA-treated animals reduced by about 42% (p<0.01) compared to that in animals treated only with 6-OHDA (Figure 9).
Quantitative real-time RT-PCR experiments show the expression levels of egl-1, ced-3, ced-4 and ced-9 using RNAs isolated from N2, 6-OHDA-treated, or n-butylidenephthalide/6-OHDA-treated animals. The data represent the mean ± SD (n = 3). An asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide/6-OHDA-treated samples (**p<0.01).
n-butylidenephthalide enhanced somatic proteasome activity by raising proteasome regulatory subunit rpn-6 expression in a transgenic C. elegans model overexpressing human α-synuclein
We hypothesized that a key aspect of the proteostasis network, the ubiquitin proteasome system, might be regulated in n-butylidenephthalide-treated OW13 animals. To evaluate whether the observed diminishing in α-synuclein accumulation in the muscle of OW13 animals was the result of elevated proteasomal activity, we analyzed 26S proteasome activity upon treatment with n-butylidenephthalide by employing a proteasome activity assay with a fluorescent substrate. As represented in Figure 10A, the level of chymotrypsin-like proteasome activity was about 14% lower in OW13 animals compared to that in N2 animals (p<0.05). The DMSO solvent had no influence on the proteasome activity of OW13 animals. n-Butylidenephthalide treatment significantly raised the chymotrypsin-like proteasome activity in OW13 animals in a dose-dependent manner. Chymotrypsin-like proteasome activity following 5 mM n-butylidenephthalide treatment was augmented by about 1.5-fold in the OW13 animals (p<0.01) (Figure 10A). These results indicate that elevated proteasome activity results in a reduction in α-synuclein accumulation and that n-butylidenephthalide treatment can increase proteasome activity in the animal model of PD. We therefore next assessed whether the proteasome activity of n-butylidenephthalide-treated OW13 animals linked with a raised expression level of the catalytically active subunits of the 20S proteasome or the regulatory particles of the 19S proteasome. The level of all subunits was not different in OW13 animals compared to that in N2 animals. The DMSO solvent had no influence on the subunit expression of OW13 animals. n-Butylidenephthalide raised the expression level of the rpn-6 of regulatory subunit. The expression level of rpn-6 following 5 mM n-butylidenephthalide treatment was enhanced by about 1.6-fold in the OW13 animals (p<0.01) (Figure 10B).
(A) n-Butylidenephthalide augments proteasome activity in the OW13 strain of C. elegans. Chymotrypsin-like activity of the proteasome was monitored by Z-Gly-Gly-Leu-AMC digestion in a day 3 adult animal extract containing equal amounts of total protein. The data represent the mean ± SD (n = 3). A hash (#) indicates significant differences between N2 and OW13 animals (p<0.05); an asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated OW13 samples (*p<0.05, **p<0.01). (B) n-Butylidenephthalide enhances the expression of rpn-6 of proteasome in the OW13 strain of C. elegans. Quantitative real-time RT-PCR experiments show the expression level of individual subunit of the 26S proteasome, using RNAs isolated from OW13 control or n-butylidenephthalide-treated animals. The data represent the mean ± SD (n = 3). An asterisk (*) indicates significant differences between the OW13 control samples and the n-butylidenephthalide-treated samples (**p<0.01).
In this research, we established strategies for assessing the therapeutic efficacy of phytocompounds in C. elegans PD models. Our methods use the advantages of the C. elegans model for drug testing, including easy and exact visualization of live DA neurons and α-synuclein accumulation. Moreover, small-scale liquid cultures significantly decrease the amount of the drug required for examination, and molecular biology methods indicate specific cellular pathways in the activity of the compound. These assays could be powerful for inexpensive, rapid examination and screening of large numbers of drugs for PD. Our data show that n-butylidenephthalide reduces DA neuron degeneration; attenuates α-synuclein accumulation; arrests lipid loss; and restores food-sensing behavior and the level of dopamine in a pharmacological or transgenic C. elegans model. Furthermore, n-butylidenephthalide raises the life span of 6-OHDA-treated animals. To the best of our knowledge, this is the first report of the antiparkinsonian ability of n-butylidenephthalide in an animal model.
