Abstract
The human β-amyloid (Aβ) cleaving enzyme (BACE-1) is a target for Alzheimer’s disease (AD) treatments. This study was conducted to determine if acacetin extracted from the whole Agastache rugosa plant had anti-BACE-1 and behavioral activities in Drosophila melanogaster AD models and to determine acacetin’s mechanism of action. Acacetin (100, 300 and 500 μM) rescued amyloid precursor protein (APP)/BACE1-expressing flies and kept them from developing both eye morphology (dark deposits, ommatidial collapse and fusion and the absence of ommatidial bristles) and behavioral (motor abnormalities) defects. The reverse transcription polymerase chain reaction analysis revealed that acacetin reduced both the human APP and BACE-1 mRNA levels in the transgenic flies, suggesting that it plays an important role in the transcriptional regulation of human BACE-1 and APP. Western blot analysis revealed that acacetin reduced Aβ production by interfering with BACE-1 activity and APP synthesis, resulting in a decrease in the levels of the APP carboxy-terminal fragments and the APP intracellular domain. Therefore, the protective effect of acacetin on Aβ production is mediated by transcriptional regulation of BACE-1 and APP, resulting in decreased APP protein expression and BACE-1 activity. Acacetin also inhibited APP synthesis, resulting in a decrease in the number of amyloid plaques.
Similar content being viewed by others
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder and the most prevalent form of dementia in developed and developing countries. Worldwide, the number of people with dementia was estimated to be 35.6 million in 2010; this figure is expected to reach 115.4 million by 20501. The worldwide societal cost of dementia, including direct and indirect care, was an estimated 604 billion USD in 2010, an increase of 288.6 billion from 315.4 billion USD in 20052. Fourteen and 40% of the people in low- and middle-income countries have dementia, respectively, which accounts for less than 1% and 10% of the total worldwide costs, respectively. However, 46% of the people in high-income countries have dementia, accounting for 89% of the costs2. AD accounts for 60–70% of dementia cases3,4. The major histopathological hallmarks of AD are neurofibrillary tangles composed of hyperphosphorylated tau protein filaments and extracellular senile plaques, which are deposits of β-amyloid (Aβ) generated via sequential proteolytic processing of the transmembrane amyloid precursor protein (APP) by two enzymes in the amyloidogenic processing pathway, namely β-secretase (β-site APP cleaving enzyme or BACE-1) and γ-secretase5,6,7.
AD is currently treated using acetylcholinesterase (AChE) inhibitors8 and N-methyl-D-aspartate (NMDA) receptor antagonists9,10. However, these treatments do not stop the disease process or prevent neuronal degeneration11. These treatments also have serious side effects9,12. BACE-1 is considered a primary target for preventing and treating AD13,14,15. Many peptidomimetics and heterocyclic compounds have been evaluated as BACE-1 inhibitors16,17,18; however, none of these have been successfully developed as anti-AD drugs. Therefore, there is a pressing need to develop new improved anti-AD agents.
Natural compounds from plant extracts have been suggested as alternative sources for anti-AD drugs. This approach is appealing, in part, because plants are sources of bioactive secondary metabolites that are perceived by the general public as relatively safe and that often act on multiple, novel target sites19,20. Certain plant preparations and their constituents are regarded as potential sources for commercial anti-AD products to prevent or treat AD. Plant-derived BACE-1 and AChE inhibitors have been well documented by Orhan18 and Mukherjee et al.21, respectively. Previous studies have shown that a methanol extract from the whole blue licorice (Korean mint) plant, Agastache rugosa (Fisch. & C.A. Mey.) O. Kuntze (Lamiaceae), possessed human BACE-1 inhibitory activity. Historically, this plant species has been used to treat cholera, vomiting and miasma22. However, no previous studies have investigated the potential use of A. rugosa for managing AD, although the phytochemistry and bioactivity of plants in the genus Agastache have been well documented by Zielinska and Matkowski22.
In this study, our aim was to assess whether the flavonoid, acacetin and two triterpenoids, maslinic acid and oleanolic acid, extracted from the whole A. rugosa plant had BACE-1 inhibitory activity in comparison with commercial organic pure acacetin and two positive controls, the cell-permeable isophthalamide, BACE-1 inhibitor IV23 and the natural BACE-1 inhibitor, epigallocatechin gallate (EGCG)24, using a fluorescence resonance energy transfer (FRET)-based enzyme assay. The effects of the most potent inhibitory constituent, acacetin, on the eclosion rate, feeding, climbing and life span of a Drosophila melanogaster mutant that co-expresses human APP and BACE-1 within the developing nervous system were evaluated. In addition, the morphological changes in the compound eyes of the transgenic flies were examined using light microscopy and scanning electron microscopy (SEM). Finally, the possible mechanism underlying the anti-AD actions of acacetin was elucidated using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses.
Results
Fluorescence resonance energy transfer-based enzyme assay-guided fractionation and isolation
The fractions obtained from solvent partitioning of the methanol extract of the whole A. rugosa plant were tested for human BACE-1inhibitory activity using a FRET-based enzyme assay (Table 1). Significant differences in inhibitory activity were observed among the fractions and were used to identify the peak activity fractions for the next step of purification. At a concentration of 1 mg/mL, the hexane-soluble fraction was the most potent inhibitor, while no inhibition was obtained using the chloroform-, ethyl acetate-, butanol-, or water-soluble fractions.
FRET-based enzyme assay-guided fractionation of the whole A. rugosa plant afforded three active compounds that were identified by spectroscopic analyses, including electron ionized mass spectrometry (EI-MS) and nuclear magnetic resonance (NMR) spectroscopy. The three BACE-1 inhibitory compounds were maslinic acid (1), oleanolic acid (2) and acacetin (3) (Fig. 1). Maslinic acid (1) was identified based on the following evidence: a white crystal; Ultraviolet (UV) (MeOH): λmax nm = 217; EI-MS (70 eV), m/z (% relative intensity): 472 [M]+ (1.9), 284 (3.0), 256 (2.8), 248 (100), 233 (8.6), 203 (73.5), 189 (8.1), 173 (3.4), 133 (10.2), 105 (5.1), 95 (4.4), 69 (6.2), 55 (5.3) (see Supplementary Fig. S1 online); 1H NMR (MeOD, 600 MHz): δ 0.79 (3H, s), 0.86 (3H, s), 0.87 (3H, s), 0.94 (3H, s), 0.99 (3H, s), 1.10 (3H, s), 1.13 (3H, s), 2.89 (1H, m), 2.98 (1H, d, J = 9.54 Hz), 3.61 (1H, ddd, J = 4.50, 2.94, 3.90 Hz), 5.21 (1H, t, J = 6.72 Hz) (see Supplementary Fig. S2 online); and 13C NMR (MeOD, 150 MHz): δ 17.2 q, 17.6 q, 18.3 q, 19.8 t, 24.5 q, 24.7 q, 24.8 t, 26.6 t, 29.4 t, 29.5 q, 30.9 s, 31.9 q, 34.1 t, 34.2 t, 34.5 t, 39.5 s, 40.6 s, 40.7 s, 43.2 s, 43.8 d, 48.3 t, 48.4 s, 48.7 t, 49.0 d, 56.9 d, 69.7 d, 84.7 d, 122.5 d, 146.9 s, 185.8 s (see Supplementary Fig. S3 online). Oleanolic acid (2) was characterized as follows: a white amorphous powder; UV (MeOH): λmax nm = 216; EI-MS (70 eV), m/z (% relative intensity): 456 [M]+ (4.3), 249 (18.8), 248 (100), 233 (6.0), 207 (17.3), 204 (11.3), 203 (59.7), 190 (10.0), 189 (10.6), 175 (7.4), 133 (15.0), 105 (6.7), 81 (6.1), 69 (8.0), 55 (7.6) (see Supplementary Fig. S4 online); 1H NMR (MeOD, 600 MHz): δ 0.77 (3H, s), 0.85 (3H, s), 0.90 (3H, s), 0.95 (3H, s), 0.97 (3H, s), 1.12 (3H, s), 1.15 (3H, s). 1.38 (2H, m), 1.41 (2H, m), 1.56 (4H, m), 2.20 (3H, m), 2.85 (1H, dd, J = 9.9, 4.5 Hz), 3.15 (1H, dd, J = 15.78, 4.4 Hz), 3.17 (1H, t, J = 14.76 Hz), 3.34 (2H, s), 4.62 (2H, s), 5.23 (1H, s) (see Supplementary Fig. S5 online); and 13C NMR (MeOD, 150 MHz): δ 16.0 q, 16.5 q, 16.5 q, 17.9 t, 21.7 t, 24.2 q, 24.3 t, 25.5 q, 26.5 t, 28.0 t, 28.9 q, 31.8 s, 33.8 t, 34.0 t, 34.2 q, 34.5 t, 35.1 s, 38.2 t, 38.3 s, 40.0 s, 40.7 d, 42.9 s, 43.0 s, 47.5 t, 47.9 d, 56.9 d, 79.8 d, 123.7 d, 145.5 s, 182.4 s (see Supplementary Fig. S6 online). Acacetin (3) was characterized as follows: yellow needles; UV (MeOH): λmax nm = 269, 315; EI-MS (70 eV), m/z (% relative intensity): 284 [M]+ (100), 283 (12.5), 241 (11.4), 152 (6.9), 132 (18) (see Supplementary Fig. S7 online); High resolution EI-MS: C16H12O5 observed: 284.0683, calculated: 284.0684; Fourier transform infrared spectroscopy (FT-IR) νmax cm–1: 3147 (-OH), 1651 (−C=O), 1605, 1560, 1503, 1428 (-C=C) (see Supplementary Fig. S8 online); 1H NMR (DMSO-d6, 600 MHz): δ 3.16 (3H, s), 6.20 (1H, d, J = 1.98 Hz), 6.51 (1H, d, J = 2.04 Hz), 6.87 (1H, s), 7.11 (2H, d, J = 8.88 Hz), 8.04 (2H, d, J = 8.88 Hz), 10.85 (1H, s), 12.92 (1H, s) (see Supplementary Fig. S9 online); and 13C NMR (DMSO-d6, 150 MHz): δ 55. 6 q, 94.0 d, 98.9 d, 103.5 d, 103.8 s, 114.6 d, 114.6 d, 122. 8 s, 128.3 d, 128.3 d, 157.3 s, 161.4 s, 162.3 s, 163.3 s, 164.2 s, 181.8 s (see Supplementary Fig. S10 online). The interpretations of the proton and carbon signals of compounds 1, 2 and 3 were largely consistent with those of Tanaka et al.25 and Dam et al.26, Hossain and Ismail27 and Gangwal et al.28 and Wawer and Zielinska29 and Miyazawa and Hisama30, respectively.
