Nude mice (weight: 26 to 28 g; age: 2 months), APP/PS1 mice (weight: 25.3 to 28.1 g; age: 8 months) and C57/6J mice (weight: 25.5 to 28.6 g) were purchased from the institute of laboratory animal sciences, CAMS & PUMC (Chinese Academy of Medical Sciences and Peking Union Medical College). APP/PS1 mice have been previously demonstrated to form amyloid plaques, which have been approved by institute of laboratory animal sciences, CAMS and PUMC. All animal experiments were performed in accordance with the China Physiological Society “Guiding Principles in the Care and Use of Animals” approved by Tianjin Medical University Animal Care and Use Committee (NO. 20130021).

H102, FITC-labeled HPYD and HPYD were synthesized using the Fmoc solid-phase synthesis method and purified by HPLC (Gill Biotechnology Company, Shanghai, China). The compounds were greater than 95% pure as measured by HPLC-MS. HPYD, a polypeptide comprising the amino acid (AA) sequence of His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp, was dissolved in normal saline.

0.5 mg of HPYD or H102 was dissolved in 1 mL of 0.2 mol/L phosphate buffer containing 100 mg/L of trypsin. The trypsin solution and polypeptide were mixed at a ratio of 1:4, and the solution was placed in a temperature controlled water bath shaker at 37°C for 0, 40, 100, 160, 220, 280, 340, and 400 min. The samples were then heated to 80°C for 10 min, and the stability of HPYD and H102 was detected at the different time points. High performance liquid chromatography (HPLC) analysis of HPYD and H102 was performed using a Phenomerex C18 column (250 mm × 4.6 μm, 5 μm; Sigma, Inc. U.S.A.). The mobile phase consisted of solvent A, 0.1% TFA in acetonitrile and solvent B, 0.1% TFA in water at a ratio of 22.5:77.5 (v/v). The sample injection volume was 20 μL. The flow rate was 1 mL/min, and the detection wavelength was 220 nm.

Aβ₁₋₄₂ freeze-dried powder was dissolved in 50 mmol/L sodium phosphate buffer solution (pH = 7.4) to a concentration of 22.15 μmol/L, and H102 and HPYD were dissolved in the same PBS solution to a concentration of 88.60 μmol/L. Aβ₁₋₄₂ solution was then mixed with H102 and HPYD respectively in equal volumes. The Aβ fibrils were grown at 37°C for 24 hrs. 10 μL solution from each group was then added to 990 μL of 3.0 μmol/L ThT solution and fluorescent intensity was measured using a VARIAN PTC-Au00-01058 fluorescence spectrophotometer (VARIAN, USA) with an excitation wavelength of 453 nm and emission of 478–486 nm.

The Aβ₁₋₄₂ samples (11.07 μmol/L 20 μL) were incubated in 37°C for 5 days, and the mixture of Aβ₁₋₄₂ (22.15 μmol/L 10 μL) with HPYD (88.61 μmol/L 10 μL) or H102 (88.61 μmol/L 10 μL) were incubated under the same conditions for 5 days. Aliquots (5 μL) of each sample was spotted onto thin carbon substrates supported by carbon film on a 300 mesh copper grid for 15 min and blotted dried. The TEM grids were negatively stained with 2% uranyl acetate for 2 min and air dried. The samples were subsequently imaged using a Hitachi H-600 transmission electron microscope.

Nude mice were anesthetized using 20% urethane (5 mL/kg), and then placed on the VFIS observation platform. Nude mice were then irradiated at 490 nm excitation without treatment, and then irradiated for 5, 15, and 30 min after nasal administration with FITC-HPYD (5.535 mg/kg). Thirty minutes after nasal administration with FITC-HPYD, the mice were euthanized and the hippocampus, cortex, olfactory bulb, heart, lung, liver, spleen and kidneys were removed. These organs were then irradiated at 490 nm excitation.

