Media and N2 supplements for cell culture were obtained from Invitrogen. 2’,7’-dichlorofluorescein (DCF) diacetate (DCFDA) was obtained from Invitrogen. Rotenone and curcumin were from Sigma (St. Louis, MO, USA) were first dissolved in DMSO and then diluted into midia or fly food before use. The final concentration of DMSO was 0.2% in the media or fly food that were not alter cell and fly growth. The pan caspase inhibitor, z-VAD-fmk was purchased from Enzyme Systems Products (Livermore, CA, USA).

SH-SY5Y human neuroblastoma cells were grown in DMEM with 10% Fetal boral serum (FBS) and antibiotic-antimycotic (100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml fungizone) solutions at 37˚C in 5% CO2/95% air. For rotenone toxicity does-response studies, SH-SY5Y cells were added rotenone at 0 - 250 nM concentrations.

To test effect of curcumin on rotenone toxicity, we used the LC 80 (the concentration causing 80% cell death) of rotenone. We pretreated cells with curcumin at 0, 0.1, 05, 1 and 5 μM concentrations for 1 h, and then exposed them to LC80 of rotenone for 24 h. We also used a pan caspase inhibitor, z-VAD (100 µM) as a positive control for curcumin protective experiments. Trypan blue exclusion was used to measure cell death by counting the number of dead (blue) and live cells in the cultures after rotenone exposure and/or curcumin treatment.

The levels of cytosolic ROS were measured by redoxsensitive fluorophore DCFDA as previously described [14]. Briefly, cells were washed with PBS, and then incubated for 45 min with DCFDA, which is initially non-fluorescent and is converted by oxidation to the fluorescent molecular DCF. DCF was then quantified using a CytoFluor Multi-well Plate Reader, Series 400 (Perseptive Biosystems) with 485 nm excitation and 538 nm emission filters.

For detection of mitochondrial superoxide, MitoSOX Red and MitoTracker Green (Invitrogen) dissolved in DMSO were used at a final concentration of 5 μmol/L and 1 μmol/L respectively, according to the protocols of the manufacturers. Briefly, cells after rotenone exposure and/or curcumin treatment were loaded with the fluorogenic probe MitoSOX Red (5 μmol/L) for 20 min. After removing MitoSOX Red and washing cells with Hanks’ balanced salt solution, fluorescent images were captured by fluorescence microscope. MitoSOX Red is a fluorescent dye specific for the detection of O2—in the mitochondria of live cells. To confirm the localization of MitoSOX Red to mitochondria, cells were also incubated with mitochondria marker (MitoTracker Green (1 μmol/L) for 20 min at the same time. The yellow fluorescence in the overlay images indicated that MitoSOX is inside of the mitochondria. Fluorescence pictures were taken by fluorescence microscopy with identical exposure settings. Ten random images from each well in a 24-well cell culture plate were taken for each parameter to obtain a mean value for statistical comparison. NIH Image J 5.0 software was used for the analysis of fluorescent intensity of MitoSOX in individual cell. Average intensity per cell was determined and more than 50 cells in each experimental group were analyzed. Each experiment was repeated three times.

Caspase-3 and caspase-9 activities were measured by a colormetric method at A405 nm as previously described [14]. Cells were harvested in cell lysis buffer (50 mM HEPES, 1 mM DTT, 0.1 mM EDTA, 0.1% CHAPS and 0.1% Triton X-100, pH 7.4). DEVD-p-nitroanilide and LEHD-p-nitroanilide were the substrates for caspase-3 and caspase-9, respectively. The experiments were performed according to the manufacturer’s protocol (Biosource International).

W1118 strain of Drosophila were grown on standard cornmeal medium at 25˚C and fresh food media were changed every 3 days. To get PD model, the rotenone (0 - 125 nM final concentration) were added freshly when the food changed.

Cohorts of 50 flies from each experimental group were monitored for survival. Flies were maintained on standard media, rotenone and rotenone with curcumin (0 - 5 mM final concentration) for untreated control, rotenone only and curcumin protect group. Mortality was scored daily and analyzed by using Kaplan-Meier survival curves. This experiment was repeated three times.

The fly locomotor ability was determined using a climbing assay (negative geotaxis assay) as described previously [15]. Cohorts of 60 flies from each genotype were subjected to the assay weekly. The tested flies were age-matched, randomly selected, anesthetized, and placed in a vertical plastic column (length, 25 cm; diameter, 1.5 cm). Flies were gently tapped to the bottom of the column. We counted and calculated the percentage of flies that could climb to, or above the median line of the cylinder in 10 seconds. Each week, the assay was repeated three times.

