A tetrapeptide from maize protects a transgenic Caenorhabditis elegans Aβ_1-42 model from Aβ-induced toxicity

Abstract

A food-derived bioactive peptide that works as an important antioxidant in vivo could be used to remedy oxidative stress-related diseases. Alzheimer's disease (AD) is influenced by the accumulation and deposition of amyloid beta (Aβ) peptides in vivo, and such accumulation may worsen under conditions of oxidative stress. This study aimed to assess whether a tetrapeptide from maize, TPM, could protect Caenorhabditis elegans against Aβ-induced disease and to clarify the possible mechanism of such protection, as well as contribute to a model of oxidative stress that influences the process of Alzheimer's disease. These parameters were tested in a C. elegans model of full-length Aβ_1-42 expression (GMC101). TPM at 10 mM alleviated Aβ-induced paralysis in GMC101 under oxidative stress and normal conditions. Further studies demonstrated that TPM can efficiently inhibit Aβ aggregation in vitro and scavenge reactive oxygen species (ROS) that accelerate the accumulation and deposition of Aβ peptides in vivo. In addition, Aβ_1-42 dimer and Aβ_1-42 trimer were down-regulated by TPM under both oxidative stress and normal conditions. Our observations lead to the hypothesis that the bioactive peptide, TPM, is a potential drug candidate that might efficiently alleviate the symptoms of AD.

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1. Introduction

Alzheimer's disease (AD), which is characterized by cognitive decline and deficits, is a complex neurodegenerative disorder typically associated with aging. AD displays a unique pathology including regional neuronal loss, amyloid plaques and neurofibrillary tangles.^1–3 According to the amyloid cascade hypothesis of Alzheimer's disease, AD is pathophysiologically linked to the accumulation of β-amyloid peptide (Aβ).^4–6 After cleavage by γ-secretase, Aβ40 and Aβ42, which shows enhanced neurotoxicity and a high propensity for aggregation, are released from APP. Aβ peptides form a β-sheet structure and aggregate to form oligomers, protofibrils, and fibrils.⁶ In particular, some types of oligomers and Aβ-derived diffusible ligands (ADDLs) containing certain multiples of monomers such as trimers and tetramers are low-molecular-weight Aβ_1-42 oligomers and are considered the most neurotoxic agents.⁴ These oligomers are proposed to bind specifically to cell surface receptors, leading to synaptic malfunction, disruption of long-term potentiation (LTP), and memory deficits.⁴ Thus, identification of drug candidates that can scavenge low-molecular-weight Aβ_1-42 oligomers is necessary.

Multiple factors in addition to the aggregation of Aβ, including the formation of neurofibrillary tangles (NFT)⁷ and oxidative stress,⁸ undeniably are involved in the pathogenesis of AD. The earliest change that occurs during the pathogenesis of AD may be oxidative stress.⁹ Oxidative stress acts as a morbigenous factor in a number of age-related diseases. Free radicals, including hydroxyl free radicals, superoxide anion free radicals, and others, can cause cell damage or other forms of damage.¹⁰ Free radicals have also been identified as a risk factor for AD. It has been proposed that free radicals may induce AD by damaging proteins, DNA, and other molecules.⁹ The brain is readily damaged by free radicals due to its high use of oxygen and its low level of antioxidants.¹¹ Increased levels of oxidative DNA damage have been found in brain tissue from AD patients.¹² Aβ can also amplify oxidative stress in vivo, and this is considered a key feature in the pathology of AD.¹² Therefore, screening of potential antioxidants for their ability to prevent Aβ-induced oxidative damage should provide an effective method of identifying powerful antioxidants that prevent Aβ-induced oxidation.

