Tocotrienol-Rich Fraction Modulates Amyloid Pathology and Improves Cognitive Function in AβPP/PS1 Mice

Preparation of TRF and PO

An in vitro assay was conducted to study the effects of TRF on Aβ₄₂ aggregation. The TRF and palm oil stripped of vitamin E (PO) were purchased from Sime Darby Foods and Beverages Sdn Bhd (Selangor, Malaysia) and Malaysian Palm Oil Board (MPOB) respectively. The TRF used consists of α-tocotrienol (196.0 mg/g), β-tocotrienol (24.0 mg/g), γ-tocotrienol (255.0 mg/g), δ-tocotrienol (75.0 mg/g), and α-tocopherol (168.0 mg/g), while vitamin E was not detected in the PO as mentioned by Nesaretnam, et al. [29]. The TRF was prepared for experimental application according to a previous study with modification [30]. Briefly, TRF or PO stock at 5% v/v was dissolved in 100% ethanol and stored at –30°C. Prior to use, 60 μL of 5% v/v TRF or PO stock was incubated with 45 μL of phosphate-buffered saline (PBS) at 37°C overnight. To obtain 1% v/v TRF or PO, 40 μL of 100% ethanol and 55 μL of PBS were added to the TRF or PO mixture, and then 100 μL of a mixture containing 100% ethanol and PBS [1:1] was added. Further dilutions were carried out using PBS to obtain concentrations of 0.001%, 0.01%, and 0.1% v/v TRF or PO.

Preparation of Aβ₄₂ aggregates

Human Aβ_1-42 peptide (Aβ₄₂, Peptide Institute, Osaka, Japan) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 1 mM [31]. The mixture was then incubated at 37°C for 1 h, aliquoted into microcentrifuge tubes and evaporated for 30 min in an evaporation chamber. The peptide residue was stored at –30°C until use. Prior to use, this HFIP-treated Aβ₄₂ was dissolved in dimethyl sulfoxide (DMSO; Wako, Osaka, Japan) to 0.5 mM, vortexed, and further diluted to 20 μM with PBS. To examine the effects of TRF and PO on Aβ₄₂ aggregation, the Aβ₄₂ solution was mixed with TRF or PO to a final concentration of 10 μM Aβ₄₂ plus 0.001%, 0.01%, or 0.1% v/v TRF or PO. As a control, PBS containing 5% ethanol was added to the Aβ₄₂ solution. The mixture was incubated at 37°C for 6, 24, and 48 h and then analyzed by gel electrophoresis, thioflavin T assay and cell viabilityassay.

Gel electrophoresis and western blotting

After incubation, samples were diluted in 4× sample buffer (62.5 mM Tris, pH 6.8, 1% sodium dodecyl sulfate (SDS), 25% glycerol) in PBS without heating to concentration of 2.25 μM. Ten-microliter of samples were loaded into 10% –20% precast polyacrylamide gels (SuperSep Ace; Wako) for electrophoresis (20 mA/gel for 65 min). After electrophoresis, samples were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Merck Millipore). Membranes were washed with Tris-buffered saline (25 mM Tris-HCl, pH 7.4, 0.9% NaCl) containing 0.1% Tween 20 (TBS-T) for 10 min, blocked with 5% skim milk in TBS-T for 60 min, washed three times with TBS-T (7 min/wash), and incubated with mouse monoclonal anti-Aβ antibody clone 6E10 (1:2000; Covance, Princeton, NJ, USA) in TBS-T at 4°C overnight. After three washes with TBS-T (7 min/wash), immunolabeled membranes were incubated with HRP-conjugated anti-mouse IgG antibody (1:20000; Jackson ImmunoResearch, West Grove, PA, USA) in TBS-T containing 0.5% skim milk for 60 min at room temperature (RT). The membranes were then washed three times with TBS-T, and labeling was visualized by chemiluminescence (SuperSignal West Pico; Thermo Scientific, Rockford, IL, USA) using a lumino image analyzer (ImageQuant LAS 4000 mini; GE Healthcare Life Sciences, Japan).

