Curcumin reduces α-synuclein induced cytotoxicity in Parkinson's disease cell model
© Wang et al; licensee BioMed Central Ltd. 2010
Received: 28 July 2009
Accepted: 30 April 2010
Published: 30 April 2010
Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinson's disease (PD) and other synucleinopathies. αS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models.
Here we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra- or extracellulary.
Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.
Parkinson's disease (PD) affects 1% of the population over the age of 65 and is the second most common progressive neurodegenerative disorder after Alzheimer's disease (AD) [1, 2]. The classical symptoms of PD include resting tremor, muscular rigidity and bradykinesia [2, 3] resulting from the progressive loss of dopaminergic neurons in the substantia nigra region of the brain [3, 4]. Intracellular inclusions known as Lewy bodies (LB) and Lewy neurites (LN), composed primarily of insoluble aggregates of ubiquitin and α-synuclein (αS), are neuropathological hallmarks of PD found in many regions of the brain and central nervous system (CNS) [4–6]. Point mutations and multiplication of the αS gene are associated with rare early onset familial forms of the disease, further implicating the role of αS in PD [7–10]. The increased degeneration of dopaminergic neurons in the substantia nigra of PD animal models correlates with increased levels of LBs and LNs in this region of the brain and strongly suggests that overexpression of αS selectively targets dopaminergic neurons [11–13]. While it is unclear why dopaminergic neurons are more susceptible to degeneration by αS, the oxidation of dopamine and exposure to neurotoxins such as rotenone [14, 15] and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [16–19] generate excessive reactive oxygen species (ROS), promoting mitochondrial complex I dysfunction [15, 20, 21] and depleting glutathione levels [22, 23] ultimately causing acute Parkinsonism in animal and cell models. In addition, overexpression of both wild type (WT) and mutant αS results in formation of cytoplasmic inclusions and degeneration of dopaminergic neurons in mouse and Drosophila models [11–13, 24].
αS is a presynaptic protein expressed at synaptic terminals in the CNS [25, 26]. While αS is a natively unfolded protein, the monomeric form can misfold and aggregate into larger oligomeric and fibrillar forms which are linked to the pathogenesis of PD. Recent studies have implicated small soluble oligomeric and protofibrillar forms of αS as the most neurotoxic species [27–30]. While previous studies provide good evidence for the intracellular toxicity of αS in PD, there is also evidence showing an extracellular component as well [27–29, 31, 32]. Monomeric and oligomeric forms of αS have been detected in blood plasma and cerebrospinal fluid of PD patients [27, 31–33], and exposure to extracellular pre-aggreated αS induces cytotoxicity in primary mesencephalic neuron-glia and human neuroblastoma cell cultures [28, 29, 34, 35].
Since generation of ROS has been correlated with onset of PD, anti-oxidants may have therapeutic value. Curcumin, a polyphenolic compound commonly used as food additives in Asian cuisine, has anti-oxidant properties and suppresses inflammatory responses of brain microglial cells [36–38]. Curcumin was also shown to have protective effects in neurodegenerative disease by either reducing inflammation and oxidative damage in AD [36–39], or by inhibiting protein misfolding and aggregation in Creutzfeld-Jakob disease  and PD [41, 42].
Given these numerous beneficial properties, curcumin shows promise as a therapeutic agent for neurodegenerative diseases. We show that curcumin can provide protection against αS-induced cytotoxicity in SH-SY5Y neuroblastoma cells by decreasing cytotoxicity of aggregated αS, reducing intracellular ROS, inhibiting caspase-3 activation and ameliorating signs of apoptosis. We also show that either extracellular addition of oligomeric αS and intracellular overexpression of αS increases generation of intracellular ROS in SH-SY5Y cells and both have similar cytotoxic effects resulting in induced caspase-3 activity and apoptosis.