The APP/PS1 transgenic mice were randomized into the model and HPYD treatment group (n = 12 for each group). C57BL/6J mice with the same genetic background and age served as the normal controls. Treatment group received intranasal administration of HPYD saline solution (33 mg/mL, 5 μL/d), while the normal control group and model group were given with the same volume of saline for 30 days.

The behavioral test was performed after 30 days of intranasal administration using the Morris Water Maze (MWM) according to our previous report (Lin et al., 2014). Briefly, the MWM test was conducted in a circular pool with a diameter of 80 and 32 cm deep. The pool was filled with 25 ± 1°C water to a depth of 1 cm higher than the escape platform. The water was made opaque white to hide the escape platform. The orientation and navigation experiments were conducted for 5 days to record the time intervals when the animals climbed onto the platform. If animal failed to find the hidden platform within 90 s, the mouse was placed back onto the platform for 20 s, and the escape latency was recorded as 90 s. The platform was placed in the center of the third quadrant and remained in the same position throughout the orientation and navigation experiment. On the 6th day, the platform was removed, and each mouse was allowed to swim freely for 90 s to record the frequency of passing the hidden platform and the original angle.

At the end of the MWM test, the mice were euthanized and their brains were removed and rapidly placed on ice. The brain tissues were then fixed in 4% paraformaldehyde solution and waxed. Tissue sections were then dewaxed, and antigen retrieval was performed using boiling citrate buffer solution. This was followed by incubating the sections in 3% H₂O₂ solution at room temperature for 10 min to block endogenous peroxidase activity. The sections were then blocked by incubating with normal goat serum at room temperature for 15 min, and subsequently diluted antibodies (Aβ: 1:100; APP: 1:100) were added and incubated overnight at 4°C. Biotin-labeled secondary antibody was added and incubated for 15 min at room temperature, followed by incubation with strept avidin-biotin complex (SABC) and DAB chromogenic reagent. Finally, the sections were counterstained with hematoxylin and observed under a microscope.

Western blot analysis was performed according to the detail procedures described previously. Primary Aβ antibody (1:200) was purchased from Abcam (USA) and anti-APP antibody (1:200) was purchased from Wuhan BOSTER Bio Company (Wuhan, China). The secondary goat anti-rabbit antibody was purchased from Sigma-Aldrich (St Louis, MO, USA).

Microarray hybridization was carried out by Shanghai GMINIX Biotech Limited Company (China) using the GeneChip® Mouse Gene 1.0 ST Array (Affymetrix, USA Scientific) with 770,317 probes. Briefly, total RNA was extracted from frozen brain tissues using the RNeasy mini kit (Qiagen, Valencia, CA) and used for cDNA synthesis using the cDNA synthesis kit (Affymetrix, Inc., USA). cDNA was labeled using the Gene Chip2 WT Terminal Labeling Kit (Affymetrix, Inc., USA), and then the labeled cDNA was hybridized to the mouse Gene chip at 45°C for 16 h. The Gene chip was then washed with wash solution A, wash solution B and deionized water, and stained using Cocktail 1 and Cocktail 2. The Gene Chip 2 Scanner 300 7G (Affymetrix Inc.) and the AGCC Scan Control software was used for date analysis. The gene ontology (GO) enrichment analysis was performed using the GOEAST software toolkit (P ≤ 0.05), and signaling pathway analysis was performed using the KEGG data software. Software Matlab 7.1 and java, was used to build and analyze the dynamic Gene networks, and network maps of the differentially expressed genes were constructed from the different states.

The Gene Expression Omnibus accession number for normal mice, model mice and HPYD mouse expression profiles is GSE104249.

RT-qPCR analysis was performed using an ABI 7500 thermocycler (Applied Biosystems) with UltraSYBR Mixture purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China). Gene transcript normalization was performed using housekeeping gene β-actin. All primers used for RT and qPCR are listed in Table 1.