Given that DA neurons are particularly sensitive to oxidative stress, reactive oxygen species are major regulators in neuronal cell apoptosis and death. 6-OHDA impairs neurons by producing reactive oxygen species such as the superoxide radical . Recently, it was demonstrated that n-Butylidenephtaline possesses anti-oxidant activities. The transcription factor Nrf2 mediates the expression of many cellular anti-oxidative stress genes. Data showed that n-Butylidenephtaline activates the Nrf2 pathway, and then protects against oxidative stress . Therefore, the neuroprotective role of n-butylidenephthalide in 6-OHDA-induced DA neuron lesion, food-sensing behavior defects, dopamine loss, and longevity shortening is possibly associated with its antioxidant activity. It has also been shown that mitochondrial damage induced by 6-OHDA causes release of cytochrome c to cytosol and the activation of caspase-3 . Caspase-3 is an important effector in apoptosis that is induced via different pathways in various mammalian cell types, mainly in the cytochrome c-dependent apoptosis pathways . In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase participate apoptosis induction, whereas the Bcl-2 homolog CED-9 arrests apoptosis. CED-9 is thought to inhibit CED-4 by blocking CED-4 accumulation in the perinuclear space in response to proapoptotic stimuli. EGL-1 antagonizes CED-9, leading to CED-4 oligomerization and triggering apoptosis through CED-3 caspase activation , . Nuclei of DA neuron of 6-OHDA-exposed C. elegans also revealed dark and chromatin condensation, characteristics that are consistent with apoptotic cells . Therefore, in this study, we demonstrated that n-butylidenephthalide lessened the expression of egl-1, which, in turn, may counter 6-OHDA-induced DA neuron apoptosis. The detailed mechanism requires further investigation.
To observe accumulation of α-synuclein, we used the C. elegans strain OW13. It was overexpressed human α-synuclein fused to yellow fluorescent protein under control of the unc-54 promoter, which directs expression to the body wall muscle cells . Muscle expression rather than neuronal expression was decided for three reasons. First, the unc-54 promoter is strong and muscle cells are the largest, most easily scored cell type, allowing for exact measurement of α-synuclein accumulation and its subcellular localization. Second, muscle expression has been employed successfully to determine modifier genes of PD in previous studies , , , . Third, the accumulation of α-synuclein in the muscle resulted in PD-like progressive decline of motility in C. elegans, indicating the in vivo toxicity of these aggregates , . In our study, we implied that the anti-accumulation effect of n-butylidenephthalide may associate with its mediation of the proteosome system. It is well known that ubiquitin-proteosome system promotes cell survival from harm under environmental stressful conditions . The 26S proteasome complex of C. elegans comprises a 20S "catalytic core", a huge protein complex that harbors the proteolytically active centres and 19S "regulatory caps", which are responsible for recognition of poly-ubiquitinated protein substrates targeted for degradation. 19S can be further divided into two distinct substructures including a ring of six homologous ATPase subunits of the AAA family (Rpt-1–6), eight essential RP non-ATPase subunits (Rpn-3, Rpn-5–9 and Rpn-11–12), two α-helical solenoid structures (Rpn-1 and Rpn-2), and two Ub receptors (Rpn-10) . The effect of n-butylidenephthalide may be linked with enhanced expression of rpn-6, a subunit of the 19S proteasome. Rpn-6 is a candidate to correct deficiencies in age-related protein homeostasis disorders. Ectopic expression of rpn-6 is sufficient to confer proteotoxic stress resistance and extend lifespan . The exact mechanisms underlying these results will require further investigation.
The abundance of lipid molecules in the central nervous system suggests that their function is not restricted to the structural components and energy of cells. Some lipids in the central nervous system are well-known to play a key role in neurotransmission. Disorders in cellular signaling have been connected to almost every neurodegenerative disease . Spatial and temporal aspects of cellular signaling activities are in part modulated by lipid components that can change protein location and scaffolding events through a dynamic control of membrane microdomains . This protective effect of n-butylidenephthalide could lessen α-synuclein accumulation, therefore decreasing lipid peroxidation. Hence, such a protective effect may regulate the disturbed arrangement of lipids and thus maintains efficient cellular signaling.