Thioflavin T (ThT) fluorescence assay

A stock solution was prepared by dissolving ThT (AnaSpec, Fremont, CA, USA) in DMSO at 5 mM. A 20 μL sample of Aβ₄₂ incubated with TRF, PO, or PBS (containing 5% ethanol) was periodically added to 180 μL of 5 μM ThT dissolved in PBS and the fluorescence measured in 96-well black plates (Greiner Bio-one, Frickenhausen, Germany) using an Infinite M200 microplate reader (Tecan, Grödig, Austria) at excitation and emission wavelengths of 450 nm and 482 nm, respectively. As a control, ThT was mixed with PBS containing only TRF or PO. The Aβ₄₂-specific fluorescence was calculated by subtracting the fluorescence intensity of the control solution. Each measurement was performed in duplicate. Data represent the means of three independent experiments.

Photo-induced cross-linking of unmodified protein (PICUP) assay

The PICUP assay was performed according to a previous study [32]. Briefly, a mixture of 50 μM Aβ_1-40 or Aβ_1-42 peptides, 2 mM tris(2,2^′-bipyridyl)dichlororuthenium(II) and 40 mM ammonium persulfate was mixed with TRF or PO at 1:1 ratio. Immediately, the mixture was cross-linked by irradiation to visible light for 1 s and then the reaction was quenched by addition of Tricine sample buffer and dithiothreitol. The samples were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions with 15–20% Tricine gel (SuperSep Ace; Wako) and the protein was visualized by silver staining with a SilverQuest Staining Kit (Invitrogen, California, USA).

Cell viability assay

Human neuroblastoma cell line SH-SY5Y (ATCC, Manassas, Virginia, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (Nacalai Tesque, Kyoto, Japan) containing 10% (v/v) fetal bovine serum in a humidified atmosphere of 5% CO₂/95% air at 37°C.

Ten-micromolar Aβ₄₂ was incubated at 37°C with 0.001%, 0.01%, and 0.1% v/v TRF or PO as described in the method above. The vehicle was served as a control containing 5% ethanol. For treatment, the mixtures were dissolved in DMEM/Ham’s F-12 (Nacalai Tesque) to final concentration of 0.3 μM Aβ₄₂ and 0.00003%, 0.0003%, 0.003% v/v TRF or PO. The final concentration of vehicle was 0.15% ethanol in medium.

Cell viability was assessed by the MTT assay with a MTT Cell Count Kit (Nacalai Tesque). The assay was based on a reduction of tetrazolium salt (MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) by dehydrogenase in viable cells into a purple formazan product. Therefore, high amount of formazan formed reflects an increase in number of living cells. The cells used in this study was seeded at 5.0×10⁴ cells per well in a 96-well microplate and incubated at 37°C in humidified atmosphere for 24 h. After incubation, the culture medium was replaced with 100 μl of new medium with or without Aβ and TRF or PO. Ten-microliter of MTT solution was added to each well after 20 h of incubation at 37°C in humidified atmosphere and further incubated for another 4 h. Then, 100 μl of solubilization solution was added to the wells and incubated at 37°C in humidified atmosphere overnight. The absorbance was measured at 570 nm with a reference wavelength of 700 nm using an Infinite M200 microplate reader (Tecan). Each measurement was performed in triplicate. Data represent the means of three independent experiments.

Animals

AβPPswe/PS1dE9 double transgenic mice (AβPP/PS1), expressing a chimeric mouse/human amyloid protein precursor (Mo/HuAPP695swe) and a mutant human presenilin 1 with deletion at exon-9 (PS1-dE9), were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained by breeding heterozygous females with wild-type (WT) males, and offspring were genotyped by polymerase chain reaction. Only heterozygous and WT male mice were used in this study. All mice were housed at 23°C under a 12-h/12-h light/dark cycle (lights on from 8:00–20:00) with ad libitum access to food and water. Experiments were conducted with approval of the Animal Care and Use Committee of Shiga University of Medical Science.

Supplementation

The TRF was dissolved at 12 mg/mL in PO. Five-month-old AβPP/PS1 mice were divided into three groups. Two groups received daily water (control, 5 mL/kg body weight) or PO as a vehicle (5 mL/kg body weight) and the other received daily TRF (60 mg/kg body weight) by oral administration. All mice received daily supplementation from 5 to 15 months of age.

