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. 2025 Nov 4;10(45):54671–54681. doi: 10.1021/acsomega.5c08051

Role of the PKA–PGC1α–BACE1 Signaling Pathway in Aluminum-Induced Neurotoxicity in PC12 Cells

Ruo-nan Wang †,‡, Fan-peng Kong †,‡, Zhuo-hui Wang †,‡, Wen-cheng Hu †,‡, Zi-yan Pei †,‡, Jing Song †, Xiao-ting Lu †, Bao-long Pan ‡,*
PMCID: PMC12631702  PMID: 41280810

Abstract

Object: This objective of this study was to investigate how aluminum affects the PKA–PGC1α–BACE1 pathway in PC12 cells and its role in neurotoxicity. Method: According to the exposure dose of aluminum maltol, PC12 cells were selected for research and divided into five experimental groups and six intervention groups. After 24 h of 8-Bromo-cAMP intervention, they were treated with Al­(mal)3 for 24 h. After the experiment, cell morphology was observed, and the cell survival rate was assessed using the Cell Counting Kit-8 (CCK-8) assay. Western blot and ELISA techniques were used to detect the expression of relevant proteins, enzyme activity, and Aβ levels. Result: Under the microscope, the number of cells in the aluminum maltol group decreased, the morphology changed, and the number of intercellular connections decreased. However, after treatment with the 8-Bromo-cAMP agonist, a significant increase in the number of cells was observed, and significant morphological changes occurred, with a gradual increase in intercellular connections. CCK-8 assays showed that cell viability gradually decreased with increasing aluminum exposure doses. Western blot showed that PKA and PGC1α expressions decreased with higher aluminum doses, while BACE1 increased; agonist treatment upregulated PGC1α and downregulated BACE1, with minimal effect on PKA; and ELISA results indicated that aluminum reduced PKA enzyme activity but increased BACE1 activity and Aβ levels. Conclusion: Exposure to aluminum inhibits the PKA–PGC1α–BACE1 signaling pathway, while PKA agonists can alleviate neurotoxicity by restoring this pathway.

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1. Introduction

Aluminum is a naturally abundant metallic element in the environment. It enters the human body through various routes, including respiration, the digestive system, and the skin. , Studies of occupational epidemiology have shown that exposure to aluminum in the workplace is associated with cognitive dysfunction, particularly impairments in spatial perception and memory. , Furthermore, animal experiments have confirmed that aluminum exposure can impair short-term memory and spatial learning abilities. Aluminum exposure is closely associated with the neuropathological features of Alzheimer’s disease (AD). AD is a progressive neurodegenerative disease characterized by cognitive decline and prominent pathological features including β-amyloid (Aβ) plaque deposition and neurofibrillary tangles (NFTs). These features lead to neuronal loss and alterations in synaptic connections. Among these features, Aβ deposition represents an early pathological event pivotal to AD onset and progression. Recently, environmental factors in AD pathogenesis have received more attention. As a ubiquitous neurotoxin linked to senile plaques, aluminum may promote Aβ deposition and thus participate in AD pathology.

Aβ is produced through the cleavage of the amyloid precursor protein by β-secretase (BACE1) and γ-secretase. Impaired clearance relative to production accelerates deposition. This, in turn, causes neuronal damage, synaptic dysfunction, and neuroinflammation, ultimately leading to cognitive decline. Aβ accumulates outside of neurons, primarily in the forms of soluble Aβ1–40 and insoluble Aβ1–42. As the rate-limiting enzyme in Aβ biogenesis, BACE1 activity determines the rate of Aβ generation. Under physiological conditions, only a minimal amount of Aβ is produced in the body. Increased expression or activity of BACE1 directly leads to increased Aβ production, while inhibiting BACE1 significantly reduces Aβ production. BACE1 activity and expression levels are regulated by various factors and mechanisms, including transcriptional factor regulation, post-translational modifications, and signaling pathway regulation. , Consequently, elucidating the regulatory mechanisms of BACE1 is scientifically significant for understanding the Aβ production pathway.

Among numerous regulatory factors, peroxisome proliferator-activated receptor α-coactivator 1α (PGC1α) critically modulates BACE1 due to its master regulatory roles in energy metabolism, mitochondrial biogenesis, and antioxidant defense, making it a key molecular link between metabolic dysregulation and neurodegeneration. PGC1α serves as a primary regulator of mitochondrial biogenesis, energy metabolism, and respiration, interacting with various transcription factors, such as nuclear respiratory factor 1 (NRF-1) and 2 (NRF-2). In studies of metabolic diseases such as hypertension, overexpression of PGC1α reduces Aβ production, particularly by regulating the expression of the rate-limiting enzyme BACE1; in vitro, PGC1α overexpression reduces BACE1 transcription and BACE1 promoter activity, while cells transfected with PGC1α siRNA exhibit the opposite effect. , Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is associated with an AD-like pathology. PKA activity is reduced by approximately 20% in the temporal cortex tissue of AD patients and by 20–40% in the cerebellum. PKA can upregulate PGC1α expression by phosphorylating cAMP response element-binding protein. Activating the PKA signaling pathway increases PGC1α mRNA and protein levels, thereby enhancing mitochondrial function and antioxidant capacity. The PKA–PGC1α signaling pathway plays a central role in energy metabolism, mitochondrial biogenesis, and antioxidant defense. The activation of this pathway inhibits BACE1 expression, reduces Aβ production, and, thereby, achieves neuroprotective effects.