Evidence suggests that chronic neuroinflammation is linked with the pathophysiology of PD . Activation of microglia and raised levels of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 have been confirmed in the substantia nigra of PD patients and in animal models of PD . It is hypothesized that activated microglia secrete high levels of proinflammatory mediators that impair neurons and further activate microglia, leading to further promoting of inflammation and neurodegeneration. Moreover, DA neurons are more sensitive to proinflammatory mediators than other cell types are . In our previous study, n-butylidenephthalide attenuated the maturation of mouse dendritic cells via blockage of IκB kinase/nuclear factor-κB activities . Therefore, n-butylidenephthalide may also be able to modulate inflammation in the brains of patients with PD and lessen DA neuron damage.
Some reports have implied that phytocompounds, including acacetin , curcumin , gastrodin , isoliquiritigenin  and quercetin , have special neuroprotective effects on DA neurons. In addition, isorhynchophylline promotes the degradation of α-synuclein in neuronal cells via inducing autophagy . The current study indicates that n-butylidenephthalide is a new member on the list of phytocompounds with these effects. Natural antioxidant phytocompound curcumin showed neuroprotective capacity in the 6-OHDA model of PD ,  and also attenuated aggregation of α-synuclein in cell model of PD , . The simpler anti-oxidant N-acetylcysteine prevents loss of DA neurons in the EAAC1-/- mouse . Oral N-acetylcysteine reduced loss of dopaminergic terminals in α-synuclein overexpressing mice model . Vitamin E also exhibited beneficial effects in PD . Our study indicates that n-butylidenephthalide, although less studied, possesses several pharmacological properties that cover with those of the more broadly investigated curcumin, N-acetylcysteine, and vitamin E in PD. Moreover, according our supplement data, n-butylidenephthalide showed neuroprotective capacity in the 6-OHDA-treated BZ555 animals as well as curcumin and N-acetylcysteine, and better than vitamin E (Figure S1). Furthermore, n-butylidenephthalide revealed the power of anti-accumulation in the OW13 animals better than curcumin, N-acetylcysteine, and vitamin E (Figure S2). Therefore, n-butylidenephthalide has better potential in pharmaceutics for PD. The available data on the potential clinical applications of n-butylidenephthalide are increasing. The in vivo influences of n-butylidenephthalide treatment have been shown in a rat model , . Further clinical trials are required to assess the suitability of n-butylidenephthalide for disease control.
These results support traditional Chinese medicine practitioners with information about the use of herbs containing n-butylidenephthalide in the treatment of neuron-related disorders. This readily available agent allows a low-cost, convenient, and highly effective means of modifying the survival and function of DA neurons. In the future, we will address the exact mechanism by which n-butylidenephthalide maintains DA neuron activity in other animal PD models, which may develop the novel antiparkinson potential of n-butylidenephthalide in the prevention and treatment of PD.
Recent reports have shown that incorporation of phytocompounds into liposome or nanoparticles improves in the oral delivery of a drug –, as these particles can avoid it degradation in the gastrointestinal tract; as a result of their individual absorption mechanism through the circulation system, these particles also shield the drug from the first-pass effect in the liver and allow continued release at the correct site of action. Such techniques will be exploited for improvements in the clinical applications of n-butylidenephthalide.
The neuroprotective effects of n-butylidenephthalide, curcumin, N-acetylcysteine and vitamin E on 6-OHDA-induced degeneration of DA neurons in C. elegans. Curcumin, N-acetylcysteine and vitamin E were purchased from Sigma-Aldrich (St. Louis, MO). The addition of 5 mM curcumin, N-acetylcysteine and vitamin E individual to the cultures containing transgenic C. elegans strain BZ555 revealed no effect on food clearance assay compared to that in control animals (data not shown). Graphical representation for fluorescence intensity of GFP expression pattern in DA neurons of transgenic C. elegans strain BZ555 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). A hash (#) indicates significant differences between 6-OHDA-treated and untreated animals (p<0.001); an asterisk (*) indicates significant differences between the 6-OHDA-treated control samples and the n-butylidenephthalide, curcumin, N-acetylcysteine or vitamin E/6-OHDA-treated samples (*p<0.05, **p<0.01).
