Male C57BL/6 mice (Jinan Pengyue, Jinan, Shandong, China) were used for this study under the animal production license SCXK(LU)2019-0003. The rearing environment was conducive to maintaining a stable room temperature and humidity, with natural ventilation, circadian rhythm, and unrestricted access to food and water. The operation and treatment of mice during the experiment adhered to the guidelines and ethical regulations of the Wannan Medical College on the humane treatment of animals. The animal ethics review number was WNMC-AWE-2023132.

Radioimmunoprecipitation assay buffer (RIPA) lysate (Beyotime, P0013C, Shanghai, China); phenylmethanesulfonylfluoride (PMSF) (Beyotime, ST2573, Shanghai, China); 5$\times$SDS sampling buffer (Beyotime, P0015L, Shanghai, China); Glial fibrillary acidic protein (GFAP) antibody (CST, #3670, Danvers, MA, USA); p-Tau antibody (Invitrogen, MN1020, Carlsbad, CA, USA); Tau antibody (Abcam, ab308439, Cambridge, UK); Ub-K48 antibody (Millipore, 05-1307, Bedford, MA, USA); B-cell lymphoma-2 (Bcl-2) antibody (Proteintech, 68103-1-Ig, Rosemont, IL, USA); Bcl-2-associated x protein (Bax) antibody (Proteintech, 50599-2-Ig, Rosemont, IL, USA); $\beta$-Actin antibody (Beyotime, AF5003, Shanghai, China); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Biosharp, BL006B, Hefei, Anhui, China); Goat anti-rabbit IgG (Beyotime, A0208, Shanghai, China); Goat anti-mouse IgG (Beyotime, A0216, Shanghai, China); Hematoxylin stain (Phygene, LA0516, Fuzhou, Fujian, China); Eosin stain (Phygene, BA-4098, Fuzhou, Fujian, China); Nissl stain (Servicebio, G1036, Wuhan, Hubei, China); Immunohistochemistry kit (OriGene, Sp-9000, Rockville, MD, USA).

Specific Pathogen Free (SPF)-grade, 71-week-old, senescent male C57B/L6 mice (Weight: 31–36 g) were screened for open field test (OFT) behavioral identification as an AD model, while 5-week-old healthy mice (Weight: 16–21 g) were used as controls. The mice were anesthetized with 1% pentobarbital sodium (Yacoo, W0091, Suzhou, Jiangsu, China), injected intraperitoneally at 15 mg/kg of body weight and subsequently sacrificed by cervical dislocation. The skull was then peeled off to remove the brain tissue, which was subsequently divided into two equal parts for the cryogenic isolation of the hippocampal tissues. One portion was immersed in a 4% paraformaldehyde solution and stored in a refrigerator at 4 °C, while the other portion was placed in a –80 °C refrigerator for subsequent experiments.

Open Field Test (OFTs) were conducted on the two groups of mice separately, recording parameters including resting time, average speed, total distance travelled, and distance travelled in the central region during exercise. The methodology of this study was as follows: First, a clean and odorless open-field experimental chamber was prepared, and a camera system (ShXinRuan, XR-XZ301, Shanghai, China) and behavioral analysis software (ShXinRuan, SuperMaze+, Shanghai, China) were set up. The numbers, dates, and other information for the control and AD groups were recorded. Subsequently, for each test, one individual mouse was carefully removed from the feeding cage, and placed in the central area of the experimental box with its back facing the experimenter, after which the experimenter immediately left the study area. Simultaneously, the video capture system was activated to document the mouse movements. The environment remained undisturbed throughout the course of the experiment, which typically lasted for a period of five minutes. At the end of the experiment, mice were returned to their feeding cages, the experimental box was cleaned, and any residual odor was eliminated. Behavioral analysis software was subsequently used to automatically calculate the average speed, total distance, and central area distance of the mice in open fields, thereby enabling the evaluation of their activity range and cognitive levels. Close attention was paid to the standardization of the experimental environment, adaptation period before the experiment, quiet environment during the experiment, and accuracy of data recording throughout the process.