All experiments were performed at least three times. Data is presented as mean ± SD. The date of escape latency from MWM were analyzed by multivariate analysis of variance (ANOVA) and the other data were analyzed by one-way ANOVA, followed by Student-Newman-Keuls test. All the analysis was performed by SPSS statistical software (version 21, IBM, Armonk, NY). P ≤ 0.05 was considered to be statistically significant (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001).

To investigate the stability of HPYD in vitro, accelerated stability test using trypsin was performed. After treatment with 100 mg/L trypsin at 37°C for 400 min, followed by heat treatment for 10 min at 80°C, the concentration of HPYD was 81.67% ± 0.42, while the concentration of H102 was only 41.07% ± 0.17, indicating that the stability of HPYD was better compared to H102 (Table 2). In addition, to compare the inhibitory effects on aggregation of Aβ₁₋₄₂ with H102, ThT fluorescence spectral analysis and TEM was performed. Results showed that HPYD could significantly inhibit the aggregation of Aβ₄₂ compared to H102 (Figures 1A,B). Taken together, this demonstrates that HPYD had better in vitro stability and inhibitory effects on Aβ₁₋₄₂ aggregation compared to H102.

HPYD, a β-sheet breaker peptide of 10 AAs, is difficult to administer orally or intravenous because it easily undergoes rapid in vivo degradation in the gastrointestinal tract and blood. Therefore, to determine whether HPYD can be administered to the brain through the olfactory pathway after nasal administration, HPYD was labeled with FITC and observed by fluorescence imagining of the brain. Before nasal administration, nude mice were irradiated at 490 nm excitation wavelength, and then were subsequently administrated with FITC-HPYD though nasal cavity and irradiated after 5, 15 and 30 min with wavelength of 490 nm. Results showed that FITC-HPYD first appeared at the brain area at 5 min after nasal administration, and the fluorescence gradually expanded with time. As shown in Figure 2A, 30 min after nasal administration, the fluorescence had spread over the whole body of the nude mouse, however, the fluorescence intensity in the brain was the strongest compared to other organs. The mice were then euthanized and the hippocampus, cortex, olfactory bulb, heart, lungs, liver, spleen and kidneys were removed at 30 min after nasal administration and irradiation. The results indicated that the fluorescence intensity of the olfactory bulb was the strongest, with the cortex and the hippocampus having the second and third strongest intensities, respectively. The other major organs showed varying degrees of fluorescence intensity. Among these organs, the lungs had the strongest intensity, followed by the liver (Figure 2B). These results demonstrated that FITC-HPYD could be delivered into the brain through nasal administration.

MWM test for behavioral analysis is a common strategy to investigate the learning and memory ability in AD mouse models. To determine the efficacy of HPYD on APP/PS1 transgenic mice, the MWM test was conducted for 6 days. First, the escape latency test was performed from Day 1 to Day 5. As shown in Figures 3A,B, the control group and the HPYD group displayed no significant differences from Day 1 to Day 5. In addition, the control group vs. the model group, or the model group vs. the HPYD group, showed no significant difference from Day 1 to the Day 3. However, significant differences were observed between the HPYD group and the model group at Day 4, and in addition, statistical significant differences between the model group and control group were observed on Day 4. The escape latency test indicated that the APP/PS1 transgenic mice, normal mice and the HPYD group mice all required time to find the platform at the start of the test, however, the learning and memory ability demonstrated significant differences after treatment with HPYD in APP/PS1 transgenic mice after 3 days of training. On the 6 day, spatial probe test was performed on the mice without the platform. As shown in Figures 3C–E, memory ability was estimated by the frequency passed the hidden platform (Figure 3C) and original angle (Figure 3D). Frequency passed the hidden platform in the target quadrant displayed significant differences between the model group and the control group, indicating that the model group had a poor ability to find the platform compared to the control group. However, the HPYD group demonstrated an improved ability to find the platform compared to the model group. No significant differences were observed between the HPYD group and the control group. Finally, the ability to locate the original angle was also investigated at the 6th day. The HPYD group was able to find the location of the original angle much quicker compared to the model group, indicating that the HPYD group mice had relatively good memory. No significant differences were observed between the control group and the HPYD group. Collectively, these results indicate that HPYD could improve the learning and memory ability in APP/PS1 transgenic mice after nasal administration of HPYD.