The anti-accumulation effects of n-butylidenephthalide, curcumin, N-acetylcysteine and vitamin E in the OW13 strain of C. elegans. Curcumin, N-acetylcysteine and vitamin E were purchased from Sigma-Aldrich (St. Louis, MO). The addition of 5 mM curcumin, N-acetylcysteine and vitamin E individual to the cultures containing transgenic C. elegans strain OW13 revealed no effect on food clearance assay compared to that in control animals (data not shown). Graphical representation for fluorescence intensity of YFP expression pattern in muscles of transgenic C. elegans strain OW13 as quantified using AxioVision software. The data represent the mean ± SD (n = 10). An asterisk (*) indicates significant differences between the control samples and the n-butylidenephthalide, curcumin, N-acetylcysteine or vitamin E-treated samples (*p<0.05, **p<0.01).
List of primers used for qPCR assays.
Conceived and designed the experiments: RHF YCW. Performed the experiments: RHF WLC YMC JEH RJL SYT YCW. Analyzed the data: RHF. Contributed reagents/materials/analysis tools: RHF HJH SPL CSC HSH WCS SZL. Wrote the paper: RHF.
- 1. Coelho M, Ferreira JJ (2012) Late-stage Parkinson disease. Nat Rev Neurol 8: 435–442.
- 2. Hansen C, Li JY (2012) Beyond alpha-synuclein transfer: pathology propagation in Parkinson's disease. Trends Mol Med 18: 248–255.
- 3. Youle RJ, van der Bliek AM (2012) Mitochondrial fission, fusion, and stress. Science 337: 1062–1065.
- 4. Blandini F, Armentero MT (2012) Animal models of Parkinson's disease. FEBS J 279: 1156–1166.
- 5. Auluck PK, Chan HY, Trojanowski JQ, Lee VM, Bonini NM (2002) Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease. Science 295: 865–868.
- 6. Snyder H, Mensah K, Theisler C, Lee J, Matouschek A, et al. (2003) Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function. J Biol Chem 278: 11753–11759.
- 7. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D (2004) Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305: 1292–1295.
- 8. Bussell R Jr, Eliezer D (2004) Effects of Parkinson's disease-linked mutations on the structure of lipid-associated alpha-synuclein. Biochemistry 43: 4810–4818.
- 9. Jensen PH, Nielsen MS, Jakes R, Dotti CG, Goedert M (1998) Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation. J Biol Chem 273: 26292–26294.
- 10. Binukumar BK, Bal A, Kandimalla RJ, Gill KD (2010) Nigrostriatal neuronal death following chronic dichlorvos exposure: crosstalk between mitochondrial impairments, alpha synuclein aggregation, oxidative damage and behavioral changes. Mol Brain 3: 35.
- 11. Jankovic J, Poewe W (2012) Therapies in Parkinson's disease. Curr Opin Neurol 25: 433–447.
- 12. Yi L, Liang Y, Wu H, Yuan D (2009) The analysis of Radix Angelicae Sinensis (Danggui). J Chromatogr A 1216: 1991–2001.
- 13. Chiu SC, Chen SP, Huang SY, Wang MJ, Lin SZ, et al. (2012) Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide. PLoS One 7: e33742.
- 14. Wei CW, Lin CC, Yu YL, Lin CY, Lin PC, et al. (2009) n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity. Acta Pharmacol Sin 30: 1297–1306.
- 15. Chen YL, Jian MH, Lin CC, Kang JC, Chen SP, et al. (2008) The induction of orphan nuclear receptor Nur77 expression by n-butylenephthalide as pharmaceuticals on hepatocellular carcinoma cell therapy. Mol Pharmacol 74: 1046–1058.
- 16. Yeh JC, Cindrova-Davies T, Belleri M, Morbidelli L, Miller N, et al. (2011) The natural compound n-butylidenephthalide derived from the volatile oil of Radix Angelica sinensis inhibits angiogenesis in vitro and in vivo. Angiogenesis 14: 187–197.
- 17. Fu RH, Hran HJ, Chu CL, Huang CM, Liu SP, et al. (2011) Lipopolysaccharide-stimulated activation of murine DC2.4 cells is attenuated by n-butylidenephthalide through suppression of the NF-kappaB pathway. Biotechnol Lett 33: 903–910.
- 18. Teng CM, Chen WY, Ko WC, Ouyang CH (1987) Antiplatelet effect of butylidenephthalide. Biochim Biophys Acta 924: 375–382.