Structures of maslinic acid, oleanolic acid and acacetin.
These compounds were identified in the whole Agastache rugosa plants in this study. The chemical formula of maslinic acid (1) is C30H48O4, with a molar mass of 472.70 g/mol; the chemical formula of oleanolic acid (2) is C30H48O3, with a molar mass of 456.70 g/mol; and the chemical formula of acacetin (3) is C16H12O5, with a molar mass of 284.26 g/mol.
In vitro BACE-1 inhibitory activity of the isolated compounds
The BACE-1 inhibitory activity of the three isolated compounds (acacetin, maslinic acid and oleanolic acid), organic pure acacetin and human BACE-1 inhibitor IV and EGCG, which were used as positive controls, were elucidated (Table 2). Based on the IC50 values, natural and pure organic acacetin had similar inhibitory activity, indicating that the activity of the methanol-extracted acacetin was purely due to acacetin. Natural acacetin was a 4.0-fold and 5.5-fold more potent inhibitor of BACE-1 than oleanolic acid and maslinic acid, respectively. The BACE-1 inhibitory activity of acacetin and EGCG did not differ significantly. Overall, these compounds were significantly less potent inhibitors of BACE-1 than BACE-1 inhibitor IV.
Effect of acacetin on age-dependent neurodegeneration, as reflected by an aberrant eye phenotype
Overexpression of human BACE-1 and APP during Drosophila eye development resulted in an aberrant rough eye phenotype in the male GMR < APP/BACE-1 flies (1-day-old) (Fig. 2B,C), particularly those with dark deposits in the eye (marked with an arrow in Fig. 2C), compared to the control male GMR-GAL4/+ flies with normal and well-organized compound eyes (Fig. 2A). Similar results were also observed with the transgenic female GMR < APP/BACE-1 flies. The effects of acacetin on the morphological defects in the eyes with dark deposits were examined (Fig. 2D). Acacetin (100, 300 and 500 μM) suppressed the ratios of female flies (6.3–8.0%) and male flies (5.0–7.0%) with dark deposits in the eyes compared to the vehicle dimethyl sulfoxide (DMSO)-fed control females (12.0%) and males (18.7%).
Effect of acacetin on the morphological defects in the compound eyes of the transgenic flies.
Human APP and BACE-1 transgenic flies (GMR < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. The morphology of the compound eyes of male and female flies (1 day old) was observed with a stereo microscope (×35). The flies with aberrant eye phenotypes were collected and the number of flies with dark deposits in the eyes was counted. (A) Control male GMR-Gal4 flies with a wild-type phenotype of a smooth surface compound eye. (B) Transgenic male GMR < APP/BACE-1 flies with a compound eye with an abnormal appearance. (C) Male GMR < APP/BACE-1 flies with morphological defects, particularly dark deposits in the eyes. (D) Quantitation of the phenotype of male and female flies showing serious morphological defects with dark deposits in the eyes. Each bar represents the mean ± SE from three independent experiments (***P < 0.001; ns, no significant difference, using Bonferroni’s multiple comparison test). The scale bars represent 20 μm.
It has been reported that co-expression of the human APP and BACE-1 genes induced the age-dependent neurodegeneration of the photoreceptor cells in Drosophila compound eyes31. To evaluate whether acacetin affected eye degeneration over time, the morphological changes in the eyes were first investigated in flies of different ages (1, 10, 20 and 30 days old). The control strain carrying GMR-GAL4 alone showed a normal, well-organized and smooth external eye surface and there were no obvious morphological changes as time elapsed (Fig. 3A1–A4). In contrast, external phenotypic changes were observed in flies co-expressing human APP and BACE-1 and these changes became more severe as time elapsed (Fig. 3B–E). In vehicle-fed flies, dark deposits were observed at the edges of the compound eyes, even in 1-day-old flies (marked with an arrow in Fig. 3B1) and the size of the deposits had increased in 10-day-old flies (marked with an arrow in Fig. 3B2). As time elapsed, the flies with dark spots in their eyes died earlier than those without the dark spots. Furthermore, the number of ommatidial fusions increased in 20-day-old fly eyes compared to the 1-day-old fly eyes (marked with asterisks in Fig. 3B3) and were further exacerbated in 30-day-old fly eyes (marked with asterisks in Fig. 3B4).
Effect of acacetin on the aberrant eye phenotype associated with age-dependent eye development.
Human APP and BACE-1 transgenic flies (GMR < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. Light micrographs. (A) The control male GMR-GAL4/+ flies (1, 10, 20 and 30 days old) had a normal and well-organized eye morphology. There were no obvious morphological changes as time elapsed. (B) The vehicle-fed male GMR < APP/BACE-1 flies exhibited dark deposits at the edge of the compound eye, even at the youngest age evaluated (marked with an arrow in Fig. 3B1); the size of the deposits had increased at 10 days (marked with an arrow in Fig. 3B2). As time passed, the flies with dark spots in their eyes tended to die earlier than the control flies and more ommatidial fusions were observed in 20-day-old fly eyes with deposits (marked with asterisks in Fig. 3B3), with further exacerbation of these fusions in 30-day-old fly eyes (marked with asterisks in Fig. 3B4). (C–E) Acacetin partially suppressed the morphological defects in the eyes. Scanning electron micrographs. (F1,G1) Thirty-day-old male GMR-GAL4/+ flies with smooth surface compound eyes, without any defects in the ommatidia size, ommatidial bristles, or fused ommatidia. (F2,G2) Vehicle-fed male GMR < APP/BACE-1 flies (30 days old) with abnormal phenotypes, characterized by the collapse (marked with arrows) and fusion of ommatidia (marked with arrowheads) and the absence of ommatidial bristles (marked with an asterisk). (F3–5,G3–5) Acacetin partially suppressed these eye defects. The scale bars represent 20 μm.
Next, GMR < APP/BACE-1 flies in the egg stage were cultured in media supplemented with acacetin (100, 300 and 500 μM) dissolved in 0.1% DMSO. Acacetin suppressed the numbers of flies with dark spots in their eyes (Fig. 2) at 1 day, although there were no obvious external structural changes in acacetin-fed fly eyes at 1 and 10 days old (Fig. 3C1–E1). However, the visible eye color changed from light red to dark red in 20- or 30-day-old flies (Fig. 3C3–E3). Furthermore, the compound eyes in these older flies had a rougher surface and more severe ommatidial fusions than those in the younger flies; this was especially pronounced for the 30-day-old flies (marked with asterisks in Fig. 3C4–E4). At concentrations of 100, 300 and 500 μM, acacetin significantly suppressed ommatidial fusion in the central side of the flies’ eyes compared to the vehicle-fed flies (marked with an asterisk in Fig. 3B3–E3). In particular, 300 and 500 μM acacetin ameliorated ommatidial fusion at the edges of the fly eyes. Although acacetin did not completely suppress the morphological changes in the eyes, it delayed this age-dependent degeneration progress.
The protective effects of acacetin on the compound eyes of the male GMR < APP/BACE-1 flies (30 days old) were examined using SEM. The control GMR-GAL4/+ flies had eyes with a smooth appearance, without any defects in the ommatidia size (Fig. 3F1) or ommatidial bristles (Fig. 3G1). In contrast, the transgenic GMR < APP/BACE-1 flies showed varying degrees of eye disorganization. The eyes of the vehicle-fed flies showed the strongest phenotypes; they were characterized by ommatidial collapse (marked with arrows in Fig. 3F2), fused ommatidia (marked with arrowheads in Fig. 3G2), a reduced ommatidia size (marked with an asterisk in Fig. 3G2) and the absence of ommatidial bristles (Fig. 3G2). These eye phenotype defects were partially suppressed in male flies treated with three concentrations of acacetin (100, 300 and 500 μM) (Fig. 3F3, 4, 5,G3, 4, 5). Remarkably, external surface collapse was not observed in flies fed 300 μM acacetin (Fig. 3F4), but there was still some collapse in flies fed 100 and 500 μM acacetin (marked with arrows in Fig. 3F3,F5). Ommatidial external surface fusion was not observed in acacetin-fed flies, but the reduced ommatidia size (marked with asterisks in Fig. 3G3) was observed, irrespective of concentration.
Protective effect of acacetin on age-dependent eye degeneration induced by co-expression of human BACE-1 and APP.
Human APP and BACE-1 transgenic flies (elav < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. Cason’s trichrome staining. (A1,A2) Staining of the photoreceptors of control male GMR-Gal4/+ flies (5 and 15days old). (B1,B2) Staining of the photoreceptors of vehicle-fed transgenic male GMR < APP/BACE-1 flies (5 and 15days old). Remarkable retinal collapse (marked with an asterisk in B1) was observed even in young flies (5 days old) and more severe external surface collapse of the photoreceptors was detected in 15-day-old flies (marked with an arrowhead in B2) compared to 5-day-old flies. (C1-E1) The photoreceptor degeneration was partially suppressed in 5-day-old acacetin-fed transgenic flies. (C2-E2) The external surface collapse of the photoreceptors was significantly suppressed in 15-day-old acacetin-fed flies. Congo red staining. (A3,A4) Amyloid plaques were not observed in the control male GMR-Gal4/+ flies (5 and 15 days old). (B3,B4) Scattered amyloid plaque deposits were apparent in the 5-day-old vehicle-treated transgenic flies (marked with arrows in B3). Many amyloid plaque deposits (marked with an arrowhead in B4) had accumulated in the 15-day-old flies. (C3-E3) Acacetin reduced the number of scattered amyloid plaque deposits in the photoreceptors of 5-day-old transgenic flies. (C4-E4) Acacetin reduced the number of amyloid plaque deposits in the 15-day-old transgenic flies. The images were observed and captured using a stereo-microscope. The scale bars represent 20 μm.
Effect of acacetin on the eclosion rate of the transgenic flies.
Human APP and BACE-1 transgenic flies (elav < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. The third instar larvae climbing on the wall were collected in vials containing fresh media. The numbers of pupae and adults were counted. (A) Co-expression of human BACE-1 and APP as along with acacetin supplementation did not affect the pupation of the male elav-Gal4/+ and elav < APP/BACE-1 flies. (B) Co-expression of human BACE-1 and APP significantly reduced the emergence of the male transgenic flies. Acacetin had no significant effect on the emergence defect. Each bar represents the mean ± SE from four independent experiments (**P < 0.01; ns, no significant difference, using Bonferroni’s multiple comparison test).
