The effects of Brazilian green propolis that contains flavonols against mutant copper-zinc superoxide dismutase-mediated toxicity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. The purpose of this study was to clarify effects of brazilian green propolis and the active ingredient against ALS-associated mutant copper-zinc superoxide dismutase (SOD1)-mediated toxicity. Ethanol extract of brazilian green propolis (EBGP) protected N2a cells against mutant SOD1-induced neurotoxicity and reduced aggregated mutant SOD1 by induction of autophagy. Kaempferide and kaempferol, the active ingredients of EBGP, also inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Both kaempferide and kaempferol significantly suppressed mutant SOD1-induced superoxide in mitochondria. Western blot analysis showed that kaempferol potentially induced autophagy via the AMP-activated protein kinase (AMPK) - the mammalian target of rapamycin (mTOR) pathway. These results suggest that EBGP containing the active ingredient against mutant SOD1-mediated toxicity is a promising medicine or health food for prevention and treatment of ALS.

neurotrophic factors 13 . However, the effect of propolis and the active components against ALS-associated mutant SOD1-mediated toxicity is not well known.
In the present study, to examine whether propolis and the active components have neuroprotective effect against mutant SOD1-induced neurotoxicity in a cellular model, we used the ethanol extract of Brazilian green propolis (EBGP). In Japan, Brazilian green propolis has recently come to be used as a health food, which is of sufficiently good quality to provide experimental reproducibility. The ethanol extract of propolis is the major form used in health food 14 . We also further investigate whether autophagy is involved in the neuroprotection of kaempferol and kaempferide against mutant SOD1-related neurotoxicity via the AMP-activated protein kinase (AMPK) -the mammalian target of rapamycin (mTOR) pathway.

Results
EBGP reduced these intracellular aggregates of SOD1 G85R and prevented SOD1 G85R -induced neurotoxicity. Currently, more than 150 types of pathogenic mutations in SOD1 gene have been identified in ALS patients 15 . Among these mutations, the pathogenic SOD1 G85R mutation has been frequently studied 16 . The transgenic mice having transgene of SOD1 G85R mutation show rapidly progressive motor neuron degeneration. The aggregate formation was confirmed in SOD1 G85R -transfected N2a cells based on previous study 8, [17][18][19] . As shown in Fig. 1A, the mCherry-fused SOD1 G85R (thereafter SOD1 G85R ) formed intracellular aggregates in approximately 15% of total transfected N2a cells, whereas mCherry-fused SOD1 WT (thereafter SOD1 WT ) was distributed evenly in the cytoplasm (Fig. 1A,C). Immunostaining was performed to confirm that the signal of mCherry was SOD1. As shown in Fig. 1B, the signal of mCherry colocalized with the signal of SOD1. Similarly to previous studies 17,18 , western blot analysis showed that triton X-100-insoluble SOD1 G85R was increased (Fig. 1D). In order to examine SOD1 G85R -mediated toxicity to differentiated N2a cells, we performed MTT assay. Although SOD1 WT did not affect cell survival rate, SOD1 G85R induced cell death (Fig. 1E). These results suggest that SOD1 G85R caused cell death via the formation of insoluble aggregates.
Next, to investigate the effect of EBGP against SOD1 G85R aggregates, we evaluated the number of intracellular SOD1 G85R aggregates. EBGP significantly reduced these intracellular aggregates of SOD1 G85R to approximately 30% of untreated cells ( Fig. 2A,B). Western blot analysis also showed that triton X-100-insoluble SOD1 G85R was significantly decreased by treatment of EBGP, although triton X-100-soluble SOD1 G85R did not change (Fig. 2C,D). In addition, EBGP prevented SOD1 G85R -induced neurotoxicity (Fig. 2E). There results suggest that the active components of EBGP have significant effects for the neuroprotection against SOD1 G85R -induced neurotoxicity.