In this study, we sought novel Aβ aggregation inhibitors and antioxidants as a basis for the development of novel AD drugs. Oligopeptides derived from plants and animals are a focus of current research because of their ability to scavenge free radicals, inhibit lipid peroxidation, and chelate metal ions in vivo.¹³ In addition, it is not difficult for oligopeptides to cross the blood-brain barrier (BBB). Peptides are able to cross the blood-brain barrier (BBB) through various mechanisms.^14,15 Researching in this area may provide an opportunity to cure neurodegenerative disease by oligopeptides. Recently, Leu–Asp–Tyr–Glu (TPM), a bioactive tetrapeptide isolated from maize, was found to possess antioxidant properties.¹⁶ TPM has been shown to reduce ROS levels in wild-type Caenorhabditis elegans N2.^16,17 This encouraged us to determine whether this oligopeptide can reduce oxidative stress-induced Aβ aggregation in vivo. If oligopeptide from maize could be used as potential drug for AD, it will be a low production cost and more safety than chemosynthesis drug. C. elegans is a convenient in vivo animal model that has been used in drug screening for potential AD therapeutics because of its ease of culturing and short life cycle.¹⁸ Transgenic C. elegans can be engineered to express human Aβ_1-42, leading to the production of amyloid aggregates and progressive paralysis.¹⁹ A new C. elegans model of full length Aβ_1-42 expression, GMC101, was reported in 2012.²⁰ We employed the transgenic C. elegans strain GMC101 to determine whether TPM could alleviate the symptoms of AD. Unlike C. elegans expressing Aβ_3-42, which displays age-dependent paralysis, the GMC101 strain displays no adverse effects on motion performance when adults are cultured at 20 °C.²⁰

In this study, we conducted experiments to determine (1) whether juglone (5-hydroxy-1,4-naphthoquinone), which can be reduced with NAD(P)H by diaphorases, produces intracellular superoxide anion and thus increases oxidative stress in vivo;^21,22 (2) whether induced oxidative stress promotes paralysis in C. elegans GMC101; (3) the optimal concentration of juglone in this oxidative stress model; and (4) the efficacy of TPM in the treatment of AD using GMC101 under oxidative stress conditions, as well as in a normal environment. Based on the results of these experiments, a hypothesis is proposed that the antioxidant properties of bioactive peptides obtained from food might contribute to the alleviation of AD.

2. Materials and methods

2.1. Reagents

H₂DCF–DA (2′,7′-dichlorodihydrofluorescein diacetate) (Sigma, St. Louis, MO, USA) was used as a fluorescent probe. Juglone (5-hydroxy-1,4-naphthoquinone; Sigma), a ROS-generating compound, was used to induce oxidative stress in worms. Thioflavin T (ThT) and thioflavin S (ThS) were purchased from Sigma-Aldrich. TPM was prepared by proteolysis of zein by the alkaline protease alcalase, followed by isolation, purification, and synthesis by ChinaPeptides Co., Ltd. (Shanghai, China).

2.2. Worm strains and maintenance

C. elegans was grown and maintained in standard nematode growth medium (NGM) seeded with Escherichia coli strain OP50 and maintained at 20 °C. Both the wild-type N2 strain and the transgenic GMC101 strain were obtained from the Caenorhabditis Genetics Center (CGC). To prepare age-synchronized animals, nematodes were transferred to fresh NGM plates on reaching maturity at 3 days of age and allowed to lay eggs for 4 h. To paralyze the worms, transgenic strain GMC101 worms that had just reached late L4 stage were transferred to a 25 °C environment. For the control animals, all treatments were the same as for the test animals.

2.3. Reductive effect of TPM on Aβ_1-42 aggregation in vitro

Thioflavin T can bind to amyloid β-sheets; following such binding, there is a characteristic shift in absorbance. Under conditions of excitation at 450 nm, the intensity of fluorescence emission at 485 nm reflects the degree of Aβ aggregation. Aβ_1-42 monomer was prepared using hexafluoroisopropanol (HFIP). To test the effect of TPM on the spontaneous aggregation of Aβ_1-42, various concentrations of TPM were added to the wells of a 96-well plate together with 50 μM Aβ_1-42 monomer and thioflavin T, and the fluorescence intensity was measured using a microplate reader (Infinite F200 Pro, TECAN) every 2 hours for 24 hours. As a control, we mixed PBS (pH 6.6) with Aβ_1-42 monomer and thioflavin T. Using fluorescence microscopy (Olympus IX51, Japan) and a Cell Imaging Multi-Mode Reader (BioTek Cytation 3, USA), we photographed the aggregation of Aβ_1-42 after incubation in a 96-well plate at 37 °C for 24 hours to provide a qualitative assessment of the effect of TPM on aggregation. The experiments were replicated three times.

2.4. Molecular docking

The 3D structure of TPM was built using the Chem 3D program. The crystal structure of Aβ42 was extracted from the Protein Data Bank (PDB, PDB ID: 1IYT). The molecular docking of TPM to Aβ42 was carried out using the program AutoDock 3.1. Images of the structures were generated using Discovery Studio (DS) Visualizer 4.0.