Acacetin has a protective effect on age-dependent neurodegeneration of the eye
Male compound eye sections were examined to demonstrate the protective effects of acacetin on the age-dependent neurodegeneration induced by co-expression of human BACE-1 and APP. Photoreceptor degeneration in the transgenic GMR < APP/BACE-1 flies depended on age, but this was not the case for the control GMR-Gal4/+ flies (Fig. 4). The GMR-Gal4/+ flies (5 and 15 days old) had well-organized photoreceptors, without age-dependent phenomena, as determined by Cason’s trichrome staining (Fig. 4A1). In contrast, photoreceptor degeneration was observed in young transgenic flies (Fig. 4B1–E1) and 15-day-old flies (Fig. 4B2–E2). In the vehicle-fed transgenic flies, remarkable retinal collapse was observed, even in young flies (5 days old) (Fig. 4B1) and external surface collapse of the photoreceptors was detected in the 15-day-old flies (marked with an arrowhead in Fig. 4B2). Acacetin (100, 300 and 500 μM) partially suppressed the photoreceptor degeneration in 5- (Fig. 4C1–E1) and 15-day-old flies (Fig. 4C2–E2). In particular, acacetin significantly suppressed the photoreceptor collapse (Fig. 4C2–E2). Although acacetin did not completely suppress photoreceptor degeneration, it delayed this age-dependent process.
Congo red staining was performed to investigate whether amyloid deposition was age-dependent or due to the effects of acacetin on these deposits. Amyloid plaques were not detected in the compound eyes of the control male GMR-Gal4/+ flies (5 and 15 days old) (Fig. 4A3). However, scattered amyloid plaque deposits (marked with arrows in Fig. 4B3) were evident in 5-day-old vehicle-fed transgenic flies and many amyloid plaque deposits (marked with arrows in Fig. 4B4) had accumulated in the 15-day-old flies. Interestingly, Congo red staining revealed that 100 and 300 μM acacetin significantly suppressed the number of amyloid deposits in 5- (Fig. 4C3) and 15-day-old transgenic flies (Fig. 4C4); however, a few amyloid plaque deposits were also detected in 5- (marked with arrows in Fig. 4E3) and 15-day-old transgenic flies (marked with arrows in Fig. 4E4) supplemented with 500 μM acacetin. Although acacetin did not completely suppress amyloid deposition in the flies’ photoreceptors, it reduced the number of amyloid deposits.
Effect of acacetin on eclosion of the transgenic flies
The pupation and emergence rates were examined to determine whether acacetin affected the development of the transgenic flies. Co-expression of human BACE-1 and APP in the nervous system did not affect pupation compared to the control elav-Gal4/+ flies. The acacetin-treated flies showed nearly normal pupation rates, irrespective of the treatment concentration (100, 300 and 500 μM) and these rates were not significantly different from those of the vehicle-fed transgenic flies (Fig. 5A). This could be due to the relatively weak promoter – elav – used to drive human APP and BACE-1 gene expression. However, the emergence rate (76%) of the vehicle-fed elav < APP/BACE-1 flies was significantly different from that (99%) of the elav-Gal4/+ flies (Fig. 5B). This finding indicates that co-expression of human BACE-1 and APP is toxic during fly development, particularly at the emergence stage. Acacetin (100, 300 and 500 μM) did not have a significant effect on the emergence of flies (82–86%) compared to the vehicle.
Effect of acacetin on the flies’ age-dependent motor abnormalities
AD is characterized by age-dependent degeneration in locomotor coordination32. In Drosophila AD models, locomotor coordination can be quantified by the negative geotaxis assay, as described by Wang et al.33 and Crowther and colleagues34. We used male flies because the climbing defects were more pronounced in males than in females. The male elav-Gal4/+ flies showed a clear age-dependent reduction in climbing, with climbing indices of 80, 66, 53 and 34% at 1, 5, 10 and 15 days postemergence, respectively (Fig. 6A–D). In contrast, the vehicle-fed male elav < APP/BACE-1 flies showed severe defects in performance, with climbing indices of 47, 39, 36 and 11% at 1, 5, 10 and 15 days postemergence, respectively (Fig. 6A–D). Acacetin improved the motor abnormalities to approximately 68% as early as 1 day after emergence, irrespective of the concentration (100, 300 and 500 μM) (Fig. 6A). The acacetin treatment resulted in a 60–66% climbing index at 5 days postemergence (Fig. 6B) and a 53–54% climbing index at 10 days postemergence (Fig. 6C). At 15 days postemergence, acacetin significantly ameliorated the climbing defects (climbing index, 27–30%) (Fig. 6D).
Effect of acacetin on the age-dependent climbing behavior of the transgenic flies.
Human APP and BACE-1 transgenic flies (elav < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. Male flies (1, 5, 10 and 15 days old) were placed in empty vials conjoined with another vial on top and manually tapped twice. After 20 s, the flies that climbed from the bottom and crossed the 9.5 cm line were counted and the climbing index was calculated as the number of flies that climbed to the top vial relative to the total number of test flies and expressed as a percentage. Climbing behavior of 1- (A), 5- (B), 10- (C) and 15-day-old male flies (D). Co-expression of human BACE-1 and APP significantly decreased the flies’ climbing activities compared to the control elav-Gal4/+ flies. Acacetin ameliorated the climbing defects of the elav < APP/BACE-1 flies compared to the vehicle-fed flies, irrespective of concentration. Each bar represents the mean ± SE from three independent experiments (***P < 0.001; **P < 0.01; *P < 0.05; ns, no significant difference, using Bonferroni’s multiple comparison test).
Effect of acacetin on fly feeding and longevity
A feeding assay was used as an index to quantify the effect of different concentrations of the active compound on the amount of food consumed by the flies35,36. The effects of acacetin on food intake were examined in transgenic male elav < APP/BACE-1 flies. Acacetin did not significantly affect feeding by transgenic flies, irrespective of the concentration (100, 300 and 500 μM) (Fig. 7A), indicating that any changes in the flies’ behaviors were due to the ingestion of compounds by food intake rather than the effect of acacetin on their appetite.
Effect of acacetin on the feeding and longevity of the transgenic flies.
Human APP and BACE-1 transgenic flies (elav < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO to determine the lifespan and median life time (T1/2). (A) To determine feeding behavior, newly emerged transgenic male flies were cultured on standard media for 3 days and then starved for 20 h. The flies were transferred into vials containing media (with 0.2% Acid red) supplemented with acacetin. The control flies were fed with media containing 0.2% Acid red and vehicle. The abdomens of flies fed with Acid red were cut and homogenized in distilled water and the optical density (OD) of the supernatant was measured at 505 nm as the index of amount of food consumed by the flies. Acacetin did not affect the feeding behavior of the flies. (B) Acacetin did not significantly prolong the transgenic flies’ lifespan compared to the vehicle-fed male transgenic and elav-Gal4/+ flies. (C) Co-expression of human BACE-1 and APP reduced the T1/2 of the male flies compared to that of the control male elav-Gal4/+ flies. Supplementing the diet with 100 μM acacetin only significantly extended the T1/2 of the male elav < APP/BACE-1 flies. Each bar represents the mean ± SE from 10 independent experiments for longevity and from three independent experiments for feeding (***P < 0.001; **P < 0.01; ns, no significant difference, using Bonferroni’s multiple comparison test).
The median life time (T1/2) is a more credible measurement of lifespan than the mean survival time37. We investigated the effects of acacetin on the lifespan and T1/2 of our Drosophila AD model, because it was previously reported that co-expression of BACE-1 and APP reduced the lifespan of the adult flies37. Acacetin (100, 300 and 500 μM) did not significantly increase the lifespan of the male elav < APP/BACE-1 flies compared to the vehicle-fed transgenic flies and control elav-Gal4/+ flies (Fig. 7B). However, co-expression of human BACE-1 and APP markedly reduced the T1/2 (26 days) of vehicle-fed flies compared to the elav-Gal4/+ flies (T1/2, 36 days) (Fig. 7C). Treatment of transgenic flies with 100 μM acacetin increased the T1/2 (31 days), while 300 and 500 μM acacetin did not significantly extend the T1/2. These findings suggest that the lowest concentration of acacetin (100 μM) has an effect on the T1/2 of elav < APP/BACE-1 flies; however, this is limited to young adults. Taken together, these data suggest that BACE-1 may not have a major function in the survival of elav < APP/BACE-1 flies, which is consistent with the conclusions of a previous study37.
Effect of acacetin on the human APP and BACE-1 mRNA levels
Many studies have focused on APP proteolysis and Aβ generation as potential targets for AD therapy38 and APP inhibitors are also used to lower the Aβ peptide levels, as described by Utsuki et al.39. To investigate whether acacetin affected the transcription of the human BACE-1 and APP genes, we analyzed the APP and BACE-1 mRNA levels in male elav < APP/BACE-1 flies (20 days old) using real-time qRT-PCR. An active compound could reduce the human APP and BACE-1 mRNA levels by indirectly inhibiting the elav promoter. To investigate this, we used flies with a UAS-GFP sequence driven by the elav promoter and treated them with acacetin. We also quantified the levels of the GFP mRNA. There were no significant differences in the GFP mRNA levels between the vehicle-fed and acacetin-fed flies (Fig. 8A). However, a significant reduction in the APP mRNA levels (69–82%) was observed in the male elav < APP/BACE-1 flies following treatment with acacetin (100, 300 and 500 μM) compared to the vehicle-treated flies (Fig. 8B). Similarly, the human BACE-1 mRNA levels were reduced to 82–89% of the control levels following acacetin treatment (Fig. 8C). Taken together, these data indicate that acacetin had no significant effect on the GFP mRNA levels, but decreased the human APP and BACE-1 mRNA levels rather than indirectly inhibiting the elav promoter.
Effect of acacetin on the expression of the human BACE-1 and APP mRNAs.
The total RNA was extracted from 30 heads (30 mg) of elav < APP/BACE-1 and elav < GFP flies (20 days old) cultured from the egg stage in polystyrene vials containing media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. Real-time qRT-PCR was performed to determine the levels of the BACE-1 and APP mRNAs. Specific BACE-1, APP and ribosomal protein 49 (rp 49) coding sequence primers were used to amplify the BACE-1, APP and rp 49 cDNAs, as described in the Methods section. (A) The elav < GFP flies were used as control flies to confirm that the effects of acacetin to reduce the APP and BACE-1 levels were not due to its inhibitory activity toward the Gal4 transcription activator. Acacetin had no significant effects on GFP mRNA expression, irrespective of concentration. (B) Acacetin significantly reduced the human APP mRNA levels. (C) Acacetin significantly reduced human BACE-1 mRNA expression in 20-day-old male flies. The mRNA expression was normalized to the constitutive expression of the mRNA for the housekeeping gene, rp 49 and analyzed by the 2–ΔΔCT method. Each bar represents the mean ± SE of duplicate samples run in three independent experiments (***P < 0.001; *P < 0.05; ns, no significant difference, using Bonferroni’s multiple comparison test).