Following necropsy, brain tissues were removed from the mice and fixed in 4% paraformaldehyde. They were then dehydrated, transparentized, paraffin-embedded and sectioned at a thickness of 5 µm. The paraffin sections were subsequently baked in an oven at 60 °C for 30 minutes in order to prevent deslaberation. The sections were then dehydrated, stained with hematoxylin for 3–5 minutes, rinsed with running water for 15–30 minutes, re-stained with eosin for 10 s, and again rinsed with running water for 15–30 minutes. Finally, the sections were sealed with neutral gum and morphological changes in the brain tissue sections were observed under a microscope (OLYMPUS BX53, Ishikawa-machi, Hachioji-shi, Japan).

Nissl staining was performed based on a previous report [14] with minor modifications. In brief, paraffin sections were sequentially treated with xylene and gradient alcohol, and washed with double-distilled water for 3 min each. The sections were then immersed in a Nissl staining solution for 2–5 minutes, rinsed until the running water was colorless, and observed under a microscope. Differentiation was subsequently performed with appropriate amounts of 0.1% glacial acetic acid, according to the staining results. The slices were then dried and sealed with neutral gum.

The immunohistochemistry employed in this study was primarily based on methodologies outlined in a previous report [15], with minor modifications. In brief, following perfusion with 4% paraformaldehyde, the brain tissue was sliced (at a thickness of 5 µm) following dehydration, transparency, and paraffin embedding. The brain slices were sequentially treated with xylene and a gradient alcohol solution, mixed with citrate buffer for microwave antigen repair, and then titrated with primary antibodies (Tau 1:500, p-Tau 1:100, and Ub-K48 1:200) and incubated in a refrigerator at 4 °C overnight. Subsequently, the slices were incubated with biotin-labelled histochemical secondary antibodies (goat anti-rabbit IgG 1:50, Goat anti-mouse IgG, 1:50). The 3,3^′-Diaminobenzidine (DAB) staining solution was finally added. Subsequently, the cells were stained with hematoxylin for 4 min, after which they were rinsed with running water for 15 to 30 min. The slices were then sealed with neutral resin, and analyzed using a microscope (OLYMPUS, BX53, Ishikawa-machi, Hachioji-shi, Japan). The numbers of cells which stained positive for Tau, p-Tau, and Ub-K48 were determined using Image J software (NIH, Image J 1.8.0, Bethesda, MD, USA), and statistical analysis was performed using GraphPad Prism 8.0.1 (Dotmatics, GraphPad Prism 8.0.1, San Diego, CA, USA).

The experimental mice in each group were anesthetized (1% Pentobarbital sodium (Yacoo, W0091, Suzhou, Jiangsu, China)), after which the brain tissue was exposed and removed. The entire procedure was conducted at a low temperature to prevent protein degradation. The brain tissues were placed in pre-cooled saline and thoroughly washed to remove blood and other impurities. Samples were then weighed on ice and the required quantities of cell lysate and protease inhibitor were added. Subsequently, the brain tissue was thoroughly ground using a tissue homogenizer (BKMAMLAB, BK-HTG80, Changsha, Hunan, China) to obtain a homogenate. In brief, the brain tissue homogenate was sonicated for a duration of three seconds using a sonicator (Tenln, TY-250Y, Yancheng, Jiangsu, China), after which it was incubated on ice for a period of thirty minutes. Subsequently, the brain tissue suspension was subjected to centrifugation at 4 °C (12,000 rpm for 15 minutes) using a high-speed centrifuge. Following centrifugation, the supernatant from the upper brain tissue protein was carefully aspirated, flash frozen in liquid nitrogen, and stored in an ultra-low temperature refrigerator (Midea, MD86L708, Foshan, Guangdong, China) at –80 °C. The extracted proteins were then quantified using a bicinchoninic acid (BCA) protein quantification kit.