2.5. Protection against Aβ-induced paralysis by TPM

All nematodes were cultured at 20 °C and developmentally synchronized from a 3 h egg-lay. We exposed worms at the L4 stage (the final larval stage) to a range of TPM concentrations for 48 h prior to shifting the temperature to 25 °C. Nematodes were scored as paralyzed if they failed to complete full body movement (i.e., a point of inflection traversing the entire body length) either spontaneously or after provoking by touch. The proportions of paralyzed individuals were calculated. Comparisons of proportions were made using a two-tailed Z-test. The experiments were replicated as indicated.

Juglone (5-hydroxy-1,4-naphthoquinone), a reactive oxygen species-generating compound, was used to induce oxidative stress in worms.²³ To optimize the oxidative stress model, nematodes that had reached the final larval stage was transferred to graded concentrations of juglone at 25 °C. The number of paralyzed worms was counted and recorded every 2 hours. The effect of TPM on AD under oxidative stress was also tested. The young adult worms were incubated with TPM (10 mM) for 48 h and then transferred to plates with juglone at 25 °C. Standard NGM plates containing water were used for the control group. The number of paralyzed worms was counted and recorded every 2 hours. Each experiment was repeated three times and conducted in a double-blind manner.

2.6. Measurement of intracellular ROS in C. elegans

Intracellular ROS in C. elegans was measured using the molecular probe H₂DCF–DA. For detection of ROS production under normal culture conditions, worms that had just reached the late L4 stage were treated with or without TPM (10 mM) for 24 h followed by a shift to 25 °C for 12 h. To measure the ROS produced under conditions of oxidative stress, worms that had just reached late L4 stage were treated with 100 μM juglone for 1 h and then treated with or without TPM (10 mM) for 23 h followed by a shift to 25 °C for 12 h. After 12 h of exposure to 25 °C, worms were washed off the plates with cold M9 buffer. The bacteria were removed by three washes and subjected to centrifugation at low speed. The worms were resuspended in M9 buffer. A 50 μL volume of the suspension (in four replicates) was pipetted into the wells of a 96-well plate and allowed to equilibrate to room temperature. A fresh 100 μM H₂DCF–DA solution was prepared in M9 buffer from 100 mM stock solution. Fifty microliters of 100 μM H₂DCF–DA were then added to each suspension. 10 mM glutathione was selected as positive control. In each plate, control wells containing nematodes from each treatment without H₂DCF–DA and wells containing H₂DCF–DA without worms were prepared in parallel.²⁴ Immediately after the addition of H₂DCF–DA, the basal fluorescence of each well was measured in a microplate reader at excitation/emission wavelengths of 485 and 538 nm. The plates were read at 20 °C every 30 min for 2 h.

2.7. Fluorescence staining of β-amyloid

The effect of TPM on Aβ aggregation was determined using thioflavin-T (ThT) and thioflavin-S (ThS) fluorescence assays. Worms that had just reached late L4 stage were treated with or without TPM (10 mM) for 24 h, shifted to a 25 °C environment for 24 h, and then fixed overnight in 4% paraformaldehyde in PBS pH 7.4 at 4 °C. The worms were then permeabilized by incubation in 5% β-mercaptoethanol in 125 mM Tris, pH 7.4 and 1% Triton X-100 for 24 h at 37 °C. The permeabilized samples were washed 2–3 times with PBS-T and stained with 0.125% thioflavin-T/thioflavin-S in 50% ethanol for 20 min, then destained by sequential washes in ethanol (50%, 75% and 90%) and observed under a fluorescent microscope (Olympus, FV1000) for the presence of amyloid plaques in the head region.²⁵ Quadruple populations were used for each determination. For fluorescence microscopy, the worms were placed on cover slips covered with 80% glycerol, and fluorescence was quantified using a Thermo Labsystems Fluoroskan Ascent microplate reader. After staining for ThT, forty control or treated adult nematodes were transferred in 100 μL of PBS to a well of a 96-well clear, flat-bottom plate. The fluorescence in each well was measured using an excitation wavelength of 450 nm and an emission wavelength of 482 nm. Quadruple populations were used for each determination.