The active component of EBGP, kaempferol and kaempferide, are involved in neuroprotection against mutant SOD1-related toxicity. Previous study reported that HPLC analysis shows the major chemical constituents of the EBGP are phenolic acids/flavonoids, such as artepillin C, baccharin, drupanin, p-coumaric, Betuletol, kaempferide and kaempferol 20 . Among these components, we chose two flavonoids, kaempferol and kaempferide (Fig. 3A). We examined effects of kaempferol and kaempferide against SOD1 G85R aggregates. As shown in representative photomicrographs, kaempferol and kaempferide remarkably reduced the intracellular aggregates of SOD1 G85R (Fig. 3B). We further examined the effect of kaempferol and kaempferide on SOD1 aggregation by using a series of concentrations based on previous studies (Fig. 3C) 21,22 . As results, kaempferol (from 0.3 µM to 10 µM) and kaempferide (from 5 µM to 50 µM) significantly decreased the aggregates. Western blot analysis also showed that triton X-100-insoluble SOD1 G85R was significantly decreased by treatment of kaempferol and kaempferide (Fig. 3D,E). Moreover, we showed that kaempferol and kaempferide prevented SOD1 G85R -induced neurotoxicity ( Fig. 3F and Supplementary Fig. S1). These results suggest that kaempferol and kaempferide are involved in neuroprotection against mutant SOD1-related toxicity.
The anti-oxidant activity is involved in the neuroprotection of kaempferol and kaempferide against mutant SOD1-related toxicity. Previous study showed that flavonoid exerts an anti-oxidant activity 23 . Because oxidative stress is well recognized as an important pathogenic event in ALS, the effects of kaempferol and kaempferide against SOD1 G85R -induced ROS were examined using MitoSOX Red, a mitochondrial-targeted superoxide sensitive fluorogenic probe 24 . The increase in MitoSOX Red fluorescence in N2a cells transfected GFP-SOD1 G85R was significantly attenuated by kaempferol and kaempferide (Fig. 4A,B). To clarify the direct scavenging effect of kaempferol and kaempferide against ROS such as hydroxyl radical (•OH), we performed electron spin resonance (ESR) analysis (Fig. 4C,D). As result, both kaempferol and kaempferide presented potent scavenging activity against •OH. These results demonstrate that the anti-oxidant activity is involved in the neuroprotection of both kaempferol and kaempferide against mutant SOD1-related neurotoxicity.
Kaempferol, but not kaempferide, reduced intracellular aggregate via the activation of autophagy. Previous study reported that flavonol induced autophagy 21 . To further investigate whether autophagy is involved in the neuroprotection of kaempferol and kaempferide against mutant SOD1-related neurotoxicity, western blot analysis was performed using LC3 antibody, a selective marker of autophagy. Western blot analysis showed that kaempferol, but not kaempferide, enhanced the formation of LC3-II (Fig. 5A,B). Western blot analysis was also performed using p62 antibody, another selective marker of autophagy. As shown in Fig. 5C-E, the expression of LC-3-II/LC-3-I was increased while that of p62 was decreased after treatment with kaempferol. In addition, the change of LC-3-II/LC-3-I and p62 levels was improved by 3-methyladenine (3-MA), an inhibitor of autophagy, indicating the autophagy is activated by kaempferol. To confirm the relationship between neuroprotective effect of kaempferol against mutant SOD1-mediated toxicity and induction of autophagy, the MTT assay was performed with 3-MA. The protective effect of kaempferol was significantly abolished by 3-MA treatment (Fig. 5F). In addition, immunofluorescence staining showed that the reduction of intracellular aggregates of SOD1 G85R caused by kaempferol was significantly abolished by 3-MA treatment (Fig. 5G,H). These results suggest that kaempferol, but not kaempferide, reduced intracellular aggregate via the activation of autophagy and then inhibited mutant SOD1-induced neurotoxicity.