Acacetin significantly reduces the Aβ levels by interfering with human APP proteolytic processing and BACE-1 expression
A western blot analysis revealed that acacetin suppressed Aβ expression, although the responses varied according to concentration. Aβ (approximately 4 kDa) was not detected in the control elav-Gal4/+ flies (Fig. 9A). Quantification of the western blots showed that 100, 300 and 500 μM acacetin decreased the Aβ levels to 71, 72 and 47% of the controls, respectively (Fig. 9B). To determine whether the anti-amyloidogenic effect of acacetin was mediated by modulating APP proteolytic processing or BACE-1 expression, the protein levels of BACE-1 and APP were analyzed. A band corresponding to BACE-1 (approximately 70 kDa) was visible in the transgenic flies, but was not detected in the control elav-Gal4/+ flies. Treatment with 100, 300 and 500 μM acacetin decreased the expression of BACE-1 (Fig. 9C). Quantification of the western blots indicated that the BACE-1 protein levels were considerably decreased to 81, 75 and 74% of the control levels using 100, 300 and 500 μM acacetin, respectively (Fig. 9D). In this experiment, the APP monomer (approximately 100 kDa) and dimer bands (approximately 200 kDa) were confirmed by the western blots, as described by Jung et al.40 and were also visible in the control elav-Gal4/+ flies, which is consistent with the previous study by Groth et al.41 (Fig. 9E). Quantification of the western blots indicated that the APP levels were also significantly reduced to 70 and 69% of the control levels by 300 and 500 μM acacetin, respectively, but 100 μM acacetin reduced the APP levels to 86%, which was not significantly different from the control APP levels (Fig. 9F). We also analyzed the effects of acacetin on the formation of the APP-alpha C-terminal fragment (αCTF) (14 kDa, marked with the green arrow in Fig. 9G) and APP-βCTF (14.5 kDa, marked with the red arrow in Fig. 9G), because the level of the APP-CTF is useful for understanding how genetic manipulation of APP processing impacts Aβ generation and accumulation. Acacetin inhibited the generation of the APP-CTF by affecting APP cleavage (approximately 80% of the control levels) (Fig. 9H). The APP intracellular domain (AICD) fragment has been shown to be involved in a variety of signaling processing, many of which are potentially relevant to AD pathology and the AICD levels are elevated in human AD brains, as described by Branca et al.42. Accordingly, we also investigated the effects of acacetin on the AICD levels. A single band was detected in both the control elav-Gal4/+ and transgenic elav < APP/BACE-1 flies and acacetin decreased the AICD levels (approximately 7 kDa) in the transgenic flies (Fig. 9G). Quantification of the western blots revealed that the AICD levels were reduced to 68, 27 and 37% of the control levels following treatment with 100, 300 and 500 μM acacetin, respectively (Fig. 9I).
Effect of acacetin on Aβ, BACE-1 and APP processing in the transgenic flies.
Human APP and BACE-1 transgenic flies (elav < APP/BACE-1) were cultured from the egg stage in polystyrene vials containing media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. Western blot analyses were performed to determine the levels of the Aβ, BACE-1, APP, APP-CTFs and AICD proteins, as described in the Methods section. (A) Aβ was immunoblotted with an Adβ, 17–24 (4G8) monoclonal antibody and each lane contained 70 μg of protein. (B) The relative amounts of total Aβ were detected by western blotting. (C) BACE-1 was probed with an anti-BACE-1 antibody and each lane contained 30 μg of protein. (D) Protein levels of human BACE-1 in the transgenic flies. (E) Human APP was detected by western blotting with an anti-APP C-terminal antibody. Each lane contained 30 μg of protein. (F) Quantification of the human APP protein levels in the transgenic flies. (G) The APP proteolytic processing products βCTF, αCTF and AICD were detected by western blotting with an anti-APP C-terminal antibody; each lane contained 30 μg of protein. (H) The levels of the CTFs resulting from APP processing in the transgenic flies. (I) The levels of the AICD fragment resulting from APP processing in the transgenic flies. β-Actin was used as a loading control. The differences in protein expression were quantified using a Molecular Imager Gel Doc XR system and normalized to the actin expression on the same membrane. Each bar represents the mean ± SE of duplicate samples of three independent experiments (***P < 0.001; **P < 0.01; *P < 0.05; ns, no significant difference, using Bonferroni’s multiple comparison test).
Discussion
The amyloid cascade hypothesis, which presumes that the deposition of amyloid plaques in the brain is the reason for the AD pathology, has promoted the development of drugs to treat AD5,43. There are three proteases involved in APP processing to produce amyloid plaques – α-secretase, β-secretase and γ-secretase—and β-secretase is a major therapeutic target in AD44. Selective phytochemicals may be used to treat AD and are of great interest because they may biodegrade to nontoxic products45. These anti-AD products can be applied to humans in the same manner as conventional drugs. Plants contain various compounds, such as alkaloids, phenols and terpenoids and these compounds, alone or in combination, contribute to BACE-1 inhibition14,15. The phytochemicals that inhibit human BACE-1 include alkaloids (e.g., epiberberi and groenlandicine, IC50 8.55 and 19.68 μM, respectively46), terpenoids (e.g., 16α-hydroxy-17-isovaleroyloxy-ent-kauran-19-oic acid and 10 other diterpenoids, IC50 18.58–92.20 μM47; bakuchiol, IC50 21.38 μM48), flavonoids (e.g., epigallocatechin gallate and other two catechins, IC50 1.6–4.5 μM24; neocorylin and other five flavonoids, IC50 0.7–10.2 μM48; kuraridin and other two chalcones, IC50 6.03–7.1949; leachianone G and six other flavonones, IC50 8.56–60.88 μM49), benzopyranoids (e.g., aloeresin D and C-2′-decoumaroyl-aloeresin G, IC50 39.0 and 20.5 μM, respectively50; imperatorin and its four derivatives, IC50 91.8–359.2 μM51), phenylpropanoids (e.g., p-coumaric acid, IC50 90 μM52), stilbenoids (e.g., resveratrol and its eight derivatives, IC50 0.34–19.80 μM53), diarylalkyls (e.g., bisdemethoxycurcumin and its two derivatives, IC50 17–340 μM33) and tannins (e.g., geraniin and corilagin, IC50 4 and 34 μM, respectively54).
In the current study, we used a FRET-based enzyme assay to identify the BACE-1 inhibitory constituents from A. rugosa whole plant extracts. The active constituents were determined to be the O-methylated flavone acacetin and the oleanane triterpenoids maslinic acid and oleanolic acid; the chemical structure of oleanolic acid differs from that of maslinic acid by the lack of a hydroxyl group at the 2-carbon position. The IC50 values of these constituents were between 88.5 and 487.6 μM, while the IC50 values of the natural compounds described above are between 0.34 and 359.2 μM. Acacetin was more potent in inhibiting BACE-1 than either maslinic acid or oleanolic acid, although it was less potent in inhibiting BACE-1 than the human BACE-1 inhibitor IV. However, the peptide-based BACE-1 inhibitor IV and other BACE-1 inhibitors are poorly absorbed when administered orally, due to their low penetrability across the blood brain barrier, which results in little pharmacological activity in vivo23. Small molecule BACE-1 inhibitors with a low molecular weight and good plasma membrane permeability are crucial for drug development. Many in vivo studies have shown that flavonoids can be absorbed by oral administration, cross the blood brain barrier and work on the central nervous system55,56. Acacetin has been reported to possess antioxidant57, anti-inflammatory58 and anticarcinogenic59 activities and have neuroprotective effects on the central nervous system. They also have therapeutic potential for treating neurological diseases associated with excitotoxicity60.
Drosophila models of amyloid toxicity and Tau have been developed to simulate the underlying pathogenesis of AD61. Several researchers have evaluated whether phytochemicals have therapeutic effects in Drosophila AD models. Caesar et al.62 studied the behavior of curcumin as a drug candidate to alleviate Aβ toxicity in five different transgenic Drosophila AD models. They reported that curcumin treatment resulted in an improved lifespan (up to 75%) and climbing activity in transgenic flies; Aβ deposition was not decreased following treatment. It has also been reported that prolonged exposure to either curcumin or bisdemethoxycurcumin can rescue the morphological defects (ommatidia atrophy at the edges of the compound eye, absence of ommatidial bristles and ommatidial fusion) in the compound eyes of GMR < APP/BACE-1 flies co-expressing APP and BACE-1 and also significantly improve locomotor coordination in the elav < BACE-1 and elav < APP/BACE-1 flies33.
In the current study, the human APP and BACE-1 genes were co-expressed under the control of the elav and gmr promoters. Some dark spots were observed in the compound eyes of the GMR < APP/BACE-1 flies. Chakraborty et al.37 reported the presence of melanotic masses on both the ventral abdomen and proboscis of elav < APP/BACE flies. The number of masses was significantly decreased in these flies after treatment with the γ-secretase inhibitor L-685,458. They suggested that the masses were composed of Aβ or induced by Aβ. A previous study showed that β-amyloid deposits were localized outside the retinas of the GMR < APP/BACE-1 flies31. According to these studies, the dark spots in the Drosophila eyes might be composed of Aβ due to the expression of the human APP and BACE-1 genes in the photoreceptor cells and secretion of Aβ by these photoreceptor cells into the projection areas31. Here, Aβ could induce the immune response by activating the Toll pathway63,64, resulting in the formation of dark spots on the surfaces of the compound eyes. We found that acacetin could rescue the morphological defects (dark deposition, photoreceptor collapse and fusion and absence of ommatidial bristles) in the eyes of the GMR < APP/BACE-1 flies that co-express BACE-1 and APP in their compound eyes. In addition, acacetin also improved the locomotor coordination in the elav < APP/BACE-1 flies and prolonged the T1/2 of the transgenic flies, without any effects on feeding behavior. These findings, together with our elucidation of the inhibitory action of acacetin on BACE-1, indicate that A. rugosa whole plant-derived materials hold promise for the development of novel, effective, naturally occurring anti-AD agents.