- 19. Ko WC, Liao CC, Shih CH, Lei CB, Chen CM (2002) Relaxant effects of butylidenephthalide in isolated dog blood vessels. Planta Med 68: 1004–1009.
- 20. Chan SS, Choi AO, Jones RL, Lin G (2006) Mechanisms underlying the vasorelaxing effects of butylidenephthalide, an active constituent of Ligusticum chuanxiong, in rat isolated aorta. Eur J Pharmacol 537: 111–117.
- 21. Chan SS, Jones RL, Lin G (2009) Synergistic interaction between the Ligusticum chuanxiong constituent butylidenephthalide and the nitric oxide donor sodium nitroprusside in relaxing rat isolated aorta. J Ethnopharmacol 122: 308–312.
- 22. Ko WC, Sheu JR, Tzeng SH, Chen CM (1998) The selective antianginal effect without changing blood pressure of butylidenephthalide in conscious rats. Planta Med 64: 229–232.
- 23. Ko WC, Charng CY, Sheu JR, Tzeng SH, Chen CM (1998) Effect of butylidenephthalide on calcium mobilization in isolated rat aorta. J Pharm Pharmacol 50: 1365–1369.
- 24. Mimura Y, Kobayashi S, Naitoh T, Kimura I, Kimura M (1995) The structure-activity relationship between synthetic butylidenephthalide derivatives regarding the competence and progression of inhibition in primary cultures proliferation of mouse aorta smooth muscle cells. Biol Pharm Bull 18: 1203–1206.
- 25. Harrington AJ, Yacoubian TA, Slone SR, Caldwell KA, Caldwell GA (2012) Functional analysis of VPS41-mediated neuroprotection in Caenorhabditis elegans and mammalian models of Parkinson's disease. J Neurosci 32: 2142–2153.
- 26. Kuwahara T, Tonegawa R, Ito G, Mitani S, Iwatsubo T (2012) Phosphorylation of alpha-synuclein protein at Ser-129 reduces neuronal dysfunction by lowering its membrane binding property in Caenorhabditis elegans. J Biol Chem 287: 7098–7109.
- 27. Jadiya P, Chatterjee M, Sammi SR, Kaur S, Palit G, et al. (2011) Sir-2.1 modulates 'calorie-restriction-mediated' prevention of neurodegeneration in Caenorhabditis elegans: implications for Parkinson's disease. Biochem Biophys Res Commun 413: 306–310.
- 28. Liu Z, Hamamichi S, Lee BD, Yang D, Ray A, et al. (2011) Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models. Hum Mol Genet 20: 3933–3942.
- 29. Schreiber MA, McIntire SL (2011) A Caenorhabditis elegans p38 MAP kinase pathway mutant protects from dopamine, methamphetamine, and MDMA toxicity. Neurosci Lett 498: 99–103.
- 30. Martinez-Finley EJ, Avila DS, Chakraborty S, Aschner M (2011) Insights from Caenorhabditis elegans on the role of metals in neurodegenerative diseases. Metallomics 3: 271–279.
- 31. Benedetto A, Au C, Avila DS, Milatovic D, Aschner M (2010) Extracellular dopamine potentiates mn-induced oxidative stress, lifespan reduction, and dopaminergic neurodegeneration in a BLI-3-dependent manner in Caenorhabditis elegans. PLoS Genet 6: e1001084.
- 32. Yao C, El Khoury R, Wang W, Byrd TA, Pehek EA, et al. (2010) LRRK2-mediated neurodegeneration and dysfunction of dopaminergic neurons in a Caenorhabditis elegans model of Parkinson's disease. Neurobiol Dis 40: 73–81.
- 33. Kamp F, Exner N, Lutz AK, Wender N, Hegermann J, et al. (2010) Inhibition of mitochondrial fusion by alpha-synuclein is rescued by PINK1, Parkin and DJ-1. EMBO J 29: 3571–3589.
- 34. Cornejo Castro EM, Waak J, Weber SS, Fiesel FC, Oberhettinger P, et al. (2010) Parkinson's disease-associated DJ-1 modulates innate immunity signaling in Caenorhabditis elegans. J Neural Transm 117: 599–604.