Kaempferol increases AMPK phosphorylation. Previous study reported that flavonol inhibited the mammalian target of rapamycin (mTOR) complex, which suppresses autophagy 22 . However, the mechanisms through which kaempferol inhibits mTOR are still unclear. To identify the signal transduction pathway mediated by kaempferol, we investigated AMPK and AKT pathways. AMPK inactivates mTOR, and AKT induced mTOR activity. Kaempferol significantly inhibited mTOR phosphorylation (Fig. 6A,B) and significantly increased AMPK phosphorylation (Fig. 6A,C). On the other hand, AKT phosphorylation was not affected by kaempferol (Fig. 6A,D). These results suggest that kaempferol potentially induces autophagy via the AMPK-mTOR pathway.

Discussion
Many studies have been conducted to elucidate the health benefits and the mechanisms of EBGP, including studies on its effects on bacteria 9 , viruses 10 , inflammation 11 , and cancer 12 . However, little has been reported on effects of EBGP for neurodegenerative disease such as ALS. Dominant mutations in SOD1 are frequent cause of inherited ALS 1 . Misfolded and aggregated SOD1 is present in both sporadic and familial ALS, although its pathobiology remains unclear 25,26 . A reduction of misfolded and aggregated SOD1 might be one of potential therapeutic approaches for ALS 17 . Therefore, in the present study, we examine whether EBGP have neuroprotective effect against SOD1 G85R -induced neurotoxicity in a cellular model for ALS. EBGP prevented SOD1 G85R -induced neurotoxicity. Brazilian green propolis has been reported to contain many biological active compounds 27 . There results suggest that the active components of EBGP play an important for the neuroprotection against SOD1 G85R -induced neurotoxicity.
Propolis is collected by honeybees from various plants. There are different types of propolis according to its botanical origin and geographical region. Brazilian green propolis has been reported to contain many biological active compounds, including cinnamic acid derivatives (e.g., artepillin C, baccharin, drupanin, and p-coumaric acid) and flavonoids (e.g., kaempferol, and kaempferide) 20,27 . Flavonoids are considered as an important part of the anti-oxidants in food. Kaempferol and kaempferide are known to have an anti-oxidative activity 23 . In addition, previous study reported that kaempferol induced autophagy, although the mechanism is unclear 21,22 . Therefore, because oxidative stress and autophagy are well recognized as an important pathogenic event in ALS, we chose two flavonoids, kaempferol and kaempferide among these components in Brazilian green propolis, and examined effects of these flavonoids against mutant SOD1-related toxicity.
In the present study, separation of protein fraction by using triton X-100 was performed according to published protocols 17,18 . We also performed immunocytochemical assay based on previous studies 8, [17][18][19] . In addition, previous studies have shown that progressive accumulation of mutant SOD1, which is correlated with a decrease of its solubility in triton X-100, is observed in the spinal cord of SOD1 tg mice, and evident aggregates of SOD1 are found at a late stage of the disease in SOD1 tg mice 28,29 . Considering the evidence of these previous studies, we selected the triton X-100 as a detergent in the present study. Numerous studies have identified significant mitochondrial abnormalities in mutant SOD1-associated ALS 24,30,31 . In addition, mutant SOD1 forms insoluble aggregates in mitochondria at the surface of outer membrane, raising the prospect of a direct cause-effect mechanism by which mutant SOD1 impact mitochondrial function 32,33 . Taken together, these studies provide reasonable evidence for the involvement of mitochondrial-produced oxidants and mitochondrial oxidative damages in mutant SOD1-related ALS. The cause and effect of increased mitochondrial produced ROS in mutant SOD1-induced mitochondrial dysfunction requires further study. However, the present results herein showing that the active components such as kaempferol and kaempferide of Brazilian green propolis inhibited mutant SOD1-induced superoxide suggest that Brazilian green propolis and the active components might be utilized to promote the therapeutic strategy for ALS.