Six neuronal DA clusters are normally present in each adult Drosophila brain hemisphere [15]. On the anterior side, there was a paired dopaminergic cluster, PAL (protocerebral anterolateral) and a paired cluster in the middle, PAM (paired anterolateral medial). On the posterior side, there were five clusters: PPM1 (unpaired), PPM2 (paired), PPM3 (paired) (protocerebral posterior medial), PPL1 and PPL2 (paired) (protocerebral posterolateral). These neurons express tyrosine hydroxylase (TH), which is an enzyme required for the biosynthesis of dopamine and often used as a marker for dopaminergic neurons. Thus, whole-mount brain immunostaining using specific anti-TH antibodies were performed to detect DA neurons as previously described [15]. Briefly, fluorescent immunostaining was performed on whole-mount dissected adult brain at 2 - 4 weeks of age in the presence or absence of curcumin treatment and/or rotenone exposure. Cohorts of six to eight flies per experimental group were used for immunostaining. The dissected brains were mounted in Vectashield (Vector Laboratories). Rabbit polyclonal anti-TH (Chemicon) and mouse monoclonal anti-TH (Immunostar) were used as the primary antibodies. Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen) were used as secondary antibodies. The numbers of DA neurons were scored in whole-mount brains under fluorescent (Zeiss LSM 250) and/or confocal microscopy (Zeiss LSM 510). We counted the TH-positive cells in all clusters except in the paired PAM cluster, because the density of the neurons in PAM was too high to allow precise quantification. Six brain sections were counted in each experimental group.

The UAS-MTSroGFP2 was expressed in drosophila pan neurons by elav-Gal4 driver. The brain dissection and redox state analysis is performed as previously reported [16]. Briefly, the brains of elav-Gal4; UASMTSroGFP2 animals were dissected in PBS and placed in mounting medium (Vector, H-1000). RoGFP2 fluorescence was collected by using a 535 nm filter following excitation at 405 nm (Oxidized roGFP2) and 488 nm (Reduced roGFP2). The 20× lens and Z-scan by 10 um stepsize was used to get the whole brain image. The Ratios of 405 nm/488 nm fluorescences were counted by ImageJ software and data were analyzed using student t-test.

Quantitative data were expressed as arithmetic means ± SEM based on at least three separate experiments. Statistically significant differences among experimental groups were analyzed by analysis of variance (ANOVA) using Sigmastart 3.1 statistical soft ware (Aspire Software International, VA). A P value (<0.05) was considered significant.

To address whether curcumin can restrain rotenoneinduced neurodegeneration, we exposed human dopaminergic neuroblastoma SH-SY5Y cells to various concentrations of rotenone for 24 h. Rotenone (Figure 1(a)) caused apoptotic cell death in a dose-dependent manner (Figure 1(b)). The LC80 (the concentration causing 80% cell death) of rotenone was 125 nM. To test the protective effect of curcumin (Figure 1(c)) on rotenone toxicity, we pretreated cells with curcumin for 1 h, and then exposed them to 125 nM rotenone for 24 h. Curcumin protected against 125 nM rotenone-induced cell death to a significant extent at a concentration of 100 nm and to a maximum extent at 1 μM (Figure 1(d)).

We generated a rotenone-based fly model using W1118 normal wild type Drosophila. We added rotenone in fly food at a concentration gradient of 5, 25, 75 and 125 μM from day 1 post-eclosion and keep the same drug level in the food throughout the lifetime. We found that rotenone caused early death in a dose-dependent manner and is significant when compared with unexposed flies (Figure 2(a)). To measure the behavioral change resulted from rotenone exposure, we employed a climbing assay (negative geotactic test) at multiple time points in their lifetime. When flies were tapped to the

bottom of a vial, nearly all untreated control flies rapidly climbed to the top of the vial on day 7 and day 14. However, flies exposed to rotenone exhibited a significant dose-dependent motor deficit in performance, indicating locomotor dysfunction (Figure 2(b)). There was a progressive, dose dependent decline in climbing ability in flies with rotenone exposure. Rotenone at 75 μM and 125 μM caused significant locomotor impairment and early mortality compared to the untreated group. Thus, we used these two concentrations to conduct further studies.