Novel object recognition test

The novel object recognition test was conducted according to a previous study with modification [33]. A circular home-built chamber was made with 38 cm in diameter, with a 22-cm high wall. The test was conducted over four consecutive days and consisted of four phases: pre-habituation, habituation, training, and testing. Wild-type mice (WT; n = 13) and AβPP/PS1 mice that received water (Cont; n = 9), TRF (n = 11), or PO (n = 10) were kept in the testing room at least 30 min before the test for acclimation. On day 1, pre-habituation was conducted by allowing each mouse to freely explore the empty chamber for 5 min. On days 2 and 3, mice were habituated in the empty chamber for 20 min. On day 4, each mouse was given a training trial followed by a testing trial. During the training trial, two identical objects were placed in opposite positions of the chamber at the same distance from the wall, and the mouse was allowed to explore the objects for 10 min before return to the cage. In the test trial conducted 1 h later, the mouse was placed again into the same chamber, but one of the identical objects had been replaced with a novel object. The mouse was allowed to freely explore the objects for 5 min before return to its cage. The objects used in this study were of different shapes and colors but identical in size and were fixed to the chamber floor to prevent movement. All objects and the chamber were cleaned with 70% ethanol after each trial.

Novel object recognition was measured by the ratio of novel object exploration time to total exploration time (familiar plus novel) or the recognition index. Exploration time was defined as the time the mouse spent within 2 cm of the object sniffing with nose directed toward the object or directly pawing or biting the object. Sitting or standing on the object was not recognized as exploration. To exclude environmental factor that may contribute to location preference independent of object familiarity, the ratio of exploration times of the two identical objects was also measured during the training trial. The formula for calculating location preference in the training trial and the recognition index in the testing trial are as follows:

Location preference = Time exploring one of the identical objects/Total time exploring both identical objects×100%

Recognition index = Time exploring novel object/(Time exploring novel object + Time exploringfamiliar object)×100%

Brain tissue preparation

Four mice from each group (water, PO, TRF) were used for biochemical analysis and histochemical staining. The rest of the mice were employed for other experiments. Mice were sacrificed at 15 months of age by cervical dislocation and brain tissue prepared as described previously [31]. In brief, the brain was removed and divided into two hemispheres. One hemisphere was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 24 h and then cryoprotected in 15% sucrose dissolved in 0.1 M PB containing 0.1% sodium azide for at least 2 days. Coronal sections were cut at 20-μm thickness using a cryostat and kept in 15% sucrose until used for immunohistochemistry or thioflavin-S staining (below). The other hemisphere was kept at –80°C until preparation of homogenate for biochemical measures of Aβ₄₀, Aβ₄₂, and Aβ oligomers. The frozen brains were cut into 1-mm thick coronal sections. To measure Aβ₄₀ and Aβ₄₂ levels, the hippocampus and cortex from the two brain sections were dissected. To measure Aβ oligomers, the hippocampus and cortex were combined because Aβ oligomers could not be detected in separate 1:3 dilutions of these regions (data not shown). Thus, we decided to use a lower dilution (1:1), which required a higher amount of brain tissue. The combination of the hippocampus and cortex enabled the detection of Aβ oligomers using an ELISA kit. Samples of the hippocampus, cortex, and the combined hippocampus and cortex were homogenized in cold TBS (1 mL/150-mg wet weight) containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Homogenized tissue was sonicated and centrifuged at 104,300× g for 60 min at 4°C. Supernatant was collected as soluble Aβ (TBS fraction) and pellet was then resuspended with 70% formic acid in water, sonicated, and centrifuged at 104,300× g for 60 min at 4°C. The recovered supernatant was neutralized with 1 M Tris base and stored at –80°C as insoluble Aβ (FA fraction). Protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules,CA, USA).