Following the extraction of tissue protein, 5$\times$SDS upsampling buffer was added, and the mixture was heated to 95 °C for denaturation. Subsequently, the proteins in the gel were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane, which was subsequently blocked with skim milk powder for a period of two hours. Subsequently, the membrane was incubated with the primary antibody (GFAP 1:1000; Tau 1:1000; p-Tau 1:1000; Ub-K48 1:1000; Bcl-2 1:1000; Bax 1:500; $\beta$-Actin 1:2000; and GAPDH 1:2000) at a temperature of 4 °C for a period of 16 hours. The membrane was then incubated with a secondary antibody (goat anti-rabbit IgG 1:1000; goat anti-mouse IgG 1:1000) at room temperature for 2 h. Use Image J software (NIH, ImageJ 1.8.0, Bethesda, MD, USA) for WB grey value quantification. The initial normalisation of the quantitative results of the WB was achieved by dividing the grey values for the target protein by those of the corresponding internal reference protein (GAPDH or $\beta$-actin). This was followed by a second round of normalisation for the target protein, whereby the normalised data for the grey values of the four sets of proteins were divided by the normalised results for the grey values of the target protein in the specially designated control group. And then statistically analyse the data in the graphpad prism software (Dotmatics, GraphPad Prism 8.0.1, San Diego, CA, USA).

Mouse brains were fixed with formaldehyde, after which the fixed brain tissue was dehydrated using 15% and 30% sucrose. Each dehydration step lasted 48 h. Following dehydration, brain tissues were stored in a –80 °C refrigerator. Frozen brain tissues were sectioned using a microtome. The blades and tissues were kept moist during sectioning to prevent any breakage or curling. Sections were finally placed on clean microscope slides in a box in the dark. Brain slices were washed with Phosphate buffered saline (PBS) to remove the fixative and contaminants, permeabilized with Phosphate-Buffered Saline with Tween 20 (PBST) containing Triton X-100, and blocked with bovine serum albumin (BSA) to minimize any non-specific staining. After sealing, specific primary antibodies (p-Tau 1:100, Ub-K48 1:200) were added to the brain slices for incubation overnight at 4 °C. The primary antibody was then washed with PBS, after which fluorescein isothiocyanate (FITC)- or tetramethylrhodamine thiocyanate (TRITC)-labeled secondary antibodies were added for incubation at room temperature for 2 h. The nuclei were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). The brain slices were then washed with PBS, and an anti-quencher was added. Finally, coverslips were added and pressed to remove air bubbles. Sealed slices were finally observed under a fluorescence microscope, and photographs were taken to record and analyze the results. Quantitative statistics of immunofluorescence positive cells were performed by counting positive cells using Image J software (NIH, ImageJ 1.8.0, Bethesda, MD, USA), and the count results were subjected to t-test using Prism software (Dotmatics, GraphPad Prism 8.0.1, San Diego, CA, USA) to calculate the significant difference between each two groups.

All experimental data were subjected to one-way analysis of variance (ANOVA) in the statistical software SPSS 26.0 (IBM Corp., Armonk, NY, USA), with the results expressed as mean $\pm$ standard deviation ($\overline{x}$ $\pm$ s). Two-by-two comparisons between groups were performed using t-test analysis. Statistical significance was set at p 0.05.

SPF grade C57B/L6 mice aged 71-weeks-old were selected as the animal model of AD, while healthy 5-week-old SPF grade C57B/L6 mice were used as the control group. Behavioral assays for the OFT were conducted separately for the two groups of mice. The results of the behavioral tests indicated that AD mice exhibited significantly longer resting times than controls (Fig. 1A,B) (p 0.05), but significantly lower average speed (Fig. 1C) (p 0.01), central area distance (Fig. 1D) (p 0.01), and total distance (Fig. 1E) (p 0.01), suggesting that AD mice developed cognitive impairment and exhibited abnormal behaviors.