An investigation of the mechanisms of action of naturally occurring anti-AD compounds may provide useful information for the development of selective anti-AD therapeutic alternatives with novel target sites62. The target sites and mechanisms underlying the anti-dementia actions of plant secondary substances have been well documented by Howes and Perry65. In the amyloidogenic pathway, APP is cleaved by BACE-1 and releases a large soluble ectodomain of APP and APP-βCTF, which is then further cleaved by γ-secretase to generate toxic Aβ13,66. The protein levels and enzymatic activity of BACE-1 are elevated in AD brains, suggesting that abnormal BACE-1 regulation may significantly contribute to AD pathogenesis67. Utsuki et al.39 screened 144 analogs of phenserine, a physostigmine analog, to identify small molecules that inhibit APP protein synthesis and the subsequent Aβ production, without possessing potent AChE inhibitory activity, using an enzyme-linked immunosorbent assay. They reported eight analogs, including posiphen, an (–)-enantiomer of phenserine, that were capable of dose-dependently reducing APP and Aβ production without causing cell toxicity. These analogs also inhibited APP synthesis, resulting in a decrease in the number of amyloid plaques. Natural flavonoids, such as myricetin and quercetin, have also been reported to be potent inhibitors of BACE-1 activity and to reduce the Aβ levels in primary cortical neurons68. Long-term treatment with the EGb761 Ginkgo biloba extract significantly lowered the APP protein levels in a transgenic AD mouse model, suggesting that the potential neuroprotective properties of EGb761 may be, at least in part, related to its APP lowering activity69. In addition, certain flavonoids and their metabolites have been shown to exert beneficial effects on neurological processes through their interaction with neuronal signaling pathways, such as the PI3K/Akt, tyrosine kinase, protein kinase C and MAPK signing pathways, as well as the nuclear factor-κB pathway. Inhibitory or stimulatory effects on these pathways are likely to have a large impact on neuronal function by modulating gene expression7.
In the current study, the real-time qRT-PCR analysis revealed that acacetin was able to reduce both the human APP and BACE-1 mRNA levels in a transgenic Drosophila AD model, without significantly affecting the GFP mRNA levels. This finding suggests that acacetin plays an important role in inhibiting human BACE-1 and APP by regulating gene transcription and it does not block binding of the transcriptional activator Gal4 to the UAS activation domain. Western blot analysis revealed that acacetin reduced Aβ production by interfering with BACE-1 activity and APP synthesis, resulting in a decrease in the levels of APP-CTF and AICD. Therefore, the protective effect of acacetin on Aβ production is mediated by transcriptional regulation of the BACE-1 and APP genes, which results in decreased APP levels and BACE-1 activity. Acacetin acts as a direct inhibitor of BACE-1 activity and regulates the expression of both APP and BACE-1. Previous studies49,68 demonstrated that flavonoids exhibited inhibitory activity against BACE-1 in cell-free and cell-based systems. The flavonoids epicatechin and epigallocatechin were reported to be potent inhibitors of APP processing70. It has also been reported that the flavonoid icariin possesses the ability to decrease amyloid deposition in a transgenic mouse model by reducing APP and BACE-1 expression71. Acacetin was reported to exert its anti-inflammatory activity by downregulating pro-inflammatory mediators via inhibition of the NF-κB signaling pathways72. Furthermore, acacetin enhanced the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which activates the classical Ras/MAPK pathway, to induce neuritogenesis and neuronal differentiation73; therefore, acacetin is a potential therapeutic agent for AD.
In conclusion, A. rugosa whole plant constituents, particularly acacetin, are potential therapeutics or lead compounds for the prevention or treatment of AD. The anti-AD action of acacetin provides an indication of at least one of the pharmacological actions of A. rugosa. Further research is needed on the practical applications of plant-derived preparations as novel anti-AD products to establish their safety profiles in humans and determine whether the activity measured in flies would also occur in vivo in humans after consumption of A. rugosa whole plant-derived products. A. rugosa is used as a wild vegetable and herbal drug in traditional therapies22. In addition, detailed tests are needed to understand how to improve the anti-AD potency and stability of the compounds isolated from A. rugosa for eventual commercial development.
Methods
Experimental groups
In our experiment, acacetin, maslinic acid and oleanolic acid isolated from the whole A. rugosa plant were tested in vitro using a FRET-based enzyme assay. Among these constituents, acacetin was tested in flies because it was a more potent inhibitor of BACE-1 than oleanolic acid or maslinic acid. The experimental groups examined for the in vivo study are illustrated in Fig. 10. The flies used in this study were cultured from the egg stage in 94 × 25 mm polystyrene Drosophila vials (Hansol Tech, Seoul, Republic of Korea (ROK)) containing standard media supplemented with acacetin in 0.1% DMSO, with the exception of the feeding assay. Based on the preliminary test results, the behavior and eye morphology of Drosophila models were determined with three concentrations (100, 300 and 500 μM) of acacetin. Newly emerged male flies were cultured on standard media supplemented with acacetin for the feeding assays. GMR-Gal4 drove the co-expression of human APP and BACE-1 in the compound eyes of the flies, while GMR-Gal/+ flies were used as the control group. The age-dependent morphological changes in the flies’ compound eyes were observed using light microscopy and SEM. The compound eyes’ phenotypes were also observed by histologic analysis; Cason’s trichrome staining was used to distinguish the histologic changes, while Congo red staining was used to detect the amyloid plaques in the compound eyes (Fig. 10A). The elav-Gal4 promoter drove the co-expression of the targeted transgenes in the nervous system; elav-Gal4/+ was used as the control group. The behavior (climbing and feeding), eclosion (pupation and emergence) rate and lifespan of the flies were also tested (Fig. 10B). In the Gal4 system, the promoter (or enhancer) drives the expression of the yeast transcriptional activator Gal4 in cell- and tissue-specific patterns and Gal4, in turn, directs the transcription of Gal4-UAS target genes in an identical pattern74. To confirm that the effects of acacetin on human APP and BACE-1 mRNA expression were not due to a promoter (or enhancer) effect in the Gal4 system, the levels of the GFP mRNA were compared in vehicle-fed and acacetin-fed male elav < GFP flies by qRT-PCR. The possible mechanisms of the anti-AD action of acacetin were elucidated using western blotting analyses (Fig. 10C).
Schematic of the experimental fly groups used in this study.
The flies used in this study were cultured from the egg stage in 94 × 25 mm polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in DMSO, with the exception of the feeding assay. Newly emerged male flies were cultured on standard media supplemented with acacetin for the feeding assay. (A) GMR-Gal4 drives the co-expression of the human BACE-1 and APP genes in the flies’ compound eyes and the morphological changes in the compound eyes of these flies were tested. The GMR-Gal4/+ flies were used as the control group. (B) The elav-Gal4 promotor drives the co-expression of the targeted transgenes in the flies’ nervous system and the behavior (climbing and feeding), eclosion rate and lifespan of the elav < APP/BACE-1 flies were tested. The elav-Gla4/+ flies were used as the control group. (C) The possible mechanism of the anti-AD action of acacetin was elucidated using qRT-PCR and western blot analyses.
Instrumental analysis
The 1H and 13C NMR spectra were recorded in MeOD or DMSO-d6 on an AVANCE 600 spectrometer (Bruker, Rheinspettem, Germany) at 600 and 150 MHz, respectively, using tetramethylsilane as an internal standard. The chemical shifts are given in δ (ppm). The UV spectra were obtained in methanol on a UVICON 933/934 spectrophotometer (Kontron, Milan, Italy), the mass spectra on a JMS-DX 303 spectrometer (Jeol, Tokyo, Japan) and the FT-IR spectra on a Nicolet Magna 550 series II spectrometer (Midac, Irvine, CA, USA). Silica gel 60 (0.063–0.2 mm) (Merck, Darmstadt, Germany) was used for column chromatography. Merck precoated silica gel plates (Kieselgel 60 F254) were used for analytical thin layer chromatography (TLC). An Isolera One medium-pressure liquid chromatograph (Biotage, Uppsala, Sweden) and an Agilent 1200 series high-performance liquid chromatograph (Agilent, Santa Clara, CA, USA) were used to isolate the active compounds.
Materials
Commercially available pure organic acacetin and bovine serum albumin (BSA) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). BACE-1 inhibitor IV and Acid red were supplied by Merck (Darmstadt, Germany) and Amresco (Cochran Road Solon, OH, USA), respectively. RIPA buffer and the mammalian cell protease inhibitor cocktail were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human BACE-1 and the fluorogenic peptide substrate Mca-SEVNLDAEFRK (Dnp) RR-NH2 were purchased from R&D Systems (Minneapolis, MN, USA). The PageRuler Prestained Protein Ladder and Spectra Multicolor Low Range Protein Ladder were purchased from Thermo Scientific (Walldorf, Germany). All of the other chemicals and reagents used in this study were of reagent-grade quality and are available commercially.
Antibodies
The primary antibodies used in this study were as follows: APP C-terminal antibody (A8717) from Sigma-Aldrich; anti-BACE-1 antibody (ab2077) and anti-actin antibody (ab1801) from Abcam (Cambridge, MA, USA); and Aβ, 17–24 (4G8) monoclonal antibody (SIG-39240) from Covance (Princeton, NJ, USA). The secondary antibodies used in this study were goat anti-rabbit IgG H&L (HRP) (ab6721–1) from Abcam and goat anti-mouse IgG-HRP (sc-2005) from Santa Cruz Biotechnology.
Plant material
Whole A. rugosa plants were purchased from the Boeun medicinal herb shop (Seoul Yangnyeongsi, Seoul, ROK). A voucher specimen (AR-WP-01) was deposited in the Research Institute of Agriculture and Life Sciences at Seoul National University.
Drosophila stocks and rearing conditions
The flies were cultured on standard cornmeal agar medium75 at 29 °C and 60% relative humidity under a 12:12 h light:dark cycle. The w1118 (stock number, 3605), UAS-APP/BACE-1 (33797), elav-GAL4 (8760) and GMR-GAL4 (1104) fly stocks used in this study were purchased from the Bloomington Drosophila Stock Center at Indiana University and the UAS-mCD8-GFP/cyo fly stock was obtained from Dr. Young Ho Koh, Ilsong Institute of Life Science, Hallym University (Anyang, Gyeonggi, ROK). The GAL4/UAS system was used to overexpress the target genes in specific tissues. The characterization of the human APP and BACE-1 transgenic flies (elav < APP/BACE-1 and GMR < APP/BACE-1) as reliable AD models is described in our previous study33.