2.8. Western blot analyses

For comparison of Aβ levels, ∼100 μL adult worms were collected in S-basal medium in triplicate and frozen in liquid N₂.²⁶ The samples were then extracted in isopyknic RIPA lysis buffer with protease inhibitor cocktail tablets (Roche), disrupted by sonication in an ice bath, and centrifuged at 12 000g for 30 min at 4 °C. A 100 μL sample of the supernatant was added to 100 μL of 2× loading buffer and boiled for 10 min. The mixture was loaded onto Tricine-SDS-PAGE gels (30% glycerol (w/v)) and electrophoresed.²⁷ The proteins present in the gels were then transferred to PVDF membranes. To enhance reactivity with the anti-Aβ_1-42 antibody, the membrane was boiled in PBS pH 7.4 for 3 min after blotting and blocked for 1.5 h at room temperature in 0.5% (w/v) skim milk. The membranes were probed overnight at 4 °C with an anti-Aβ_1-42 antibody (D3E10) (Cell Signaling Technology).²⁸ Standard enhanced chemiluminescence was then performed.

2.9. Statistical analysis

One-way ANOVA was used to compare three or more groups. When the result was significant (p < 0.05), Tukey's HSD test was applied to test for differences between individual groups. A p value of <0.05 was considered statistically significant. All of the data were analyzed with Origin 8.0 software (Northampton, MA). The standard error of the mean is indicated by bars in the figures.

3. Results

3.1. TPM alleviates Aβ-induced paralysis in transgenic C. elegans

To determine whether TPM specifically protects C. elegans against Aβ-associated toxicity in vivo, GMC101 transgenic worms, which express and accumulate full-length Aβ_1-42, were used.¹⁸ In these experiments, we found that the paralyzed fraction of TPM-treated nematodes was lower than the paralyzed fraction in the control group, as shown in Fig. 1. Worms treated with 10 mM, 1 mM and 0.1 mM TPM all showed a reduction in the proportion of animals exhibiting Aβ-induced paralysis; the magnitude of reduction was 13.3%, 7.7% and 3.4%, respectively (Fig. 1).

Fig. 1 TPM alleviates paralysis in GMC101 in vivo. Each curve is based on three individual experiments. TPM at 10 mM was found to be the most effective when cultures were exposed to compound for 48 h prior to the initiation of the assay at 25 °C. The time required for paralysis of 50% of the animals in the 10 mM TPM treatment group is 31.39 hours, much longer than that of the control group, and the effect of TPM on alleviating paralysis is dose-dependent. The bars represent the mean ± S.E.M. *P < 0.05.

The time required for paralysis of 50% of the animals to occur in the 0.05 mM, 1 mM and 10 mM TPM treatment groups was 27.96 hours, 29.28 hours and 31.39 hours, respectively. The 1 mM and 10 mM TPM treatment groups showed longer 50% paralysis time than the control group, for which the time was 28.03 hours. These results suggest that the process of paralysis in GMC101 is alleviated by TPM in a dose-dependent manner.

3.2. TPM inhibits the aggregation of Aβ_1-42 in vitro

The results described above suggest that TPM can alleviate paralysis in the transgenic C. elegans strain GMC101. However, how did TPM relieve the animals' paralysis? First, we tested the effects of various concentrations of TPM on the aggregation of Aβ_1-42 in vitro. As shown in Fig. 2A, TPM had an inhibitory effect on the aggregation of Aβ_1-42 at all tested concentrations, and the effect was dose-dependent. Comparing the data at 8 hours and 24 hours (Fig. 2A), it is clear that 1 mM and 10 mM TPM both significantly inhibited the aggregation of Aβ_1-42 and that there was only a small increase in the number of aggregates in the 1 mM and 10 mM TPM treatment groups at 24 hours.

Fig. 2 TPM reduces the aggregation of Aβ_1-42 in vitro in a dose-dependent manner. (A) All tested concentrations of TPM inhibited the agglomeration of Aβ aggregations compared with the control group, in which 50 μM Aβ with no TPM was used. (B) TPM at 10 mM had the greatest impact on reducing Aβ aggregation after “shock incubating” for 8 hours at 37 °C. (C) After shock incubating for 24 hours at 37 °C, 1 mM and 10 mM TPM strongly inhibited the aggregation of Aβ, and the fluorescence intensity increased only slightly. (B) Compared with the control group, the TPM treatment groups displayed fewer fluorescent spots after incubation for 24 hours at 37 °C. (C) Higher magnification of the aggregation of Aβ_1-42 shows that exposure to a high concentration of TPM can reduce the formation of fibers. The bars represent the mean ± S.E.M. *P < 0.05.