Fig. 1.

Open-field test (OFT) assay in Alzheimer’s disease (AD) mice. (A) Open field behavioral tests were conducted on control (Upper panel) and AD (Lower panel) mice, to record the resting time, average speed, total distance travelled, and distance travelled in the central region. (B–E) The resting time (B) of AD mice was significantly longer than that of the control group (n = 6, p 0.05), while the average speed (C) (n = 6, p 0.01), distance travelled in the central region (D) (n = 6, p 0.01), and total distance travelled (E) (n = 6, p 0.01) of AD mice were significantly shorter than that of the control group. Significance symbols indicate: *p 0.05, **p 0.01. Con, Control gruop.

Several studies have previously demonstrated that GFAP is indicative of glial cell activation, and thus functions as a valuable diagnostic marker for the early detection of AD. This study employed naturally aged 71-week-old SPF-grade C57BL6 mice as an animal model of AD, with healthy 5-week-old SPF C57BL6 mice serving as the control group. Brain tissues were obtained from six mice in each group, with the substantia nigra region precisely localized, and the expression of GFAP protein in the brain was detected via immunoblotting. The results demonstrated that the substantia nigra region of mice exhibited a high level of GFAP protein expression (Fig. 2A,B) (p 0.05), indicating the occurrence of AD. For all original Western Blot figures in Fig. 2A, see the Supplementary Materials. This further suggests that the naturally senescent 71-week-old mice in this study were successfully modelled for AD.

Fig. 2.

Significant increase in GFAP content in the substantia nigra region of 71-week-old mice, indicating successful AD modelling. (A) The results of immunoblotting demonstrated a significant elevation of GFAP in the substantia nigra region of the brain of 71-week-old mice. (B) Quantitative analysis of the grey scale values of GFAP expression in the substantia nigra region of the brains of mice in the AD group (A) revealed a significant (n = 3, p 0.001) increase in the substantia nigra region of 71-week-old mice compared to control 5-week-old mice. Significance symbols indicate: *p 0.05, **p 0.01. GFAP, Glial fibrillary acidic protein.

In this study, the number and morphology of neurons in the hippocampal region of both control and AD groups were observed using hematoxylin and eosin (HE) and Nissl staining. The results of HE staining revealed that the neurons in the CA1 region of the hippocampus of control group mice were neatly and regularly arranged in terms of morphology, while the nuclei of the cells were rounded and full, with darker staining (Fig. 3A, left panel). The cells of the hippocampus in the CA1 region of AD mice exhibited aberrant morphology, disorganized arrangement, and extensive intercellular spaces, with pale staining (Fig. 3A, right panel). The boundaries between the cell and nuclear membranes were not clearly delineated, while the cell nuclei exhibited deep staining and evidence of nuclear rupture in the AD hippocampus (Fig. 3A, right panel).

Fig. 3.

Morphological and quantitative analysis of neuronal cells in the hippocampus of AD mice. (A) Results of hematoxylin and eosin (HE) staining demonstrated that the neurons in the CA1 area of the hippocampus of the control group mice were neatly arranged, with a regular morphology, rounded nuclei, darker coloring, and tightly packed cells. The cells in the CA1 area of the hippocampus of mice in the AD group exhibited abnormalities, including an irregular and sparse arrangement, large gaps between cells, lighter coloring, and unclear boundaries of the cell and nuclear membranes. (B) Nissl staining further demonstrated a significant reduction in the number of neurons in the CA1, CA2 and CA3 regions of mice in the AD model group. (C) Statistical analysis of Nissl staining results of the AD group compared with the normal group (n = 6, p 0.01). Significance symbols indicate: **p 0.01. Scale bar equals 50 μm or 200 μm. The arrows in the Con group refer to normal neuronal cells, while the arrows in the AD group refer to neuronal cells that are abnormally trending towards apoptosis.