- 35. Asikainen S, Rudgalvyte M, Heikkinen L, Louhiranta K, Lakso M, et al. (2010) Global microRNA expression profiling of Caenorhabditis elegans Parkinson's disease models. J Mol Neurosci 41: 210–218.
- 36. Roodveldt C, Bertoncini CW, Andersson A, van der Goot AT, Hsu ST, et al. (2009) Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/alpha-synuclein complex by Hip. EMBO J 28: 3758–3770.
- 37. Ruan Q, Harrington AJ, Caldwell KA, Caldwell GA, Standaert DG (2010) VPS41, a protein involved in lysosomal trafficking, is protective in Caenorhabditis elegans and mammalian cellular models of Parkinson's disease. Neurobiol Dis 37: 330–338.
- 38. Caldwell KA, Tucci ML, Armagost J, Hodges TW, Chen J, et al. (2009) Investigating bacterial sources of toxicity as an environmental contributor to dopaminergic neurodegeneration. PLoS One 4: e7227.
- 39. Bargmann CI (1998) Neurobiology of the Caenorhabditis elegans genome. Science 282: 2028–2033.
- 40. van der Goot AT, Zhu W, Vazquez-Manrique RP, Seinstra RI, Dettmer K, et al. (2012) Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation. Proc Natl Acad Sci U S A 109: 14912–14917.
- 41. Lee JY, Song J, Kwon K, Jang S, Kim C, et al. (2012) Human DJ-1 and its homologs are novel glyoxalases. Hum Mol Genet 21: 3215–3225.
- 42. Zheng M, Gorelenkova O, Yang J, Feng Z (2012) A liquid phase based C. elegans behavioral analysis system identifies motor activity loss in a nematode Parkinson's disease model. J Neurosci Methods 204: 234–237.
- 43. Vidal-Gadea AG, Pierce-Shimomura JT (2012) Conserved role of dopamine in the modulation of behavior. Commun Integr Biol 5: 440–447.
- 44. Kuwahara T, Koyama A, Koyama S, Yoshina S, Ren CH, et al. (2008) A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in alpha-synuclein transgenic C. elegans. Hum Mol Genet 17: 2997–3009.
- 45. Tucci ML, Harrington AJ, Caldwell GA, Caldwell KA (2011) Modeling dopamine neuron degeneration in Caenorhabditis elegans. Methods Mol Biol 793: 129–148.
- 46. De Jesus-Cortes H, Xu P, Drawbridge J, Estill SJ, Huntington P, et al. (2012) Neuroprotective efficacy of aminopropyl carbazoles in a mouse model of Parkinson disease. Proc Natl Acad Sci U S A 109: 17010–17015.
- 47. Cao P, Yuan Y, Pehek EA, Moise AR, Huang Y, et al. (2010) Alpha-synuclein disrupted dopamine homeostasis leads to dopaminergic neuron degeneration in Caenorhabditis elegans. PLoS One 5: e9312.
- 48. Slone SR, Lesort M, Yacoubian TA (2011) 14-3-3theta protects against neurotoxicity in a cellular Parkinson's disease model through inhibition of the apoptotic factor Bax. PLoS One 6: e21720.
- 49. Harrington AJ, Knight AL, Caldwell GA, Caldwell KA (2011) Caenorhabditis elegans as a model system for identifying effectors of alpha-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease. Methods 53: 220–225.
- 50. Yacoubian TA, Slone SR, Harrington AJ, Hamamichi S, Schieltz JM, et al. (2010) Differential neuroprotective effects of 14-3-3 proteins in models of Parkinson's disease. Cell Death Dis 1: e2.
- 51. Jadiya P, Khan A, Sammi SR, Kaur S, Mir SS, et al. (2011) Anti-Parkinsonian effects of Bacopa monnieri: insights from transgenic and pharmacological Caenorhabditis elegans models of Parkinson's disease. Biochem Biophys Res Commun 413: 605–610.
- 52. Locke CJ, Fox SA, Caldwell GA, Caldwell KA (2008) Acetaminophen attenuates dopamine neuron degeneration in animal models of Parkinson's disease. Neurosci Lett 439: 129–133.