To assess whether rotenone induces degeneration in dopaminergic neurons, brains from flies with rotenone exposure were dissected and subjected to whole-mount brain immunostaining using specific anti-TH antibodies. We counted the TH-positive cellsin all the clusters (the details are described in the methods section) except the paired PAM cluster, be cause the density of the neurons in PAM was too high to allow precise quantification. In unexposed control flies, at 2 week of age, the DA clusters did not change significantly in number of TH-positive cells or their morphology (Figures 2(c) and (d)). However, at 2 week of age, flies exposed to rotenone showed a striking decrease of anti-TH-positive staining in all the DA clusters, indicating that rotenone induced a loss of DA neurons in the brain.

To test the effect of curcumin in vivo, we added curcumin at 0, 0.2, 1, 5, 10 mM in fly food from 1 day post-eclosion throughout the lifetime. The flies were also exposed to 75 μM rotenone for the same time span. The fly survival and climbing activity were monitored weekly. 1 mM curcumin significantly improved fly survival (Figure 3(a)) and prevented locomotor impairment induced by rotenone (Figure 3(b)). Increase of curcumin concentration in the fly food to more than 1 mM did not further enhance its protective effect.

To further assess whether curcumin reduces dopaminergic neuron loss, flies exposed to 75 μM rotenone

were treated with 1 mM curcumin. At 2 weeks of age, flies exposed to rotenone showed a striking decrease in anti-TH-positive staining in all DA clusters. However, curcumin significantly reduced rotenone-induced dopaminergic neuron loss in these flies (Figures 3(c), (d)).

To further study the protective mechanism of curcumin, we measured intracellular ROS using the redoxsensitive fluorophore 2’,7’-dichlorofluorescein (DCF) diacetate (DCFDA). Nonfluorescent DCFDA is converted by oxidation to the fluorescent molecule DCF. We found that the level of DCF in SH-SY5Y cells treated with 125 nM rotenone was more than four folds higher than that in untreated cells (Figure 4(a)). However, this increase in DCF fluorescence was largely reversed by curcumin. To further explore whether curcumin alters mitochondrial ROS, we used MitoSOX, a specific dye for mitochondrial ROS. MitoSOX signal was localized in MitoTracker-stained organelles, indicating that the MitoSOX staining was specific to mitochondria. Mitochondrial ROS increased dramatically after 4 h exposure to rotenone (Figures 4(b), (c)). Pretreatment with curcumin for 1 h significantly reduced the rotenone-induced increase in mitochondrial ROS (Figures 4(b), (c)).

To test the antioxidant effect of curcumin in vivo, we employed a genetically encoded ratiometric redox sensor roGFP2 [16]. RoGFP2 is a ratiometric redox indicator, i.e. the ratios of fluorescence intensity from excitation wavelength 405 nm (Figure 5(a), oxidized state) to 488 nm (Figure 5(a), reduced state) can indicate the ROS level. This sensor allows us to detect real time ROS level in different tissues or cell types by using different GAL4 drivers in drosophila. The mitochondrial target sequence (MTS) were inserted upstream of roGFP2 that can specifically target roGFP2 to mitochondria. The pan neural driver elav-GAL4 was used to generate elav-GAL4; UAS-MTSroGFP2 flies and express roGFP2 in mitochondria of all the neurons. To observe the mitochondrial ROS level in neu rons, we dissected the whole brain of elav-GAL4; UAS-MTSroGFP2 flies on day 7 and day 14 after their exposure to 75 μM rotenone with or without 1 mM curcumin in the presence. The exposure to rotenone caused an increase in ROS level compared to the vehicle control. When flies were rescued with 1 mM curcumin, their ROS level was significantly reduced compared to that of flies only treated with rotenone (Figure 5(b)).

Rotenone can activate caspase cascade to induce

apoptosis [5]. Consistent with precious findings, we found that 125 nM rotenone increased both caspase-3 and caspase-9 activities (Figures 6(a) and (b)) in our model. Caspase-3 and caspase-9 activity were in creased after exposure to rotenone for 2 h and reach peak after 12 h. To examine whether curcumin alters rotenone-induced caspase-3 and caspase-9 activation, we pretreated the cells with 500 nM curcumin for 1 h, and followed 125 nM rotenone exposures for 24 h. We used a pan caspase inhibitor, 100 µM z-VAD as a positive control that can inhibit both caspase-3 and caspase-9 activities and protect against cell death. Like z-VAD, curcumin significantly reduced rotenone-induced caspase-3 and caspase-9 activation (Figure 6(c) and (d)).