Nissl staining revealed a significant reduction in the number of neurons in the CA1, CA2, and CA3 regions of AD mice (Fig. 3B, right panel), as well as a significant reduction in the number of neural progenitor cells in the CA3 region (Fig. 3B, right panel, Fig. 3C) (p 0.01). These findings were significantly different from those in the normal 5-week group (Fig. 3B, left panel; Fig. 3C) (p 0.01), indicating that neuronal cells in the hippocampal region of AD mice undergo apoptosis.

Tau, p-Tau, and Ub-K48 levels in the hippocampal regions of the brains of control and AD mice were examined by immunohistochemistry, with results demonstrating that Tau did not undergo significant changes in neurons within the hippocampal region of AD mice (Fig. 4A). In contrast, we observed a notable increase in the number of neuronal cells that stained positive for p-Tau (Fig. 4B) and Ub-K48 (Fig. 4C), accompanied by a significant increase in their expression levels (Fig. 4D). These findings indicate that p-Tau interacts with Ub-K48, potentially contributing to neuronal apoptosis in the hippocampus.

Fig. 4.

Increased number of K48-linked ubiquitin chain (Ub-K48) and p-Tau positive cells in the hippocampal region of AD mice. (A) Immunohistochemical staining for Tau protein in the hippocampal region of control and AD mice; (B) Immunohistochemical staining for Phosphorylated Tau (p-Tau) protein in the hippocampal region of control and AD mice; (C) Immunohistochemical (IHC) staining for Ub-K48 in the hippocampus of control and AD mice are shown; (D) Statistical analysis of IHC staining results of the AD group compared with the Con group (p-Tau, n = 6, p 0.01; Ub-K48, n = 6, p 0.01). Significance symbols indicate: ^n⁢sp $>$ 0.05, *p 0.05, **p 0.01. Scale bar equals 200 μm.

Subsequent analyses involved examination of the alterations in the protein expression levels of Tau, p-Tau, and Ub-K48 in the hippocampal regions of the brains of control and AD mice by immunoblotting. The results demonstrated no significant difference in Tau expression in the hippocampus and cortex between normal and AD mice (Fig. 5A,B). In contrast, p-Tau expression was significantly higher in the hippocampal region of AD mice than in the Con group (Fig. 5C–E) (p 0.01). Further, we observed Ub-K48 in the hippocampal and cortical regions of both control and AD mice (Fig. 5F), with no significant differences observed between groups (Fig. 5F,G, right panel). The level of Ub-K48 was markedly increased in the hippocampal region of the mouse brain in AD model mice (Fig. 5F,G, left panel) (p 0.01). For all original Western Blot figures in Fig. 5A,C,F, see the Supplementary Materials. These results were in general agreement with the immunohistochemistry results, and clearly indicated that the lesions in the brains of AD mice are primarily due to the accumulation of Ub-K48 and p-Tau proteins in neuronal cells in the hippocampus. In contrast, the biochemical pathways in other areas of the brain, such as the cortical area, appeared to be similar to those in the normal AD mice (Fig. 5A–G, right panel).

Fig. 5.

Protein expression levels in mouse hippocampal regions. (A,B) Tau was expressed in mouse hippocampal and cortical regions, as detected by immunoblotting, with no differences between groups. (C,D) Significant differences in the expression of p-Tau in the AD model groups were observed in the hippocampal region of the mouse brain, as detected by immunoblotting (n = 3, p 0.01), with no difference observed in the cortical region. (E) Quantitative comparative statistical analysis of the ratio of p-Tau to Tau (n = 3, p 0.01). (F,G) Ub-K48 was observed in the hippocampal region of the mouse brain, and exhibited a significant elevation in the AD group compared to the control group (n = 3, p 0.01). Additionally, K48 ubiquitination was observed in the cortical region of the mouse brain in both the normal and AD model groups, although no significant differences were observed. Significance symbols indicate: ^n⁢sp $>$ 0.05, **p 0.01.