Bioassay-guided fractionation and isolation
Air-dried whole A. rugosa plants (5 kg) were pulverized, extracted with methanol (3 × 15 L) at room temperature for 2 days and filtered. The combined filtrate was concentrated to dryness by rotary evaporation at 40 °C to yield approximately 455 g of a black tar. The extract (400 g) was sequentially partitioned into hexane- (75.4 g), chloroform- (55.1 g), ethyl acetate- (27.6 g), butanol- (54.5 g) and water-soluble (187.4 g) portions for the subsequent bioassays. The organic solvent-soluble portions were concentrated under vacuum at 40 °C and the water-soluble portion was concentrated at 50 °C. To isolate the active constituents, 0.1–2 mg/mL of each A. rugosa whole plant-derived fraction was tested in a FRET-based enzyme assay, as described by Wang et al.33 and Lv et al.50.
The hexane-soluble fraction (10 g) was the most biologically active fraction (Table 1) and medium-pressure liquid chromatography (MPLC) was performed using an Isolera apparatus equipped with a UV detector at 254 and 365 nm and a SNAP column cartridge (340 g silica gel) with a column volume of 510 mL (Fig. 11). Separation was achieved with a gradient of hexane and ethyl acetate (100:0, 90:10, 80:20, 70:30, 40:60, 50:50, 30:70 and 10:90 by volume) and finally with methanol (1 L) at a flow rate of 50 mL/min to provide 48 fractions (each approximately 180 mL). The column fractions were monitored by TLC on silica gel plates developed with a hexane and ethyl acetate (6:4 by volume) mobile phase. Fractions with similar Rf values on the TLC plates were pooled and the spots were detected by spraying the plate with 2% sulfuric acid. This separation procedure was repeated seven times. Active fractions 17–20 (H2) and 28–33 (H4) were obtained. Fraction H2 was separated by MPLC with a UV detector and a column cartridge (100 g silica gel) with a column volume of 132 mL by elution with a gradient of hexane and ethyl acetate (100:0, 90:10, 80:20, 70:30, 60:40, 50:50 and 30:70 by volume) and finally with 500 mL methanol at a flow rate of 30 mL/min to provide 235 fractions (each approximately 22 mL). The column fractions were monitored by TLC on silica gel plates, as stated previously. Active fractions 1–34 (H21) and 35–120 (H22) were obtained. Fraction H21 was separated by MPLC with a UV detector and a column cartridge (25 g silica gel) with a column volume of 33 mL by elution with a gradient of chloroform and ethyl acetate (100:0, 95:5, 90:10, 85:15, 80:20, 70:30 and 50:50 by volume) and finally with 300 mL methanol at a flow rate of 25 mL/min to provide 79 fractions (each approximately 22 mL). Fraction H22 was separated by MPLC with a UV detector and a column cartridge (25 g silica gel) with a column volume of 33 mL by elution with a gradient of hexane and ethyl acetate (100:0, 95:5, 90:10, 85:15, 80:20, 70:30, 60: 40, 40:60 and 30:70 by volume) and finally with 300 mL methanol at a flow rate of 25 mL/min to give 116 fractions (each approximately 22 mL). A preparative high-performance liquid chromatograph was used to separate the constituents from active fractions 37–65 (H213) from H21 and fractions 33–48 (H222) from H22. The column was a 7.8 mm i.d. × 300 mm μBondapak C18 (Waters, Milford, MA, USA) with a mobile phase of methanol and water (80:20 and 95:5 by volume) at a flow rate of 1 mL/min. Chromatographic separation was monitored using a UV detector at 217 and 216 nm, respectively. Finally, two active constituents, 1 (7.26 mg) from H213 and 2 (12.52 mg) from H222, were isolated at retention times of 13.15 and 16.86 min, respectively. Fraction H4 was recrystallized in methanol at –20 °C to afford active constituent 3 (81.4 mg).
Procedures to isolate the BACE-1 inhibitory constituents.
The Agastache rugosa whole plant methanol extract was sequentially partitioned into hexane-, chloroform-, ethyl acetate-, butanol- and water-soluble portions. The hexane-soluble fraction was the most biologically active fraction and MPLC was performed. Each fraction (0.1–2 mg/L) was tested in a FRET-based enzyme assay to isolate the active compounds from the fraction.
FRET-based enzyme assay
The methods of Wang et al.33 and Lv et al.50 were used, with slight modifications, to assess the BACE-1 inhibitory activity of the test compounds. Briefly, assay mixtures containing 1 μL of 0.5 μg/μL recombinant human BACE-1, 0.75 μL of a 2.5 μg/μL fluorogenic peptide substrate, 47.25 μL of 50 mM sodium acetate (pH 4.5) and the isolated compounds (1–1000 μg/mL) in 2% DMSO were preincubated for 1 h at 25 °C in darkness, followed by the addition of 16.6 μL of 2.5 M sodium acetate to terminate the reaction. The fluorescence intensity was measured at room temperature using a SpectraMAX Gemini XS plate reader (Molecular Devices, Sunnyvale, CA, USA) at 355 nm excitation and 405 nm emission. The inhibition percentage was determined using the following equation: % inhibition = 100−[(FS − FS0)/(FC − FC0)] × 100, where FS and FS0 are the fluorescence of the samples at 60 min and 0 min and FC and FC0 are the fluorescence of the control at 60 min and 0 min, respectively50. The results are expressed as the means ± standard errors (SEs) of triplicate samples from three independent experiments.
Light and scanning electron microscopy of the adult eyes
GMR < APP/BACE-1 flies were cultured from the egg stage in polystyrene vials containing standard media supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO, based on the preliminary test results. The controls received 0.1% DMSO only. The male flies (1, 10, 20 and 30 days old) were anesthetized on ice and placed on a microscope slide at room temperature for light microscopy. The morphology of the compound eyes was observed using an EZ4 HD stereo microscope (×35) equipped with an Integrated 3.0 Mega-Pixel CMOS camera (Leica, Hicksville, NY, USA). The flies with aberrant eye phenotypes were collected and the number of the flies with dark deposits in the eye was counted.
For SEM, the anesthetized male flies (30 days old) were attached to a copper mount using silver paint as a conducting adhesive, as described previously31. They were then put directly into the viewing chamber of an SEM without prior coating, as described by Hartman and Hayes76. The external surface morphology of the compound eye was visualized using a Supra 55VP field-emission scanning electron microscope (Carl Zeiss, Jena, Germany) operated at 15 kV.
Histologic analysis
Cason’s trichrome staining was performed, as described by Wang et al.33 and Perumalsamy et al.77. Briefly, the heads of 5- and 15-day-old male GMR < APP/BACE-1 flies, cultured in vials as stated previously, were fixed in a 4% paraformaldehyde buffer solution (pH 7.4) overnight at 4 °C and paraffin-embedded preparations of the fly heads were then sectioned at 10 μm thickness using a HM 340E rotary microtome (Thermo Scientific Microm, Walldorf, Germany). The sections were dried at 40 °C overnight, subsequently deparaffinized with CitriSolv (Fisher Scientific, Fair Lawn, NJ, USA) and rehydrated using a series of ethanol solutions in phosphate-buffered saline (PBS). The rehydrated paraffin sections were soaked in Carson’s trichrome solution for 15 min and the slides were gently swashed in tap water and subsequently rinsed three times in distilled water (DW). The excess water was removed with tissue paper and the samples were mounted using Vectashield H-1000 mounting medium (Vector Laboratories, Burlingame, CA, USA).
Congo red staining was performed to detect the amyloid plaques in the Drosophila AD model. The paraffin-embedded sections described above were deparaffinized and then stained using NovaUltra Special Stain Kits (IHC World, Woodstock, MD, USA) according to the manufacturer’s instructions. Finally, the sections were dehydrated and mounted with mounting medium. The images were observed and captured using an EZ4 HD stereo microscope.
Measurement of the fly eclosion rate
The pupation and emergence rates were determined according to the method of Tamura et al.78. The elav < APP/BACE-1 flies were cultured from the egg stage in vials, as stated previously. Groups of 50 third instar larvae climbing on the wall were transferred into vials containing fresh media. The treated and control (0.1% DMSO only) flies were grown under the same conditions as those used for fly maintenance. The numbers of pupae and adults were determined. All treatments were repeated four times using 50 larvae per replicate.
Lifespan assay
Groups of 20 newly emerged male elav < APP/BACE-1 flies, cultured in vials as stated previously, were separately transferred to vials containing media supplemented with acacetin (100, 300 and 500 μM) every 4 days. The controls received 0.1% DMSO. The median life time (T1/2) was defined as the time when the survivor function equaled 50%, because median survivorship is a more reliable index than mean survival time, as described by Chakraborty and colleagues37. All treatments were repeated 10 times using 20 males per replicate.
Climbing assay
The climbing activity assay was performed, as described by Wang et al.33 and Crowther et al.34. Groups of 20 male elav < APP/BACE-1 flies (1, 5, 10 and 15 days old), cultured in vials as stated previously, were separately placed in an empty vial conjoined with another vial on top and manually tapped twice. After 20 s, the flies that climbed from the bottom and crossed the 9.5 cm line were counted and the climbing index was determined as the number of flies that climbed to the top vial relative to the total number of test flies and expressed as a percentage. The controls received 0.1% DMSO. All trials were repeated five times using 20 males per replicate.
Feeding assay
The adult feeding assay was performed according to the methods of Wang et al.33 and Bahadorani et al.79. Groups of 15 newly emerged male elav < APP/BACE-1 flies were cultured on standard media for 3 days and then starved for 20 h in vials containing three layers of Whatman no. 2 filter paper (Whatman, Maidstone, UK) soaked in DW. The flies were then transferred into vials containing media (with 0.2% Acid red) supplemented with acacetin (100, 300 and 500 μM) in 0.1% DMSO. The controls were fed with media supplemented with 0.2% Acid red and 0.1% DMSO. After 2 h of feeding, the abdomens were isolated and homogenized in 1 mL DW. After centrifugation (5000 rpm, 25 °C, 5 min), the optical density (OD) of the supernatant was measured at 505 nm, because this OD value is considered to index the amount of food intake by flies, as described by Min and Tatar80. All treatments were repeated three times using 15 males per replicate.