- 53. Fu RH, Wang YC, Chen CS, Tsai RT, Liu SP, et al.. (2013) Acetylcorynoline attenuates dopaminergic neuron degeneration and alpha-synuclein aggregation in animal models of Parkinson's disease. Neuropharmacology doi:10.1016/j.neuropharm.2013.08.007.
- 54. Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
- 55. Yang X, Zhang P, Wu J, Xiong S, Jin N, et al. (2012) The neuroprotective and lifespan-extension activities of Damnacanthus officinarum extracts in Caenorhabditis elegans. J Ethnopharmacol 141: 41–47.
- 56. Voisine C, Varma H, Walker N, Bates EA, Stockwell BR, et al. (2007) Identification of potential therapeutic drugs for huntington's disease using Caenorhabditis elegans. PLoS One 2: e504.
- 57. Nass R, Hall DH, Miller DM 3rd, Blakely RD (2002) Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans. Proc Natl Acad Sci U S A 99: 3264–3269.
- 58. Sawin ER, Ranganathan R, Horvitz HR (2000) C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway. Neuron 26: 619–631.
- 59. Kuwahara T, Koyama A, Gengyo-Ando K, Masuda M, Kowa H, et al. (2006) Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans. J Biol Chem 281: 334–340.
- 60. Hoogewijs D, Houthoofd K, Matthijssens F, Vandesompele J, Vanfleteren JR (2008) Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol Biol 9: 9.
- 61. Vilchez D, Morantte I, Liu Z, Douglas PM, Merkwirth C, et al. (2012) RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489: 263–268.
- 62. Shvadchak VV, Yushchenko DA, Pievo R, Jovin TM (2011) The mode of alpha-synuclein binding to membranes depends on lipid composition and lipid to protein ratio. FEBS Lett 585: 3513–3519.
- 63. Bove J, Perier C (2012) Neurotoxin-based models of Parkinson's disease. Neuroscience 211: 51–76.
- 64. Saw CL, Wu Q, Su ZY, Wang H, Yang Y, et al.. (2013) Effects of natural phytochemicals in Angelica sinensis (Danggui) on Nrf2-mediated gene expression of phase II drug metabolizing enzymes and anti-inflammation. Biopharm Drug Dispos DOI: 10.1002/bdd.1846.
- 65. Hwang CK, Chun HS (2012) Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons. Biosci Biotechnol Biochem 76: 536–543.
- 66. Charriaut-Marlangue C (2004) Apoptosis: a target for neuroprotection. Therapie 59: 185–190.
- 67. Pourkarimi E, Greiss S, Gartner A (2012) Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction. Cell Death Differ 19: 406–415.
- 68. Hyman BT, Yuan J (2012) Apoptotic and non-apoptotic roles of caspases in neuronal physiology and pathophysiology. Nat Rev Neurosci 13: 395–406.
- 69. van Ham TJ, Thijssen KL, Breitling R, Hofstra RM, Plasterk RH, et al. (2008) C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging. PLoS Genet 4: e1000027.
- 70. Hamamichi S, Rivas RN, Knight AL, Cao S, Caldwell KA, et al. (2008) Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson's disease model. Proc Natl Acad Sci U S A 105: 728–733.
- 71. Karpinar DP, Balija MB, Kugler S, Opazo F, Rezaei-Ghaleh N, et al. (2009) Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson's disease models. EMBO J 28: 3256–3268.
- 72. Usenovic M, Knight AL, Ray A, Wong V, Brown KR, et al. (2012) Identification of novel ATP13A2 interactors and their role in alpha-synuclein misfolding and toxicity. Hum Mol Genet 21: 3785–3794.
- 73. van Ham TJ, Holmberg MA, van der Goot AT, Teuling E, Garcia-Arencibia M, et al. (2010) Identification of MOAG-4/SERF as a regulator of age-related proteotoxicity. Cell 142: 601–612.
- 74. Nedelsky NB, Todd PK, Taylor JP (2008) Autophagy and the ubiquitin-proteasome system: collaborators in neuroprotection. Biochim Biophys Acta 1782: 691–699.
- 75. Yu Z, Kleifeld O, Lande-Atir A, Bsoul M, Kleiman M, et al. (2011) Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome. Mol Biol Cell 22: 911–920.