We subsequently strived to ascertain the relationship and localization of p-Tau and Ub-K48 in neuronal cells within the hippocampal region. Immunofluorescence double-labeling experiments were performed to ascertain the localization and interrelationship of p-Tau and Ub-K48 in neuronal cells in the hippocampal region, with findings demonstrating that p-Tau and Ub-K48 were co-localized in the hippocampal region of AD mice (Fig. 6). These findings indicated that the formation of Ub-K48 ubiquitin chains may be accompanied by the accumulation of p-Tau.

Fig. 6.

Immunofluorescence of Ub-K48 and p-Tau-specific antibodies in the hippocampus of mice. (A) Compared with the control group, mice in the AD group showed a significant increase in co-localization of Ub-K48 and p-Tau in the hippocampal region. Scale bar equals 200 μm. (B) Statistical analysis of Immunofluorescence (IF) staining results of the AD group compared with the Con group (Ub-K48, n = 6, p 0.01; p-Tau, n = 6, p 0.05). Significance symbols indicate: *p 0.05, **p 0.01. DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.

Finally, we sought to ascertain the impact of the accumulation of Ub-K48 and p-Tau proteins on neuronal cells in the hippocampal region of AD mice. The expression of the apoptosis-related factors Bax and Bcl-2 was examined in both the hippocampal and cortical regions of AD mice using immunoblotting. The results demonstrated that the expression of Bcl-2 protein in the hippocampal region of the brains of AD mice decreased (Fig. 7A,B) (p 0.01), whereas the expression of the pro-inflammatory protein Bax increased (Fig. 7C,D) (p 0.05). For all original Western Blot figures in Fig. 7A,C, see the Supplementary Materials. The Bcl-2/Bax ratio was significantly lower than that in the control group (Fig. 7E) (p 0.05). These findings indicated that neuronal cells in the hippocampal regions of AD mice underwent inflammation and exhibited signs of apoptosis.

Fig. 7.

Neuronal cells in the hippocampal region of AD mice showing signs of apoptosis. (A) Immunoblotting experiments showed a significant reduction in the expression of Bcl-2 in the hippocampal region of the brain in AD mice. (B) A quantitative statistical analysis of grayscale values of immunostained bands in (A) was conducted (n = 3, p 0.01). (C) Immunoblotting showed an increase in the expression of the pro-inflammatory protein Bax in neuronal cells in the hippocampal region of AD mice. (D) Quantitative statistical analysis of the grayscale values of the immunoblotted bands in (B) (n = 3, p 0.05). (E) Quantitative comparative statistical analysis of the ratio of Bcl-2 to Bax (n = 3, p 0.05). Significance symbols indicate: * p 0.05, ** p 0.01. Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated x protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Overall, the above findings collectively delineate a pathway of action orchestrated by the Ub-K48-p-Tau-Bcl-2/Bax pathway in AD pathogenesis. In essence, p-Tau is unable to undergo degradation due to impairment of the UPS, leading to its accumulation in neuronal cells within the hippocampal region of AD. This, in turn, results in a cellular inflammatory response. Subsequently, a substantial quantity of inflammatory factors is released, accompanied by a notable expression of the pro-inflammatory protein Bax. The Bcl-2/Bax ratio was significantly lower than that of the control group, which promoted neuronal cell apoptosis and ultimately contributed to the development of AD (Fig. 8).

Fig. 8.

A graphical summary of the potential mechanistic pathways of the Ub-K48 in AD development. Given that p-Tau cannot be degraded due to damage to the ubiquitin-proteasome system (UPS), it accumulates in the AD hippocampus. This results in the release of numerous inflammatory factors along with Bax and Bcl-2, which promote cell death and contribute to AD. The model drawings for this graphic summary were created using Adobe Illustrator 2020 (Adobe lnc., 24.2, San Jose, CA, USA).