We selected 24 hours as a time point for observation of the effect of TPM on Aβ_1-42 aggregation. Using fluorescence microscopy and a Cell Imaging Multi-Mode Reader (BioTek Cytation 3, USA), we photographed the aggregation of Aβ_1-42 after incubation of the treated animals at 37 °C for 24 hours. It can easily be observed in the images that the TPM-treated animals display fewer and smaller fluorescent spots (Fig. 2B), indicating that the aggregation of Aβ_1-42 was inhibited by TPM treatment. The micrographs were taken at higher magnification show that the level of fibrosis due to Aβ_1-42 aggregation was significantly decreased by treatment with TPM (Fig. 2C). High concentrations of TPM inhibited the formation of amyloid fibers in vitro. Overall, the results indicate that the aggregation of Aβ_1-42 is dose-dependent and that TPM has a strong inhibitory effect on the aggregation of Aβ_1-42 in vitro.

3.3. Molecular docking analysis

To elucidate the possible mechanism of the observed prevention of Aβ_1-42 aggregation by TPM and to intuitively examine the interaction between TPM and Aβ_1-42, molecular docking simulations of TPM to Aβ_1-42 were performed. The final simulated configurations of the docking simulation of TPM and Aβ_1-42 are shown in Fig. 3A and B. There are two possible configurations of the Aβ_1-42-TPM complex which were named as result A and result B.

Fig. 3 Molecular docking model of Aβ42 monomer binding to TPM. (A) The TPM molecule is bound to Aβ_1-42 via two intermolecular H bonds between TPM and Lys28/Val24. (B) The TPM molecule is bound to Aβ_1-42 via two intermolecular H bonds between TPM and Lys28.

It can be observed in the simulation that the TPM molecule is bound to Aβ_1-42 at the turn structure of Aβ_1-42 and that the complex is stabilized either by two intermolecular H bonds between TPM and Lys28/Val24 as well as by hydrophobic interactions between TPM and Gly25/Ala21 (result A, Fig. 3A) or by two intermolecular H bonds between TPM and Lys28 as well as by the hydrophobic interaction between TPM and Val39/Leu34/Met35/Val24/Ala21/Phe20 (result B, Fig. 3B). The free energy, internal energy and entropy of result A at 25 °C are −1362.95 kcal mol⁻¹, 1.28 kcal mol⁻¹ and 4.58 kcal mol⁻¹ K⁻¹, respectively; while the free energy, internal energy and entropy of result B at 25 °C are −1362.22 kcal mol⁻¹, 2.02 kcal mol⁻¹ and 4.58 kcal mol⁻¹ K⁻¹, respectively. Notably, the greatest number of H bonds are formed between TPM and Lys28, an amino acid in the turn structure of Aβ_1-42. Furthermore, the interaction of TPM and Aβ_1-42 occurs mostly between amino acids in the turn structure. It is thus possible that molecular interaction between TPM and Aβ_1-42 could prevent misfolding of Aβ_1-42 and thus inhibit the aggregation of Aβ_1-42.

3.4. The inhibitory effect of TPM on Aβ aggregation

We also investigated the action of TPM on Aβ_1-42 aggregation to gain insight into the mechanism of this interaction. The ThT/ThS staining results indicated that worms treated with TPM have fewer of the light spots that represent Aβ_1-42 aggregations in the head region (Fig. 4A and B). The data from the ThT/ThS confocal laser scanning microscopy experiments showed that the 10 mM TPM-treated group displayed the lowest fluorescence intensity relative to the control group (Fig. 4A and B). This demonstrates that GMC101 treated with 10 mM TPM has less Aβ_1-42 aggregation in body wall muscle cells. Quantification of the ThT fluorescent staining showed that TPM treatment at 10 mM significantly down-regulated Aβ_1-42 aggregation density by 19.5% in transgenic GMC101 (Fig. 4C); at the same time, the ThS fluorescence staining results showed that 10 mM treatment down-regulated Aβ_1-42 aggregation density by 11.2% in transgenic GMC101 (Fig. 4D). The results indicate that TPM alleviates paralysis by reducing Aβ_1-42 aggregation in nematode muscle cells.

Fig. 4 TPM efficiently inhibits amyloid aggregation. (A) Thioflavin T fluorescence staining of β-amyloid showed that the 10 mM TPM-treated group had the lowest fluorescence intensity and the fewest light spots. (B) Thioflavin S fluorescence staining of β-amyloid in GMC101. The group treated with 10 mM TPM displayed the lowest fluorescence intensity and the fewest light spots. (C, D) ThT/ThS fluorescence intensity was quantified in the control and 10 mM TPM-treated GMC101 groups, with four experiments in each group. Forty worms were used in each experiment. All experiments were performed three times.