The propensity of a flavonoid to inhibit free-radical mediated events is governed of the chemical structure. Previous study showed that flavonoids with 3-hydroxy (OH) group play a positive role in anti-oxidative activity 23 . Kaempferol and kaempferide are contained in 3-OH group. Therefore, the chemical structure of flavonoid having the 3-OH group may contribute to the anti-oxidative activity against mutant SOD1-induced ROS. On the other hand, kaempferol, but not kaempferide, induces autophagy via the AMPK-mTOR pathway in the present study. The difference in the chemical structure of flavonoid is only functional group in the B ring between Scientific RepoRts | 7: 2882 | DOI:10.1038/s41598-017-03115-y kaempferol and kaempferide. Kaempferol and kaempferide frame with the B ring 4′-OH group and 4′-methoxy group, respectively (Fig. 3). There are differences in beneficial actions such as anti-oxidative activity according to  23 . Therefore, kaempferol having 4′-OH group in the B ring may be related to the autophagy, although further studies are needed to elucidate the relation between the chemical structure of flavonoid and the autophagy. On the other hand, to the best of our knowledge, there is no report that flavonoids including kaempferol and kaempferide bind to mutant SOD-1 like a chemical chaperone. In the present study, it is unknown whether kaempferol and kaempferide directly bind to mutant SOD1. It remains in future study. Further research is required to provide more detail of interaction experiments using recombinant mutant SOD1.
In the present study, kaempferol increased AMPK phosphorylation to inhibit mTOR phosphorylation, and subsequent induced autophagy. Previous study demonstrated that AMPK was inactivated both in the in vitro and in vivo models of SOD1-associated ALS and that the reduced activation of AMPK by mutant SOD1 was recovered by cystain C treatment 17 . In addition, resveratrol protective effects were associated with increased expression and activation of AMPK in spinal cords of SOD1 G93A mice 32 . Moreover, riluzole, an approved ALS drug, leads to AMPK phosphorylation 33 . However, several previous studies demonstrated that the reduced activated of AMPK improved the mutant SOD1-induced motor neuron death in vitro and the motor activity of neurons in vivo 34 . These results suggest that an imbalance in energy metabolism should be considered an important factor in both progression and potential treatment of ALS and that AMPK play a key role in the disease. Therefore, kaempferol as well as EBGP containing the active ingredient against mutant SOD1-mediated toxicity will be an efficient source of new treatments and prevention for ALS.
N2a cells were maintained in Dulbecco's modified Eagle medium (DMEM, Wako Pure Chemical Industries, Ltd., Osaka Japan) containing 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Inc., Waltham, MA, USA) under a humidified atmosphere of 5% CO 2 at 37 °C. The cells were passaged by trypsinization every 3-4 days. Transient expression of each plasmid in N2a cells was accomplished with Lipofectamine 2000 according to the manufacturer's protocol (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Aggregation rate analysis. After 24 h of transfection of each vector to N2a cells, the cells were treated with or without EBGP (a gift from the Api Co., Ltd., Gifu, Japan), kaempferol (Wako Pure Chemical Industries Ltd., Osaka, Japan), or kaempferide (INDOFINE Chemical Company) in the presence or absence of 3-methyladenine (3-MA) for 24 h. Subsequently, the cells were washed with PBS for 5 min twice and fixed with 4% paraformaldehyde for 15 min. Fluorescent microscopy images were acquired using a confocal fluorescence microscope (LSM700, Carl Zeiss, Jena, Germany) and the images were analyzed using ZEN software (Carl Zeiss). Aggregates were defined as bright non-homogeneities with the cytoplasm. To quantify aggregate formation, the intensity of the even and diffuse fluorescence was set as threshold and the number of cells in each field showing fluorescence intensities over the threshold was counted using imaging software. Following the threshold analysis, the presence of aggregates was confirmed by eye in each field, based on previous studies 8, 17-19 . Western blot analysis. After 24 h of transfection to N2a cells each vector, the cells were treated with or without EBGP, kaempferol, or kaempferide. The cells were lysed with TNE lysis buffer (50 mM Tris-HCl (pH. 7.4), 150 mM NaCl, 1 mM ethylenediamineteraacetic acid, protease inhibitor cocktail) containing 1% Triton-X and then were centrifuged at 15,000 × g for 5 min at 4 °C. The supernatant of the lysate was defined as Triton soluble fraction. After centrifuge, the remaining deposition was resuspended with TNE lysis buffer containing 2% sodium dodecyl sulfate (SDS) (defined as Triton-insoluble fraction). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific Inc.) with bovine serum albumin (BSA) as a standard. Lysates were mixed with sample buffer containing 10% 2-mercaptoethanol, and subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The 20 µg of protein was applied to SDS-PAGE in order to separate proteins as certain molecular weight. SDS-PAGE was performed under constant voltage at 200 V at room temperature for 40 min. The separated proteins in poly acrylamide gel were transferred to PVDF membrane in transfer buffer (0.3% Tris, 1.44% glycine, 20% methanol) under constant voltage at 100 V at 4 °C for 90 min. The membranes were incubated with 5% BSA (Wako) for 60 min, and then with following primary antibodies for overnight: mouse (1:1000, Cell Signaling Technology). After the primary antibody reaction, membrane was incubated in the second antibody (goat anti-rabbit antibody conjugated with HRP (1:2500, Santa Cruz Biotechnology), goat anti-mouse HRP antibody conjugated with HRP (1:2500, Santa Cruz Biotechnology)). The membrane was incubated in ECL prime (GE Healthcare, Buckinghamshire, UK) to generate the chemiluminescence from HRP antibodies. The chemiluminescence was detected by using LAS3000 mini (Fuji film, Tokyo, Japan). The band density was measured by ImageJ software (NIH, New York, NY, USA). Neurotoxicity assays. N2a cells were seeded at 2.0 × 10 5 cells/ml in 96-well plates in DMEM containing 10% FBS. After transfection of each plasmid, the cells were differentiation for 48 h in low glucose (1.0 g/l glucose) DMEM supplemented with 2% FBS and 2 mM N,N-dibutyladenosine 3′,5′-phosphoric acid (dbcAMP; Nacalai tesque, Kyoto, Japan) with or without EBGP, kaempherol, or kaempheride in the presence or absence of 3-MA. The number of live cells was estimated by Cell Counting Kit-8, following the manufacturer's instructions (Wako Pure Chemical Industries Ltd.). Briefly, reagent was added into wells and the plate was incubated at 37 °C for 4 h.
The optical density of formazan was detected at 450 nm by GloMax ® (Promega, Madison, WI, USA) for calculating cell viability. The wavelength of 600 nm was used as reference.
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 10 min. And cells were permeabilized with 0.1% tritonX/PBS for 30 min at RT. The 2% goat serum was used for blocking reaction for 60 min. Samples were incubated with primary antibody (rabbit anti-SOD1 antibody, 1:150, Enzo Life Sciences, Inc, Farmingdale, NY, USA) at 4 °C overnight. And we incubated cells with second antibody (goat anti-mouse antibody Alexia 555, (Thermo Fisher Scientific)) at RT for 30 min. Samples were observed by using confocal imaging system (Zeiss LSM 700).

ROS production.
To detect mutant SOD1 induced ROS production, we used a Red mitochondrial superoxide indicator, MitoSOX ® Red (Thermo Fisher Scientific). N2a cells were prepared in uncoated glass-bottomed microwells. After transfection of each plasmid, MitoSOX ® Red was added to the cell culture to a final concentration of 5 μM to the cells and incubate for 30 min at 37 °C following the manufacturer's instructions (Thermo Fisher Scientific). Fluorescent microscopy images were acquired using a confocal fluorescence microscope (LSM700, Carl Zeiss) and the images were analyzed using ZEN software (Carl Zeiss).
Statistical analysis. Data are given as the mean ± standard error of the mean (SEM). The significance of differences was determined by Student's t test or an analysis of variance (ANOVA). Further statistical analysis for post hoc comparisons was performed using the Bonferroni/Dunn test (StatView, Abacus, Baltimore, MD, USA). A p-value of less than 0.05 was considered to be statistically significant.