Immunohistochemical and thioflavin-S staining

All staining was performed using free-floating coronal brain sections fixed in 4% paraformaldehyde in 0.1 M PB. The brain sections were washed with 0.1 M PBS containing 0.3% Triton X-100 (PBS-T, pH 7.4) and then incubated in 0.3% hydrogen peroxide containing 0.1% sodium azide in PBS-T for 20 min at RT to eliminate endogenous peroxidase activity. After incubation, the sections were washed three times with PBS-T (10 min/wash) and blocked with 2% bovine serum albumin (BSA) in PBS-T for 30 min at RT. Blocked sections were then incubated with rabbit polyclonal antibodies against the N-terminal of human Aβ (1:500; Immuno-Biological Laboratories) or ionized-calcium binding adapter molecule 1 (Iba1, 1:5000; Wako) in PBS-T containing 0.2% BSA overnight at 4°C. After several washes with PBS-T, immunolabeled sections were incubated with biotinylated anti-rabbit IgG antibody (1:1000; Vector Laboratories, Burlingame, CA, USA) for 60 min at RT. Following several washes with PBS-T, sections were incubated with avidin–biotin complex solution (Vectastain ABC Elite kit, 1:3000; Vector Laboratories) for 60 min at RT, washed several times with PBS-T, and the immunostaining visualized by reaction with 3,3^′-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) and nickel ammonium. The sections were then mounted onto glass slides, dehydrated in an increasing ethanol series, and cover slipped with Entellan mounting medium (Merck Millipore). Images were captured using a DP27 digital camera (Olympus, Tokyo, Japan).

Thioflavin-S staining was performed according to a previous study [34]. Briefly, the brain sections from all supplemented groups were mounted onto glass slides, dehydrated with 70% and 80% ethanol for 1 min each and stained with freshly made and filtered 1% thioflavin-S (Chroma-Gesellschaft Schmid, Kongen, Germany) dissolved in 80% ethanol for 15 min in the dark. As a control, slices were subjected to the same staining protocol but omitting thioflavin-S. The slides were then washed with 80% and 70% ethanol for 1 min each, rinsed twice in distilled water, mounted with Immuno-Mount (Thermo, Pittsburgh, PA, USA), cover slipped, and dried for at least 2 h in the dark. The slides were examined with an upright Olympus BX61 microscope (Olympus, Japan) equipped with an FITC filter set (U-MWB; band-pass excitation 450–480 nm, dichroic mirror 505 nm, long-pass emission peak 515 nm).

Enzyme-linked immunosorbent assay

Levels of Aβ₄₀ and Aβ₄₂ in the cortex and hippocampus were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Wako) according to the manufacturer’s protocol. Briefly, 100 μL of homogenate was pipetted onto the primary antibody-coated microplate and incubated overnight at 4°C. Homogenate was then removed from wells and the plates rinsed five times with washing solution. Then, a 100-μL secondary antibody solution was added to each well. Plates were sealed and incubated at 4°C for 1 h for Aβ₄₂ detection and 2 h for Aβ₄₀ detection. After five rinses with washing solution, chromogen was added to each well, and blue color was developed in the dark. The reaction was terminated by addition of 100 μL stop solution, and absorbance was measured at 450 nm using an Infinite M200 microplate reader (Tecan). The levels of Aβ₄₂ and Aβ₄₀ were calculated using standard curves.

The oligomeric form of Aβ peptide was measured with ELISA kit from Biosensis (Thebarton, Australia). A 100-μL TBS fraction was added to the pre-coated microplate well and incubated for 24 h at 4°C. After washed for five times with washing solution, 100 μL of detection antibody was added to each well and the plate was incubated for 1 h on a shaker (140 rpm) at room temperature. After five rinses, streptavidin-HRP conjugate was added into each well and the plate was incubated for 30 min on a shaker at room temperature. Then, a 100-μL TMB solution was added to the well after five rinses and incubated for 8 min at room temperature in dark. The reaction was stopped by addition of 100 μL stop solution into each well. The absorbance was measured at 450 nm using an Infinite M200 microplate reader (Tecan). The levels of Aβ oligomers were calculated using a standard curve.

Evaluation of plaques

Aβ- and Iba-1-positive areas in the cortex and hippocampus were viewed with an Olympus BX50 microscope (Olympus, Tokyo, Japan) and images captured with a digital camera (DP27, Olympus). Images were analyzed using ImageJ 1.48 V (National Institutes of Health; USA). First, images were transformed to eight-bit grayscale. The threshold was set to 110, and the percent immunoreactive area was obtained. Four mice from each group were subjected to immunohistochemical staining, and for each mouse, five brain sections wereanalyzed.

Thioflavin-S-positive plaques were evaluated according to previous studies with modification [35, 36]. Images were captured at four different locations within the cortex and hippocampus at ×20 magnification. The images were transformed to eight-bit grayscale format using ImageJ. The calibration was set at 2.58 (distance in pixels), 1.00 (known distance), 1.0 (pixel aspect ratio), and μm (unit length) and “global” was activated. The threshold was set by comparison with the original images because of differences in background staining across sections. The number of plaques was counted setting the size to 100–10000 μm², in a defined area of 0.2 mm². Four mice from each group were used for thioflavin-S staining, and two brain sections were analyzed per mouse.