HPYD is a novel β-sheet breaker peptide that could inhibit Aβ₁₋₄₂ aggregation in vitro. To determine the effect of HPYD on Aβ in vivo, HPYD was administrated though the nasal cavity into the brain. Tissues were then harvested after 6 days for immunohistochemistry and western blot analysis. As shown in Figure 4A, the hippocampus CA1 and cortex had higher expression of Aβ in the model group mice compared to control group mice, while Aβ was lower in the HPYD group compared to the model group. No significant differences were observed between the control group and the HPYD group. In addition, western blot analysis showed that Aβ was highly expressed in the hippocampus and cortex of the model group mice compared with the control group, while Aβ was expressed at a lower level in the HPYD group mice compared to the model group mice (Figure 4B). There were no statistical significant differences between the HPYD group mice and control group mice. APP, the precursor of Aβ, is upregulated in the brain tissues of AD patients. To determine whether HPYD had an effect on APP expression, we determined the expression of APP using immunohistochemistry and western blot (Figures 4B,C). Results showed that APP was upregulated in the brain tissues of the model group mice compared to the control group mice, while it was downregulated in the HPYD group mice compared with the model group mice. The expression of APP in the HPYD group mice was similar to the control group. These results indicate that HPYD can reduce Aβ and APP protein levels in APP/PS1 transgenic mice.

To investigate the effect of HPYD on gene expression of APP/PS1 transgenic mice, the brain tissues of the control group, model group and HPYD group mice were harvested at the termination of the MWM test. Total RNA from brain tissues were isolated and microarray analysis was performed by Shanghai GMINIX Biotech Limited. Based on microarray analysis, 119 genes with significant differences were selected for further analysis (Figure 5A). Among the selected genes, 15 genes were markedly down-regulated in the model group compared to the control group, however, the expression levels of these genes were restored to normal levels after treatment with HPYD. Additionally, the majority of the selected genes were up-regulated in the model group compared to the control group, which were restored to normal levels after treatment with HPYD. These differentially expressed genes were then analyzed by GO analysis to predict their function. The most enriched GO terms are shown in Figure 5B, and includes; protein binding, extracellular exosome, immune system, inflammatory reaction and gluco-lipid metabolism. KEGG pathway analysis showed that these genes were involved in many inflammatory response pathways, including viral carcinogenesis, RIG-I-like receptor signaling and NOD-like receptor (NLR) signaling pathway (Figures 5C,D). Co-expression network analysis revealed gene-function relationships of the differential gene expressions, including CDK1, PLA2G4A, APP and SCN9A, which were reported to be involved in AD (Ling et al., 2003; Schaeffer et al., 2010; Hilgeroth et al., 2014). In addition, many inflammatory related genes, such as CLEC4D and CLEC5A were differentially expressed. These two genes are members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily that play important roles in inflammation and immune response (Wu et al., 2013; Wilson et al., 2015). Other genes of interest include; Inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) that mediates non-canonical activation of the NLRP3 inflammasome (Schmid-Burgk et al., 2015). NAIP2 and NAIP5, belong to the NAIP/NLRC4 inflammasomes, and play important physiological roles in antibacterial defense and inflammation (Diebolder et al., 2015; Zhang et al., 2015). In addition, 18 genes associated with AD or inflammatory responses were selected for validation by RT-qPCR. There was a good concordance in the expression levels of these genes by RT-qPCR and microarray (Figure 6). Taken together, the above results indicated that dysregulated expression of several genes in the model group mice could be restored to normal levels after treatment with HPYD, indicating that HPYD may be a potential therapeutic candidate for treatment of AD.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.