Curcumin protects SH-SY5Y cells against extracellular αS-induced cytotoxicity
Curcumin neutralizes αS-induced cytotoxicity in SH-SY5Y cells
LDH (% control)
Viability (% control)
Tris buffer (control)
αS + curcumin
Extracellular addition of αS generates excessive ROS
Curcumin inhibits caspase-3 activity and apoptosis induced by extracellular αS
Curcumin reduces ROS and cytotoxicity induced by intracellular overexpression of αS
A key pathological feature of PD is the formation of cytoplasmic inclusions containing ubiquitin and αS known as LBs and LNs in the dopaminergic neurons of the substantia nigra region of the brain [3, 4]. The many factors that influence αS aggregation and the subsequent downstream cytotoxic events that lead to neuronal cell death are being actively studied. Several point mutations in the αS gene which correlate to rare familial early-onset PD and rapid progression of the disease [7–9] accelerate aggregation of αS and favor formation of nonfibrillar oligomeric forms. Recent studies have suggested that soluble oligomeric and protofibrillar structures are the toxic species [27–30], and that these forms can permeabilize plasma membranes, alter intracellular function, induce oxidative stress and trigger apoptosis in cells [30, 44, 45]. Epidemiological studies have also suggested that exposure to environmental agents such as neurotoxins and pesticides [16, 17] cause an increase in oxidative damage to the cells by suppressing mitochondrial complex I activity and reducing glutathione levels [22, 23], thereby increasing the risk for PD.
Oxidative stress plays a major role in aging and is associated with several neurodegenerative diseases including PD , where an increase in ROS accompanies αS aggregation and degeneration of dopaminergic neurons [15, 47, 48]. Intracellular overexpression of αS generates excess ROS and causes oxidative stress to the cells [23, 46], leading to disruption in redox homeostasis cell metabolism, free radical generation, lipid peroxidation, cholesterol and protein oxidation [46, 49]. Excess ROS causes plasma membrane damage, mitochondrial dysfunction, defects in the glutathione peroxidase expression and reduction in glutathione levels, all of which render the brain more susceptible to oxidative stress [46, 49, 50]. In this study, we find that extracellular addition of oligomeric αS and intracellular overexpression of αS in SH-SY5Y cells both increase ROS levels by almost 2-fold. The αS-induced increase in ROS levels in our current study shows similar oxidative damage to the SH-SY5Y cell as previous MPP+ PD cell models, where MPP+ selectively targets and degenerates dopaminergic neurons due to excess generation of ROS [13, 15, 18]. Prolonged exposure to MPP+ and other neurotoxins has been shown to activate caspase-3 [16, 19, 51], an important effector caspase in the final apoptotic cascade leading to cell death. If oxidative stress exacerbates the etiology of PD, then agents that can simultaneously attenuate ROS damage and suppress caspase-3 activation may hold promise for the treatment of PD and other neurodegenerative diseases.
Here we show that curcumin, a natural phenolic food additive, effectively inhibits activation of caspase-3 (Fig. 2B) and ameliorates signs of apoptosis (Fig. 3) induced by extracellular addition of oligomeric αS to SH-SY5Y cells. We also demonstrated that curcumin reduces intracellular overexpression of αS and reduces ROS generation [15, 46, 48].
Overexpression and abnormal accumulation of oligomeric αS is key in the pathogenesis of PD [14, 48, 52], and numerous studies suggest that there is both an intra- and extracellular component to αS toxicity in PD [12, 24, 31, 32, 53]. We recently demonstrated that an anti-oligomeric αS antibody fragment binds oligomeric αS on the surface of SH-SY5Y cells, verifying the presence of intracellularly produced oligomeric αS on external cell membrane surfaces . Here we show that extracellular addition of oligomeric αS induces similar cytotoxic effects as intracellular overexpression of αS, and that these αS-induced cytotoxic effects are similar to those reported in MPTP Parkinsonian models. We also show that curcumin can significantly reduce the cytotoxicity induced by extracellular or intracellular αS aggregates, suggesting it may have value for treating PD. Since extracellularly added curcumin provides protection even against intracellularly induced αS toxicity, our results suggest that there is a significant extracellular or cell surface component of αS-induced toxicity in PD models, which is consistent with a recently published report of interneuronal transmission of extracellular αS pathology in neuronal cells . However, additional studies are needed to further elucidate the mechanism of αS-induced cytotoxicity and its subsequent pathogenesis and progression to induced-apoptosis in PD.