Data analysis

Statistical analysis was performed using Graphpad Prism 6 (GraphPad Software; La Jolla, CA, USA). Data are presented as mean±standard error of mean (S.E.M.). Group mean was compared by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test for multiple comparisons.

In vitro Aβ₄₂ fibrillogenesis was inhibited by TRF

We investigated direct interactions between Aβ and TRF in in vitro experiments. Firstly, SDS-polyacrylamide gel electrophoresis was carried out to study the effect of TRF on the profiles of Aβ₄₂ aggregates. Incubation of Aβ₄₂ with vehicle (PBS containing 5% ethanol) at 37°C for 24 h resulted in a large amount of high molecular weight Aβ₄₂ aggregate at the top of the gel and little “smear” down the length of the gel, indicating that high molecular weight Aβ₄₂ aggregate was too large to enter the gel (Fig. 1A). A similar gel staining pattern was observed for Aβ₄₂ incubated with 0.1% v/v PO. In contrast, a darker smear and more intense bands of low molecular weight Aβ₄₂ aggregates, which are small enough to enter the gel, were observed in gels loaded with Aβ₄₂ incubated in 0.1% v/v TRF. We further examined the dose-dependent effect of TRF on this disruption of high molecular weight Aβ₄₂ fibril formation. Indeed, as the concentration of TRF was increased from 0.001% to 0.01% and 0.1%, the intensity of lower molecular weight fibrils increased, and trimers and tetramers appeared. These results strongly suggest that TRF can disrupt the formation of high molecular weight Aβ₄₂aggregates.

Next, we investigated the effect of TRF on the formation of fibrillar Aβ₄₂ in vitro through ThT fluorescence assay after 0-, 6-, 24-, and 48-h incubation. The fluorescence intensity of Aβ₄₂ incubated alone (5% ethanol in PBS) increased progressively with incubation time (Fig. 1B). Consistent with immunoblotting, formation of Aβ₄₂ fibrils was significantly reduced in the presence of 0.1% v/v TRF after 24 h (p < 0.01) and 48 h (p < 0.05) compared with Aβ₄₂ incubated alone and compared with PO after 24 h (p < 0.05). Further, fluorescence intensity decreased progressively at increasing TRF concentration (0.001%, 0.01%, and 0.1% v/v), with the reduction significant at 0.001% v/v after 48 h (p < 0.05), at 0.01% v/v after 24 and 48 h (p < 0.05 and p < 0.01, respectively), and at 0.1% after 24 and 48 h (p < 0.01) compared with Aβ₄₂ incubated alone.

Amyloid-β oligomerization was inhibited by TRF under cell-free conditions

We examined the potential of TRF to inhibit Aβ oligomer formation under cell-free conditions using PICUP method. As shown in Fig. 2A, the cross-linked mixtures of 25 μM Aβ₄₀ in PBS (control) resulted in oligomer formation from dimer to trimer, although uncross-linked samples were visualized as 1 band representing the monomeric form of Aβ. Cross-linked mixtures containing 25 μM Aβ₄₀ with 0.001% v/v TRF showed a slight reduction of Aβ₄₀ oligomer forms (dimer and trimer). Furthermore, 0.1% TRF markedly inhibited Aβ₄₀ oligomer formation. Similarly, cross-linked mixtures containing 25 μM Aβ₄₂ with TRF showed a dose-dependent reduction in Aβ₄₂ oligomers. The Aβ₄₀ and Aβ₄₂ oligomer formations were not changed by PO treatment. In addition, we conducted PICUP experiment using glutathione-S-transferase (GST) to investigate the specificity of TRF on inhibiting PICUP oligomer formation. TRF did not inhibit GST oligomerization in PICUP assay (data not shown). Therefore, this finding confirmed the specificity of TRF to inhibit Aβ oligomerization.