Real-time reverse transcription-PCR analysis
Real-time qRT-PCR with SYBR Green dye was performed to determine whether acacetin affected the expression levels of the human APP and BACE-1 mRNAs in the transgenic flies. The elav < APP/BACE-1 and elav < GFP flies were cultured from the egg stage in vials, as stated previously. The controls received 0.1% DMSO only. The total RNA was extracted from 30 heads (30 mg) of male elav < APP/BACE-1 and elav < GFP flies (20 days old) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The residual genomic DNA was removed using RQ1 RNase-Free DNase (Promega, Fitchburg, WI, USA) and 1 μg of the total RNA from each sample was used for complementary DNA (cDNA) synthesis with an oligo (dT) 12–18 primer (Invitrogen, Carlsbad, CA, USA) according to the protocol of the SuperScript III Reverse Transcriptase Kit. Real-time qRT-PCR was performed in 96-well plates using the StepOne Plus Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). Each reaction mixture consisted of 10 μL of the SYBR Green PCR Master Mix Kit (Applied Biosystems), 2 μL of the forward and reverse primers (5 pmol each), 25 ng of the cDNA and diethylpyrocarbonate water for a final volume of 20 μL. The oligonucleotide PCR primer pairs are listed in Table 3 and were purchased from Bioneer (Daejeon, ROK). The cycling program was an initial hold at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s and extension at 72 °C for 30 s. The mRNA expression levels of the target genes were normalized to the mRNA expression level of the housekeeping gene rp 49 that encodes the Drosophila ribosomal protein 49 and analyzed by the 2–ΔΔCT method using the StepOne Software v2.3 and DataAssist Software (Applied Biosystems). The results are expressed as the means ± SEs of duplicate samples from three independent experiments.
Western blot analysis
The elav < APP/BACE-1 flies were cultured from the egg stage, as described previously. The lysates were obtained from the heads of 50 male flies (20 days old) by placing the heads in 100 μL RIPA buffer (pH 8.0) containing a 1% protease inhibitor cocktail37. The controls received 0.1% DMSO. The lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The protein content of the supernatant was determined using a Bradford Protein Assay kit (Sigma-Aldrich) and BSA was used as the standard. The total proteins (30 μg/sample for APP and BACE-1 detection; 70 μg/sample for Aβ detection) were mixed with an equal volume of 5× sample buffer81 containing 40 mM of DL-dithiothreitol, boiled for 10 min and then loaded onto 8, 10 and 12% sodium dodecyl sulfate-polyacrylamide gels using a Mini-Protean 3 electrophoresis cell (Bio-Rad, Hercules, CA, USA) for APP, APP-CTF (APP-αCTF and APP-βCTF) and AICD, BACE-1 and Aβ detection, respectively. After electrophoresis at 110 V in 2 h, the proteins from the gels were transferred onto a polyvinyl difluoride membrane (Pall Corporation, Pensacola, FL, USA) using an electroblotting apparatus. The membrane was then blocked with 5% skim milk (BD Difco, Flanklin Lakes, NJ, USA) in PBS containing 0.1% (v/v) Tween-20 (0.1% PBS-T) at room temperature for 1 h and further incubated overnight at 4 °C with a 1:4,000 dilution of an anti-APP C-terminal antibody to detect the cleaved APP product, a 1:1,000 dilution of anti-BACE-1 and a 1:1,000 dilution of anti-amyloid (4G8). After washing with 0.1% PBS-T three times at 10 min intervals, the membranes were further incubated for 2 h with a goat anti-rabbit IgG H&L (HRP) secondary antibody at a1:4,000 dilution for APP and BACE-1 and a goat anti-mouse lgG-HRP at a 1:5,000 dilution for β-amyloid. Finally, after washing with 0.1% PBS-T three times with a 10 min interval between washes, the membranes were developed with an ECL chemiluminescence reagent (Amersham Bioscience, Buckinghamshire, UK) and immediately exposed to a CP-PU X-ray film (AGFA, Mortsel, Belgium). The differences in protein expression were quantified using a Molecular Imager Gel Doc XR system (Bio-Rad, Hercules, CA, USA) and normalized to actin expression on the same membrane. The results are expressed as the means ± SEs of duplicate samples from three independent experiments.
Data Analysis
The fifty percent inhibitory concentration (IC50) was defined as the concentration of the compound that resulted in a 50% loss of BACE-1 activity. The IC50 values were determined using GraphPad Prism 5.1 software (GraphPad Software, La Jolla, CA, USA). The IC50 values for the treatments were considered significantly different from one another when their 95% confidence limits (CLs) did not overlap. The significance of the differences between the mean values was determined using a one-way or two-way analysis of variance (ANOVA) (GraphPad Prism 5.1 software).
Additional Information
How to cite this article: Wang, X. et al. Effects and possible mechanisms of action of acacetin on the behavior and eye morphology of Drosophila models of Alzheimer's disease. Sci. Rep. 5, 16127; doi: 10.1038/srep16127 (2015).
References
Prince, M. & Jackson, J. World Alzheimer Report. Alzheimer’s Disease International. (2009) Available at https://www.alz.co.uk/research/files/WorldAlzheimerReport.pdf (Accessed: 20th February 2015).
Wimo, A. & Prince, M. World Alzheimer Report. The global economic impact of dementia. Alzheimer’s Disease International. (2010) Available at http://www.alz.org/documents/national/world_alzheimer_report_2010.pdf (Accessed: 25th February 2015).
Burns, A. & Iliffe, S. Alzheimer’s disease. BMJ 338, b158 (2009).
World Health Organization. Dementia Fact sheet N°362. (2015) Available at http://www.who.int/mediacentre/factsheets/fs362/en/ (Accessed: 5th March 2015).
Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353–356 (2002).
Suh, Y. H. & Checler, F. Amyloid precursor protein, presenilins and alpha-synuclein: molecular pathogenesis and pharmacological applications in Alzheimer’s disease. Pharmacol. Rev 54, 469–525 (2002).
Baptista, F. I., Henriques, A. G., Silva, A. M. S., Wiltfang, J. & da Cruz e Silva, O. A. B. Flavonoids as therapeutic compounds targeting key protein involved in Alzheimer’s disease. ACS Chem. Neurosci 5, 83–92 (2014).
Birks, J. Cholinesterase inhibitors for Alzheimer’s disease. Cochrane Database of Systematic Reviews. Art. No.: CD005593. doi: 10.1002/14651858.CD005593. Issue 1 (2006).
Molinuevo, J. L., Lladó, A. & Rami, L. Memantine: targeting glutamate excitotoxicity in Alzheimer’s disease and other dementias. Am. J. Alzheimers Dis. Other Demen 20, 77–85 (2005).
Danysz, W. & Parsons, C. G. Alzheimer’s disease, β-amyloid, glutamate, NMDA receptors and memantine – searching for the connections. Br. J. Pharmacol 167, 324–352 (2012).
Paris, D. et al. Flavonoids lower Alzheimer’s Aβ production via an NFκB dependent mechanism. Bioinformation 6, 229–236 (2011).
Thompson, S., Lanctôt, K. L. & Herrmann, N. The benefits and risks associated with cholinesterase inhibitor therapy in Alzheimer’s disease. Expert Opin. Saf. 3, 425–440 (2004).
Cole, S. L. & Vassar, R. The Alzheimer’s disease β-secretase enzyme, BACE 1. Mol. Neurodegener 2, 22 (2007).
Ghosh, A. K., Gemma, S. & Tang, J. beta-Secretase as a therapeutic target for Alzheimer’s disease. Neurotherapeutics 5, 399–408 (2008).
Mancini, F., De Simone, A. & Andrisano, V. Bata-secretase as a target for Alzheimer’s disease drug discovery: an overview of in vitro methods for characterization of inhibitors. Anal. Bioanal. Chem 400, 1979–1996 (2011).
Thompson, L. A., Bronson, J. J. & Zusi, F. C. Progress in the discovery of BACE inhibitors. Curr. Pharm. Des 11, 3383–3404 (2005).
John, V. Human beta-secretase (BACE) and BACE inhibitors: progress report. Curr. Top. Med. Chem 6, 569–578 (2006).
Orhan, I. E. Current concepts on selected plant secondary metabolites with promising inhibitory effects against enzymes linked to Alzheimer’s disease. Curr. Med. Chem 19, 2252–2261 (2012).
Rates, S. M. K. Plants as source of drugs. Toxicon 39, 603–613 (2001).
Raskin, I. et al. Plants and human health in the twenty-first century. Trends Biotechnol 20, 522–531 (2002).
Mukherjee, P. K., Kumar, V., Mal, M. & Houghton, P. J. Acetylcholinesterase inhibitors from plants. Phytomedicine 14, 289–300 (2007).
Zielinska, S. & Matkowski, A. Phytochemistry and bioactivity of aromatic and medicinal plants from the genus Agastache (Lamiaceae). Phytochem. Rev 13, 391–416 (2014).
Vassar, R. BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease. Alzheimers Res. Ther 6, 89 (2014).
Jeon, S. Y., Bae, K., Seong, Y. H. & Song, K. S. Green tea catechins as a BACE1 (β-secretase) inhibitor. Bioorg. Med. Chem. Lett 13, 3905–3908 (2003).
Tanaka, J. C. A., Vidotti, G. J. & da Silva, C. C. A new tormentic acid derivative from Luehea divaricata Mart. (Tiliaceae). J. Braz. Chem. Soc 14, 475–478 (2003).
Dam, N. P., Dung, T. D., Van Long, L. H. & Phi Phung, N. K. Four triterpenoids from Hedyotis tenelliflora (Rubiaceae) growing in Vietnam. J. Chem 48(4B), 250–254 (2010).
Hossain, M. A. & Ismail, Z. Isolation and characterization of triterpenes from the leaves of Orthosiphon stamineus. Arabian J. Chem 6, 295–298 (2013).
Gangwal, A., Parmar, S. K. & Sheth, N. R. Triterpenoid, flavonoids and sterols from Lagenaria siceraria fruits. Der Pharmacia Lettre 2, 307–317 (2010).
Wawer, I. & Zielinska, A. 13C CP/MAS NMR studies of flavonoids. Magn. Reson. Chem 39, 374–380 (2001).
Miyazawa, M. & Hisama, M. Antimutagenic activity of flavonoids from Chrysanthemum morifolium. Biosci. Biotechnol. Biochem 67, 2091–2099 (2003).
Greeve, I. et al. Age-dependent neurodegeneration and Alzheimer-amyloid plaque formation in transgenic Drosophila. J. Neurosci 24, 3899–906 (2004).
Ali, Y. O., Escala, W., Ruan, K. & Zhai, R. G. Assaying locomotor, learning and memory deficits in Drosophila models of neurodegeneration. J. Vis. Exp 49, e2504 (2011).
Wang, X. et al. Effects of curcuminoids identified in rhizomes of Curcuma longa on BACE-1 inhibitory and behavioral activity and lifespan of Alzheimer’s disease Drosophila models. BMC Complement. Altern. Med 14, 88 (2014).