αS was prepared and purified in our lab as previously described [28, 54]. Purified αS was lyophilized and stored at -80°C until further use. Stocks of the lyophillized αS were first dissolved in DI water and subsequent dilutions were made in Tris buffer (25 mM Tris, 150 mM NaCl, pH 7.4). The various forms of αS samples (70 μM) were prepared by dissolving the αS stock in Tris buffer. Monomeric αS samples were utilized immediately after dilution with Tris buffer, oligomeric αS were generated by incubating the samples at 37°C for 5-7 days (without shaking) while predominantly fibrillar morphologies of αS were generated by incubation at 37°C for up to 30 days (without shaking). αS morphologies were verified by AFM before use. All other chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich, MO) and used as is without further treatment unless otherwise specified.
Co-incubation of curcumin with pre-formed αS samples
Curcumin stocks (1 mg/mL) were prepared in dimethyl sulfoxane (DMSO) and stored at -20°C in dark conditions until use. Curcumin was diluted to 140 μM with Tris buffer in a 2:1 molar ratio of curcumin to pre-formed αS sample.
Atomic Force Microscopy
A 10 uL aliquot of each sample was applied to a piece of freshly cleaved mica, incubated at room temperature for 10 minutes, rinsed with DI water and dried under a gentle stream of N2 gas. Topographic AFM images were acquired using OTESPA tips (k = 40 N/m, fo = 300-kHz) (Veeco, Santa Barbara, CA) at scan rates of 2 Hz with 512 × 512 pixel resolution on a Nanoscope IIIa TM-AFM (Veeco, Santa Barbara, CA). AFM images were analyzed with the scanning probe imaging processor software (SPIP, Image Metrology) to generate height distribution plots, as previously described .
PAGE and silver staining
Oligomeric αS samples, with and without curcumin were separated on a 10% Tris/Tricine native PAGE and developed using Pierce silver stain kit according to manufacturer's protocol. (Thermo Scientific, Rockford, IL).
Cell culture and transient transfection of SH-SY5Y cells
SH-SY5Y-human neuroblastoma cells were maintained and grown as described previously [28, 29]. Transient transfection of SH-SY5Y cells was performed using TransFast™ transfection reagent according to the manufacturer's protocol (Promega, Madison, WI) with slight modification. SH-SY5Y cells were grown for 4 days (50-65% confluency) in a 6-well plate in vitro before transfection. A transfection mixture consisting of a 1 μg aliquot of wildtype α-synuclein/eGFP (WTsynEGFP) fusion protein plasmid DNA (Clontech, Palo Alto, CA) and TransFast™ reagent (1:2 v/v) in serum free media was pre-incubated in the dark for 15 min at room temperature before addition to the cells. Cell culture media was removed and the transfection mixture (500 μL) was added to each well and incubated for 1 hr at 37°C, followed by addition of complete media with serum (500 μL). The culture plates were incubated and grown in a 5% CO2 atmosphere at 37°C for 48 hrs. A 10 μL aliquot of curcumin (4 μM final concentration) was added 48 hr post-transfection and the cells were incubated for another 24 hr before analysis. Previous studies have shown that the α-synuclein fusion protein aggregates similarly to α-synuclein alone, and that eGFP expression does not induce toxicity .
Cytotoxicity by lactate dehydrogenase assay
Cytotoxicity of samples towards SH-SY5Y cells was measured using a lactate dehydrogenase (LDH) assay as described . Cells were seeded (2 × 104 cells/mL) in a 96-well plate 24 hr prior to the following treatment conditions: (a) pre-formed oligomeric αS (2 μM), (b) co-incubated samples of αS (2 μM) with curcumin (4 μM), (c) curcumin (4 μM) and (d) Tris buffer control. After incubating cells with each treatment for 48 hr, cytotoxicity of each sample was determined by measuring the reduction of iodonitrotetrazolium salt by LDH enzyme using a Wallac 1420 plate reader (Perkin Elmer, USA) at 490 nm and 650 nm. The values were expressed as a percentage of the Tris buffer control. Experiments were repeated a minimum of three times.