TRF-treated Aβ aggregates did not promotes SH-SY5Y cells viability

We investigated the cell toxicity of Aβ₄₂ aggregates that were prepared in the presence of TRF during the aggregation process. To evaluate cell toxicity, 10 μM Aβ₄₂ were incubated for 24 h at 37°C to form aggregates, in the presence or absence of 0.1% v/v TRF or PO, and then the Aβ₄₂ aggregates were treated in SH-SY5Y cells for 24 h at final concentrations of 0.3 μM Aβ₄₂ and 0.003% v/v TRF or PO. A significant reduction of cell viability approximately up to 56% was detected in 0.3 μM Aβ₄₂ aggregates, compared to vehicle (p < 0.001) (Fig. 3A). No significant difference was found in cell viability of 0.3 μM Aβ₄₂ aggregates prepared in the presence or absence of TRF or PO compared to 0.3 μM Aβ₄₂ aggregates alone. In addition, no significant difference was found in cell viability in the treatments with TRF (Fig. 3B) and PO (Fig. 3C) at final concentration of 0.00003%, 0.0003%, and 0.003% v/v, compared tovehicle.

TRF supplementation improved cognitive impairment in AβPP/PS1 mice

Novel object recognition test was conducted to study the effect of TRF supplementation on behavioral disorder of AβPP/PS1 mice. The test was performed according to the schematic diagram shown in Fig. 4. All mice spent equal time exploring the identical objects during the training trial, indicating no location preference (Fig. 5A). Mice were then tested in novel object recognition after 60 min by measure the time spent exploring a new object that replaced one of the two identical (familiar) objects. Wild-type mice spent a significantly longer time exploring the novel object compared with the familiar object (79% recognition index), indicating preserved memory for the familiar object. On the contrary, AβPP/PS1 mice supplemented with water (control) showed a significantly lower recognition index (approximately 60%), indicative of AD-related cognitive decline. In contrast, AβPP/PS1 mice administered 60 mg/kg TRF daily showed a recognition index comparable with WT mice (80%). Administration of PO slightly enhanced the recognition index compared with controls, but the increase did not reach significance. This finding demonstrates a therapeutic effect of TRF on age-related cognitive decline in AD model mice (Fig. 5B).

TRF reduced Aβ deposition in the brain of AβPP/PS1 mice

Immunohistochemical staining with anti-Aβ N-terminal antibody was measured in the brains of AβPP/PS1 mice to assess the effects of control, PO, and TRF supplementation on Aβ deposition (Fig. 6A). The immunoreactive area was uniformly higher in cortex than hippocampus (Fig. 6B). Hippocampal deposition of Aβ was slightly reduced in TRF-supplemented mice, although the difference was not significant (Fig. 6B, left), while a significant reduction was observed in the cortex of TRF-supplemented mice compared with control and PO groups (p < 0.05; Fig. 6B, right).

Thioflavin-s staining was also conducted to investigate fibrillar-type Aβ deposition in brain sections [35, 37]. Representative images of hippocampus and cortex stained with thioflavin-s are presented in Fig. 6C. Mice receiving daily TRF exhibited fewer thioflavin-s-positive Aβ deposits in both brain regions compared with control and PO groups. Semiquantitative analysis demonstrated a significant reduction in the number of amyloid plaques in the hippocampus and cortex of TRF group mice compared with control and PO groups (p < 0.05; Fig. 6D).

Microglial activity was not affected by TRF

Immunohistochemical staining for Iba1 was performed to evaluate microglial activation in the brain sections of WT mice and control, PO-treated, and TRF-treated AβPP/PS1 mice (Fig. 7A). Control AβPP/PS1 group mice exhibited significantly higher Iba1-immunoreactivity in the hippocampus and cortex compared with WT (p < 0.01), but there were no differences in staining among the control, PO, and TRF groups (Fig. 7B).

Aβ levels were not affected by TRF supplementation

Finally, we examined the effects of TRF supplementation on Aβ levels in brain homogenates. The levels of soluble Aβ₄₀, Aβ₄₂, and Aβ oligomers in the TBS fraction from hippocampus and cortex did not differ between mouse groups (Fig. 8A–E). There were no statistically significant differences in the level of Aβ oligomers between groups; however, the level of Aβ oligomers appears to increase in the TRF group. Similarly, levels of insoluble Aβ₄₀ and Aβ₄₂ did not differ (Fig. 8F–I), although the TRF group displayed a trend for lower Aβ₄₀ and Aβ₄₂ in cortex compared with the control group (Fig. 8G, I).