3.5. TPM alleviates Aβ-induced paralysis under conditions of oxidative stress in GMC101

To determine how oxidative stress influences paralysis in C. elegans, we conducted a test using juglone as a reactive oxygen species-generating compound. Using GMC101 cultured without juglone as a control, nematodes were exposed to a range of juglone concentrations. Larval nematodes were prepared as described above. After transfer of the worms to 25 °C for 42 h, we found that, except for the group treated with 20 μM juglone, the animals under oxidative stress induced by juglone became paralyzed significantly faster than the control animals (Fig. 5A). This indicates that oxidative stress promotes paralysis. To eliminate the negative influence of juglone on nematode growth, we exposed the nematodes to the same range of concentrations of juglone at 20 °C. The results showed that 100 μM and 150 μM juglone impaired the growth of C. elegans, as shown in Fig. 5B. Thus, the optimal concentration of juglone used in the oxidative stress model of paralysis in GMC101 is 50 μM.

Fig. 5 TPM alleviates Aβ-induced paralysis under conditions of oxidative stress in GMC101. (A) Juglone-induced oxidative stress accelerated paralysis at 25 °C in a dose-dependent manner. (B) At 20 °C, 20 μM and 50 μM juglone had no effect on nematode growth. (C) Oxidative stress accelerated the development of paralysis at 25 °C, whereas 10 mM TPM significantly alleviated paralysis under oxidative stress. (D) The time required for 50% paralysis to occur in the juglone treatment group was approximately 24 hours, much shorter than in the control group, and TPM treatment extended the time required for 50% paralysis to occur. Each curve is based on three individual experiments. *P < 0.05.

The effects of TPM on alleviating paralysis under oxidative stress were next studied. Under conditions of oxidative stress, TPM at 10 mM significantly decreased the ratio of paralysis in GMC101 by 18.7% (Fig. 5C). The time required for 50% paralysis to occur in the juglone treatment group was 23.99 hours, whereas TPM treatment extended the time required for 50% paralysis to 26.76 hours (Fig. 5D). Compared with the results shown in Fig. 1, the data clearly show that TPM is more effective under conditions of oxidative stress. As excessive ectogenic stimulation makes AD more and more common, TPM could be an effective candidate to relieve symptoms of AD.

3.6. Effect of TPM on intracellular ROS levels in vivo

To further understand the mechanism by which TPM inhibits Aβ-induced paralysis, the free-radical scavenging ability of TPM was evaluated to determine whether this ability is the mechanism responsible for alleviating Aβ-induced paralysis in transgenic C. elegans. We previously reported that TPM exhibits a ROS-scavenging ability in C. elegans N2.¹⁴ Herein, as shown in Fig. 6, TPM administered at 10 mM significantly inhibited the production of ROS in transgenic C. elegans strain GMC101 as detected in a 120 min time course assay (Fig. 6A) as well as 10 mM glutathione. In the next assay, TPM (10 mM) pretreatment effectively reduced ROS accumulation in juglone (100 μM)-treated GMC101 (Fig. 6B). Under oxidative stress, TPM has a better effect on scavenging ROS than glutathione. These results show that TPM is a powerful free radical scavenger in vivo and that its ROS-scavenging activity can contribute to its paralysis-alleviating action.

Fig. 6 Free-radical-scavenging effect of TPM in vivo. (A) 10 mM TPM and 10 mM glutathione decreased ROS accumulation in GMC101 under normal culture conditions. (B) 10 mM TPM and 10 mM glutathione decreased ROS accumulation in GMC101 under conditions of juglone-induced oxidative stress. Each curve is based on three individual experiments. Error bars indicate SE. **P < 0.01.

3.7. The inhibitory effect of TPM on a series of Aβ_1-42 oligomers in GMC101

The effect of TPM on Aβ_1-42 oligomer formation was also investigated. Tris-Tricine gels were used to identify the Aβ species. The PVDF membranes were treated with enhanced chemiluminescent substrate, and the chemiluminescent signals were captured using an Integrated Automatic Chemiluminescent Imaging and Analysis System (SmartChemi, Sage, China). The results are shown in Fig. 7. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for western blotting (Fig. 7A and B). GMC101 worms grown under conditions of oxidative stress displayed more Aβ_1-42 aggregation than GMC101 worms grown in a normal environment (Fig. 7A and B). TPM reduced the aggregation of Aβ_1-42 oligomers both under conditions of oxidative stress and in the normal environment (Fig. 7A and B). Consistent with the paralysis results, the higher concentration of TPM was more effective. However, the result shown in Fig. 7A indicates that TPM has little effect on high-molecular-weight Aβ_1-42 oligomers. Because low-molecular-weight Aβ_1-42 oligomers are considered the most neurotoxic agents,⁴ the effect of TPM on reducing the formation of low-molecular-weight Aβ_1-42 oligomers could contribute to the action of TPM.