Cell viability by resazurin reduction assay
Cell viability was determined using a resazurin reduction assay . Viable cells convert resazurin (blue) to resorufin (pink), and the degree of cell death can be measured directly by either absorbance or fluorescence spectrometry. Resaruzin stocks (10 mM) were made in DMSO and kept at -20°C until use when they were diluted to a 100 μM working solution with Tris buffer. Cells were seeded (5 × 104 cells/mL) in a 48-well plate 24 hr prior to exposure to the treatment conditions described above. Following treatment for 48 hr, cell culture media was removed and the cells were resuspended with 200 μL Tris buffer. An aliquot (10 μL) of resaruzin (20 μM final concentration) was added to each well and incubated at 37°C for an additional 3 hr. Absorbance of resorufin was measured at 560 nm and 600 nm. Cell viability of each sample was calculated by subtracting the background OD600 nm from OD560 nm and reported as a percentage of the Tris buffer control.
Measurement of intracellular ROS formation
The formation of intracellular ROS was measured using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA) as described . The cells were seeded (2 × 104 cells/mL) in a 96-well plate and were incubated for 48 hrs prior to ROS measurement with the conditions described above. After treatment, the cells were washed twice and resuspended in 100 μL Tris buffer. DCFH-DA (10 μM final concentration) was added to each well and the cells were incubated for 1 hr at 37°C in dark conditions. The fluorescence intensity of dichlorofluorescein (DCF, the oxidized species of DCFH-DA) was measured using a fluorescence spectrophotometer with excitation wavelength of 485 nm and emission wavelength of 535 nm.
Determination of caspase-3 activity
Caspase-3 activity was determined using the Caspase-3/CPP32 colorimetric assay kit following the manufacturer's protocol (BioVision, Inc., CA). Since caspase-3 is a pre-apoptotic marker, measurements of caspase-3 activity were taken after 24 hr incubation with the various treatments to ensure proper detection. Briefly, cells (106 cells/mL) were exposed to different treatments as described above for 24 hr, detached and lysed on ice for 10 min. The supernatant was removed and the total protein concentration of each sample was determined using a bicinchoninic acid assay (BCA, Pierce, Rockford, IL). Cell lysate was then diluted to 150 μg with lysis buffer for each assay. An equal loading amount of lysate (50 μL) was mixed with 50 μL of 2× reaction buffer with 10 mM dithiothreitol (DTT) and 5 μL DEVD-pNA substrate (200 μM) and incubated at 37°C for 1 hr. The absorbance of released p-nitroanilide (p NA) was measured at 405 nm using a plate reader. The increase in caspase-3 activity was determined by comparing the absorbance of the treated sample with the absorbance of the Tris buffer control sample.
Fluorescence microscopy and nuclear staining
WTsynEGFP-transfected cells were evaluated 48 hr post-transfection using a Nikon TE300 fluorescence microscope at an excitation wavelength of 488 nm with a 40× magnification objective. For nuclear staining, untransfected SH-SY5Y cells were seeded on glass coverslips and allowed to attach for 24 hr. The cells were fixed with 4% paraformaldehyde for 25 min, washed twice in cold Tris buffer, and stained with Hoechst 33342 (10 μg/mL) for 15 min. Nuclear morphology was observed using a 100× magnification objective. Images were captured and processed by MetaMorph software (Molecular Devices, USA). Cells stained by Hoechst 33342 with diffused nuclei were scored as viable, while cells with reduced nuclei, condensed chromatin, and increased fluorescence were considered apoptotic.
Data was presented as mean ± SE from at least three independent experiments. Statistical analysis was evaluated using either Student's t-test or using a one-way ANOVA followed by Bonferoni post-hoc test for all pair-wise comparison. A p-value of < 0.05 was considered as significant.
This research was supported in part by grants from the Michael J. Fox Foundation, the Arizona Alzheimer's Research Consortium, American Health Assistance Foundation and the ASU Graduate and Professional Student Association Research Grant. The authors would like to thank Dr. Page Baluch and the WM Keck Imaging facility at ASU for the assistance with fluorescence microscopy and Mr. Philip Schulz for protein purification.
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