- 76. Maccarrone M, Bernardi G, Agro AF, Centonze D (2011) Cannabinoid receptor signalling in neurodegenerative diseases: a potential role for membrane fluidity disturbance. Br J Pharmacol 163: 1379–1390.
- 77. Sebastiao AM, Colino-Oliveira M, Assaife-Lopes N, Dias RB, Ribeiro JA (2013) Lipid rafts, synaptic transmission and plasticity: impact in age-related neurodegenerative diseases. Neuropharmacology 64: 97–107.
- 78. Hirsch EC, Hunot S (2009) Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol 8: 382–397.
- 79. Rogers J, Mastroeni D, Leonard B, Joyce J, Grover A (2007) Neuroinflammation in Alzheimer's disease and Parkinson's disease: are microglia pathogenic in either disorder? Int Rev Neurobiol 82: 235–246.
- 80. Collins LM, Toulouse A, Connor TJ, Nolan YM (2012) Contributions of central and systemic inflammation to the pathophysiology of Parkinson's disease. Neuropharmacology 62: 2154–2168.
- 81. Kim HG, Ju MS, Ha SK, Lee H, Kim SY, et al. (2012) Acacetin protects dopaminergic cells against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neuroinflammation in vitro and in vivo. Biol Pharm Bull 35: 1287–1294.
- 82. Du XX, Xu HM, Jiang H, Song N, Wang J, et al. (2012) Curcumin protects nigral dopaminergic neurons by iron-chelation in the 6-hydroxydopamine rat model of Parkinson's disease. Neurosci Bull 28: 253–258.
- 83. Kumar H, Kim IS, More SV, Kim BW, Bahk YY, et al. (2013) Gastrodin protects apoptotic dopaminergic neurons in a toxin-induced Parkinson's disease model. Evid Based Complement Alternat Med 2013: 514095.
- 84. Zhang ZJ, Cheang LC, Wang MW, Lee SM (2011) Quercetin exerts a neuroprotective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish. Int J Mol Med 27: 195–203.
- 85. Lu JH, Tan JQ, Durairajan SS, Liu LF, Zhang ZH, et al. (2012) Isorhynchophylline, a natural alkaloid, promotes the degradation of alpha-synuclein in neuronal cells via inducing autophagy. Autophagy 8: 98–108.
- 86. Zbarsky V, Datla KP, Parkar S, Rai DK, Aruoma OI, et al. (2005) Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson's disease. Free Radic Res 39: 1119–1125.
- 87. Ramassamy C (2006) Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets. Eur J Pharmacol 545: 51–64.
- 88. Pandey N, Strider J, Nolan WC, Yan SX, Galvin JE (2008) Curcumin inhibits aggregation of alpha-synuclein. Acta Neuropathol 115: 479–489.
- 89. Ahmad B, Lapidus LJ (2012) Curcumin prevents aggregation in alpha-synuclein by increasing reconfiguration rate. J Biol Chem 287: 9193–9199.
- 90. Berman AE, Chan WY, Brennan AM, Reyes RC, Adler BL, et al. (2011) N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse. Ann Neurol 69: 509–520.
- 91. Clark J, Clore EL, Zheng K, Adame A, Masliah E, et al. (2010) Oral N-acetyl-cysteine attenuates loss of dopaminergic terminals in alpha-synuclein overexpressing mice. PLoS One 5: e12333.
- 92. Etminan M, Gill SS, Samii A (2005) Intake of vitamin E, vitamin C, and carotenoids and the risk of Parkinson's disease: a meta-analysis. Lancet Neurol 4: 362–365.
- 93. Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, et al. (2006) The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem 99: 1251–1262.
- 94. Tan W, Li Y, Chen M, Wang Y (2011) Berberine hydrochloride: anticancer activity and nanoparticulate delivery system. Int J Nanomedicine 6: 1773–1777.
- 95. Onishi H, Machida Y (2005) Macromolecular and nanotechnological modification of camptothecin and its analogs to improve the efficacy. Curr Drug Discov Technol 2: 169–183.
- 96. Fu RH, Liu SP, Ou CW, Huang CM, Wang YC (2010) Spatial control of cells, peptide delivery and dynamic monitoring of cellular physiology with chitosan-assisted dual color quantum dot FRET peptides. Acta Biomater 6: 3621–3629.