Fig. 7 Effect of TPM on Aβ_1-42 oligomer formation. (A) GAPDH was used as an internal control in this assay. TPM reduced the aggregation of low-molecular-weight Aβ_1-42 oligomers under conditions of oxidative stress induced by juglone. (B) TPM reduced the aggregation of low-molecular-weight Aβ_1-42 oligomers under normal conditions but had little effect on the aggregation of high-molecular-weight Aβ_1-42 (76 kD) oligomers. Using Image J software, we quantitated the Aβ species (8, 12 and 76 kD). (C) Different concentrations of TPM (10 mM/1 mM) had different effects on Aβ_1-42 dimer formation. 10 mM TPM treatment reduced Aβ_1-42 oligomers (8 kD) by 26.4% and 26.2% under normal and oxidative stress conditions, respectively. When TPM was used at 1 mM, the amount of Aβ_1-42 oligomers (8 kD) was reduced by 6.6% and 7.0% under these conditions. (D) TPM at 10 mM reduced the amount of Aβ_1-42 oligomers (12 kD) by 17.5% and 28.9% under oxidative stress and normal conditions, respectively, whereas TPM at 1 mM reduced the amount of Aβ_1-42 oligomers (12 kD) by 9.6% and 20.2% under these conditions. (E) TPM had little effect on the aggregation of high-molecular-weight Aβ_1-42 oligomers; treatment with 10 mM and 1 mM TPM reduced the amount of Aβ_1-42 oligomers (76 kD) by 5.0% and 1.8%, respectively. The bars represent the mean ± S.E.M. *P < 0.05.

The image analysis of these experimental results are shown in Fig. 7C–E. Administration of 10 mM TPM reduced the amount of Aβ_1-42 oligomers (8 kD) and Aβ_1-42 oligomers (12 kD) produced in the normal environment by 26.2% and 28.9%, respectively, whereas under oxidative stress conditions it reduced the amount of Aβ_1-42 oligomers (8 kD) and Aβ_1-42 oligomers (12 kD) by 26.4% and 17.5%, respectively (Fig. 7C and D). The results shown in Fig. 7E indicate that TPM does not significantly interfere with the aggregation of high-molecular-weight Aβ.

4. Discussion

Many bioactive peptides of short length (e.g., 2–9 amino acids)²⁹ are known to be associated with various physiological actions.³⁰ TPM has been reported to exhibit longevity-improving effects in the nematode C. elegans under stress conditions.¹⁶ However, TPM has not been studied as a drug candidate that could efficiently alleviate AD. In this study, we found that TPM significantly prolonged the time to paralysis in the transgenic C. elegans strain GMC101 in a normal environment or under oxidative stress conditions and that it showed a dose-dependent effect. Because TPM, which works as an effective antioxidant, significantly prolonged the lifespan of C. elegans, we considered oxidative stress as a possible precursor to AD. We used juglone as an oxidant to determine whether oxidative stress could enhance the symptoms of AD. Our research indicated that worms treated with 20 μM or 50 μM juglone displayed normal activity at 20 °C (Fig. 5B), indicating that treatment with juglone at these concentrations is not harmful to the growth of GMC101 at 20 °C. However, in worms treated with 50 μM juglone at 25 °C, the level of paralysis increased. These results show that oxidative stress induced by juglone promotes the process of paralysis and that 50 μM is the optimal concentration of juglone for use in the oxidative stress model of GMC101.