Crowther, D. C. et al. Intraneuronal Aβ, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer’s disease. Neuroscience 132, 123–135 (2005).
Deshpande, S. A. et al. Quantifying Drosophila food intake: comparative analysis of current methodology. Nat. Methods 11, 535–540 (2014).
Carvalho, G. B., Kapahi, P. & Benzer, S. Compensatory ingestion upon dietary restriction in Drosophila melanogaster. Nat. Methods 2, 813–815 (2005).
Chakraborty, R. et al. Characterization of a Drosophila Alzheimer’s disease model: pharmacological rescue of cognitive defects. PLoS ONE 6, e20799 (2011).
Haass, C. Take five—BACE and the gamma-secretase quartet conduct Alzheimer’s amyloid beta-peptide generation. EMBO J 23, 483–488 (2004).
Utsuki, T. et al. Identification of novel small molecule inhibitors of amyloid precursor protein synthesis as a route to lower Alzheimer’s disease amyloid-beta peptide. J. Pharmacol. Exp. Ther 318, 855–862 (2006).
Jung, J. I. et al. Independent relationship between amyloid precursor protein (APP) dimerization and γ-secretase processivity. PLoS ONE 9, e111553 (2014).
Groth, C., Alvord, W. G., Quiñones, O. A. & Fortini, M. E. Pharmacological analysis of Drosophila melanogaster γ-secretase with respect to differential proteolysis of Notch and APP. Mol. Pharmacol 77, 567–574 (2010).
Branca, C. et al. Pharmacological targeting of the β-amyloid precursor protein intracellular domain. Sci. Rep 4, 4618 (2014).
Karran, E., Mercken, M. & De Strooper, B. The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discov 10, 698–712 (2011).
De Strooper, B., Vassar, R. & Golde, T. The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat. Rev. Neurol 6, 99–107 (2010).
Fabry, W., Okemo, P. & Ansborg, R. Activity of East African medicinal plants against Helicobacter pylori. Chemotherapy 42, 315–317 (1996).
Jung, H. A. et al. Anti-Alzheimer and antioxidant activities of Coptidis Rhizoma alkaloids. Biol. Pharm. Bull 32, 1433–1438 (2009).
Jung, H. A. et al. Cholinesterase and BACE1 inhibitory diterpenoids from Aralia cordata. Arch. Pharm. Res 32, 1399–1408 (2009).
Choi, Y. H. et al. In vitro BACE-1 inhibitory phenolic components from the seeds of Psoralea corylifolia. Planta Med 74, 1405–1408 (2008).
Jung, H. A., Yokozawa, T., Kim, B. W., Jung, J. H. & Choi, J. S. Selective inhibition of prenylated flavonoids from Sophora flavescens against BACE1 and cholinesterases. AM. J. Chin. Med 38, 415–429 (2010).
Lv, L. et al. BACE1 (beta-secretase) inhibitory chromone glycosides from Aloe vera and Aloe nobilis. Planta Med 74, 540–545 (2008).
Marumoto, S. & Miyazawa, M. β-secretase inhibitory effects of furanocoumarins from the root of Angelica dahurica. Phytother. Res 24, 510–513 (2010).
Youn, K. & Jun, M. Inhibitory effects of key compounds isolated from Corni fructus on BACE1 activity. Phytother Res 26, 1714–1718 (2012).
Choi, C. W. et al. In vitro BACE1 inhibitory activity of resveratrol oligomers from the seed extract of Paeonia lactiflora. Planta Med 77, 374–376 (2011).
Youn, K. & Jun, M. In vitro BACE1 inhibitory activity of geraniin and corilagin from Geranium thunbergii. Planta Med 79, 1038–1042 (2013).
Jäger, A. K. & Saaby, L. Flavonoids and the CNS. Molecules 16, 1471–1485 (2011).
Faria, A. et al. Flavonoid metabolites transport across a human BBB model. Food Chem 149, 190–196 (2014).
Cholbi, M. R., Paya, M. & Alcaraz, M. J. Inhibitory effects of phenolic compounds on CCl4-induced microsomal lipid peroxidation. Experientia 47, 195–199 (1991).
Pan, M. H., Lai, C. S., Wang, Y. J. & Ho, C. T. Acacetin suppressed LPS-induced up-expression of iNOS and COX-2 in murine macrophages and TPA-induced tumor promotion in mice. Biochem. Pharmacol 72, 1293–1303 (2006).
Shen, K. H. et al. Acacetin, a flavonoid, inhibits the invasion and migration of human prostate cancer DU145 cells via inactivation of the p38 MAPK signaling pathway. Mol. Cell. Biochem 333, 279–291 (2010).
Lin, T. Y., Huang, W. J., Wu, C. C., Lu, C. W. & Wang, S. J. Acacetin inhibits glutamate release and prevents kainic acid-induced neurotoxicity in rats. PLoS ONE 9, e88644 (2014).
Prüßing, K., Voigt, A. & Schulz, J. B. Drosophila melanogaster as a model organism for Alzheimer’s disease. Mol. Neurodegener 8, 35 (2013).
Caesar, I., Jonson, M., Nilsson, K. P. R., Thor, S. & Hammarström, P. C. promotes A-beta fibrillation and reduces neurotoxicity in transgenic Drosophila. PLoS ONE 7, e31424 (2012).
Lemaitre, B. et al. Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila. EMBO J 14, 536–545 (1995).
McGeer, E. G. & McGeer, P. L. The importance of inflammatory mechanisms in Alzheimer disease. Exp. Gerontol 33, 371–378 (1998).
Howes, M. J. & Perry, E. The role of phytochemicals in the treatment and prevention of dementia. Drugs Aging 28, 439–468 (2011).
Tian, Y., Bassit, B., Chau, D. & Li, Y. M. An APP inhibitory domain containing the Flemish mutation residue modulates γ-secretase activity for Aβ production. Nat. Struct. Mol. Biol 17, 151–158 (2010).
Chen, Y. et al. Antidiabetic drug metformin (GlucophageR) increases biogenesis of Alzheimer’s amyloid peptides via up-regulating BACE1 transcription. Proc. Natl. Acad. Sci. USA 106, 3907–3912 (2009).
Shimmyo, Y., Kihara, T., Akaike, A., Niidome, T. & Sugimoto, H. Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features. Biochim. Biophys. Acta 1780, 819–825 (2008).
Augustin, S. et al. Effect of a short- and long-term treatment with Ginkgo biloba extract on amyloid precursor protein levels in a transgenic mouse model relevant to Alzheimer’s disease. Arch. Biochem. Biophys 481, 177–182 (2009).
Cox, C. J. et al. Dietary (−)-epicatechin as a potent inhibitor of βγ-secretase amyloid precursor protein processing. Neurobiol. Aging 36, 178–187 (2015).
Zhang, L. et al. Icariin decreases the expression of APP and BACE-1 and reduces the β-amyloid burden in an APP transgenic mouse model of Alzheimer’s disease. Int. J. Biol. Sci 10, 181–191 (2014).
Kim, H. G. et al. 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 (2012).
Nishina, A. et al. Neurite outgrowth in PC12 cells stimulated by components from Dendranthema × grandiflorum cv. “Mottenohoka” is enhanced by suppressing phosphorylation of p38MAPK. Evid. Based Complement Alternat. Med. 2013, 403503 (2013).
Phelps, C. B. & Brand, A. H. Ectopic gene expression in Drosophila using GAL4 system. Methods 14, 367–379 (1998).
Ren, C., Finkel, S. E. & Tower, J. Conditional inhibition of autophagy genes in adult Drosophila impairs immunity without compromising longevity. Exp. Gerontol 44, 228–235 (2009).
Hartman, H. & Hayes, T. L. Scanning electron microscopy of Drosophila. J. Hered 62, 41–44 (1971).
Perumalsamy, H. et al. Novel histopathological and molecular effects of natural compound pellitorine on larval midgut epithelium and anal gills of Aedes aegypti. PLoS ONE 8, e80226 (2013).
Tamura, T. et al. Ku70 alleviates neurodegeneration in Drosophila models of Huntington’s disease. PLoS ONE 6, e27408 (2011).
Bahadorani, S., Bahadorani, P., Phillips, J. P. & Hilliker, A. J. The effects of vitamin supplementation on Drosophila life span under normoxia and under oxidative stress. J. Gerontol. A Biol. Sci. Med. Sci 63, 35–42 (2008).
Min, K. J. & Tatar, M. Drosophila diet restriction in practice: do flies consume fewer nutrients? Mech. Ageing Dev 127, 93–96 (2006).
Bollag, D. M., Rozycki, M. D. & Edelstein, S. J. Protein Methods (Wiley-Liss, 1996).
Acknowledgements
This work was supported by Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ010487) from the Rural Development Administration (RDA) of the Republic of Korea and by the Brain Korea 21 PLUS program through the National Research Foundation of Korea funded by the Ministry of Education of the Korean Government to Y.-J. Ahn.
Author information
Authors and Affiliations
Contributions
Conceived and designed the experiments: X.W. and Y.-J.A. Performed the experiments: X.W. and H.P. Analyzed the data: X.W., H.W.K., Y.-E.N. and Y.-J.A. Wrote the paper: X.W. and Y.-J.A. Supervised the project: Y.-J.A. Interpreted the spectroscopic data: X.W., H.P. and Y.-E.N. Critically revised the manuscript: X.W., H.W.K. and Y.-J.A. Read and approved the final manuscript: X.W., H.P., H.W.K., Y.-E.N. and Y.-J.A.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Wang, X., Perumalsamy, H., Kwon, H. et al. Effects and possible mechanisms of action of acacetin on the behavior and eye morphology of Drosophila models of Alzheimer’s disease. Sci Rep 5, 16127 (2015). https://doi.org/10.1038/srep16127
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep16127
This article is cited by
-
Metabolic engineering in Streptomyces albidoflavus for the biosynthesis of the methylated flavonoids sakuranetin, acacetin, and genkwanin
Microbial Cell Factories (2023)
-
Isolation and structure elucidation of pyridine alkaloids from the aerial parts of the Mongolian medicinal plant Caryopteris mongolica Bunge
Scientific Reports (2021)
-
Amyloid β aggregation inhibitory activity of triterpene saponins from the cactus Stenocereus pruinosus
Journal of Natural Medicines (2021)
-
Neuroprotective effect of Salvia splendens extract and its constituents against AlCl3-induced Alzheimer’s disease in rats
Advances in Traditional Medicine (2020)
-
Nutrigenomics as a tool to study the impact of diet on aging and age-related diseases: the Drosophila approach
Genes & Nutrition (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