Moreover, we found that the effect of TPM on ROS generation, similar to that of other bioactive peptides produced from plants and animals,¹³ is the same in the transgenic C. elegans strains employed in this study (Fig. 6A and B) as in wild-type animals¹⁶ under both stress conditions and normal environmental conditions. This should contribute to the positive effect of TPM on the prevention of paralysis. However, not all ROS scavengers, including L-ascorbic acid, can alleviate paralysis.³⁰ L-Ascorbic acid has the ability to reduce superoxide production but cannot alleviate paralysis.⁵ Oxidative stress induced by Aβ_1-42 may contribute to the neurodegenerative process that occurs in Alzheimer's disease.¹² Tissue injury can itself induce ROS generation.³¹ This is due to the complex mechanism of Alzheimer's disease, which is still poorly understood.³² Therefore, we cannot be sure whether oxidative stress induces AD or whether oxidative stress occurs after amyloid accumulation.^33,34 Even if oxidative stress is not the primary trigger of AD, it still can lead to accelerated oligomeric deposition and ultimately aggravate the severity of the illness. TPM can scavenge ROS in vivo and prevent oxidation in GMC101 because of its proton donor capacity, which may derive from the amino acid sequence of TPM, Leu–Asp–Tyr–Glu. Tyr has a phenolic hydroxyl group that can function as a proton donor that can quench free radicals.³⁵ In addition, the carboxylic acid moieties in Asp and Glu have electronic absorption effects that decrease the density of the electron cloud around the phenolic hydroxyl group, resulting in the release of protons.¹⁷ TPM is effective at scavenging free radicals and inhibiting amyloid aggregation because of its capacity to act as a proton donor and the stability of its structure.

To further understand the mechanism of TPM's effect, we investigated the effect of TPM (Leu–Asp–Tyr–Glu) on Aβ_1-42 aggregation in vitro. We measured the fluorescence intensity of Aβ_1-42 aggregation as a function of incubation time in vitro (Fig. S1†). We found that TPM significantly inhibited the agglomeration of Aβ aggregations. According to Lührs et al.³⁶ and Irie et al.,³⁷ Aβ peptides form a β-sheet structure in which Met35 can form radical species that show neurotoxicity through interactions with the Tyr10 phenoxy radical via intermolecular hydrogen bonding and aggregate to form oligomers, protofibrils and fibrils. Our molecular docking simulations of Aβ_1-42 binding to TPM indicated visually that the molecular interactions between TPM and Aβ_1-42 are focused on the amino acids in the turn region, including Phe20/Ala21/Val24/Gly25/Lys28/Leu34/Met35/Val39, and that binding of TPM to Aβ_1-42 thus impedes the formation of β-sheet structures and diminishes the neurotoxicity of Aβ aggregates. Because small peptides can be absorbed directly in the intestine 2 to 3 times more rapidly than amino acids and proteins and thus exert their biological functions more rapidly,³⁸ TPM may rapidly and effectively interact with Aβ_1-42 in GMC101 nematodes, inhibiting its aggregation and thus alleviating paralysis.

Our fluorescence staining results demonstrated that GMC101 indeed contains intracellular Aβ_1-42 deposits. Oligomers of Aβ_1-42 specifically accumulated in the muscle cells of paralyzed GMC101 worms.^18,39 Our ThT/ThS staining results indicated that worms treated with TPM contained fewer Aβ aggregations than control animals. Moreover, in western blot experiments, we found that TPM protected C. elegans from Aβ damage by reducing the aggregation of low-molecular-weight Aβ_1-42 oligomeric species such as Aβ_1-42 dimers and trimers. More detailed experiments are ongoing to detect specific interactions that may occur between TPM and Aβ deposits. TPM scavenged ROS in vivo, thereby preventing oxidation in GMC101 as well as directly inhibiting Aβ aggregation. It thus reduced the intracellular deposition of Aβ. By reducing the deposition of Aβ aggregates, TPM can slow the progression of AD-induced paralysis.

5. Conclusions

We created an oxidative stress model for the transgenic C. elegans strain GMC101. Using this model, we observed that oxidative stress induced by juglone could accelerate the process of paralysis and that TPM, a multifunctional bioactive peptide obtained from maize, could alleviate Aβ-induced paralysis. We showed that TPM not only has antioxidant effects but can also influence Aβ aggregation in vivo, thereby reducing the oligomeric aggregation of Aβ. We also showed clearly that TPM protects C. elegans from Aβ damage by reducing the number of oligomeric Aβ species, as observed by western blotting. This reduction in Aβ aggregation leads to the stabilization of misfolded proteins, thereby increasing the nematode's health and protecting the transgenic C. elegans strain GMC101 against paralysis. Therefore, TPM is a potential therapeutic agent for use in the treatment of Alzheimer's disease.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (No. 81271697, 81571791, and 31571017), the National “Significant New Drug Creation” Science and Technology Major Program (No.2012ZX09503001-003), the Specialized Research Fund for the Doctoral Program of Higher Education (20100061120077), and the Project of Science and Technology Department of Jilin Province, China (No. 20130206069GX).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06130c

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