Most existing studies have focused on investigating the effects of aluminum on BACE1 protein expression and transcriptional regulation. However, the precise molecular mechanisms underlying the aluminum-mediated BACE1 modulation remain elusive. Based on the aforementioned evidence, we hypothesize that aluminum exposure may inhibit PKA activity and expression. This would reduce the level of PGC1α expression and impair mitochondrial function and antioxidant defense. Simultaneously, it would lift the inhibitory effect on BACE1 and influence Aβ production. This study investigates the effects of aluminum on proteins and enzyme activities related to the PKA–PGC1α–BACE1 signaling pathway through cellular experiments. These experiments provide potential strategies for intervening in aluminum-associated neurodegenerative pathologies.

2. Materials and Methods

2.1. Experimental Cells

The PC12 cell line (pheochromocytoma-derived cell line) was purchased from Wuhan Shang’en Biotechnology Co., Ltd. (product number: SNL-124). The cell line was cultured in Dulbecco’s modified Eagle's medium (high-glucose DMEM) containing 10% fetal bovine serum and 1% penicillin–streptomycin antibiotic. The cell culture environment was maintained at 37 °C in a constant-temperature incubator with 5% CO2 saturation humidity.

2.2. Cell Viability Assay

The cell viability of PC12 cells was determined by using the Cell Counting Kit-8 (CCK-8). First, PC12 cells were seeded at a density of 6000 cells per well in a 96-well plate, with 100 μL of culture medium added to each well, and cultured overnight. The next day, the original medium was replaced with fresh DMEM containing different concentrations of Al­(mal)3 (0 μM, 50 μM, 100 μM, 200 μM, 400 μM, and 800 μM), and the cells were returned to the incubator for further culture for 24 h. Subsequently, 100 μL of CCK-8 working solution was added to each well, and the cells were placed in the incubator for another hour. Finally, the absorbance was measured at 450 nm by using a microplate reader. The cell viability was expressed as the ratio of the absorbance values of the treated group to the control group.

2.3. Cell Toxicity Testing and Grouping

PC12 cells were seeded in sterile culture dishes. Once cell confluence reached 60–70%, a preprepared maltol aluminum solution was added to construct the following experimental groups: a control group with 10% DMEM, a maltol group, and low-, medium-, and high-dose Al­(mal)3 groups at concentrations of 100, 200, and 400 μmol/L. The cells were exposed to the solution for 24 h, after which cell samples were collected.

The intervention experimental cells were divided into six groups: control group (10% DMEM), maltol group, aluminum exposure group (200 μmol/L Al­(mal)3), control + agonist group (10% DMEM +1 mM 8-Bromo-cAMP), maltol + agonist group (maltol +1 mM 8-Bromo-cAMP), and aluminum exposure + agonist group (200 μmol/L Al­(mal)3+1 mM 8-Bromo-cAMP). After the agonist was applied to the intervention group for 24 h, aluminum exposure was continued for 24 h, and cell samples were collected.

2.4. Protein Extraction and Western Blot Analysis

2.4.1. Protein Extraction

The protein extractant, proteinase inhibitor, and phosphatase inhibitor were combined at a ratio of 98:1:1 to create the working solution for protein extraction. This solution is used to extract the total protein from PC12 cell samples. Protein quantification analysis was performed using the BCA protein assay kit according to the instructions. Subsequently, the protein samples were mixed with 5× loading buffer at a ratio of 4:1, treated with a boiling water bath for 3 min, and stored at −80 °C for later use.

2.4.2. Western Blot Analysis

First, 5 μg of the protein sample was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, the separated proteins are transferred to a poly­(vinylidene fluoride) (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk powder at room temperature for 2 h. Next, the target proteins were incubated with specific primary antibodies, including β-Actin (1:3000), Tubulin (1:5000), PKA (1:2000), PGC1-α (1:1000), and BACE1 (1:1000), at 4 °C overnight. After three washes with Tris-buffered saline (TBST), the membranes were incubated with horseradish peroxidase (HRP)-labeled rabbit anti-IgG (1:3000) and HRP-labeled mouse anti-IgG (1:3000) as secondary antibodies. After three additional washes with TBST, the protein intensity values were detected, and images were captured using a gel electrophoresis image analysis system.

2.5. Enzyme-Linked Immunosorbent Assay Experiments

PC12 cells were inoculated in sterile culture dishes. When the cell confluence reached 60–70%, they were exposed to a solution of maltol aluminum for 24 h. Subsequently, the cells were centrifuged at 3000 rpm for 10 min to remove particles and polymers, and the cell supernatant was collected. The activities of PKA and BACE1 in each group of PC12 cells, as well as the levels of Aβ1–40 and Aβ1–42, were, respectively, detected using enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Jingmei Biological Technology Co., Ltd.). The procedure was operated strictly in accordance with the steps in the product manual. First, sample wells and standard wells were set up. Fifty microliters of standard substances of different concentrations were added to the enzyme-labeled plate precoated with antibodies. Then, 10 μL of the sample to be tested and 40 μL of sample diluent were added to each sample well. Finally, 100 μL of HRP-conjugate reagent was added to each well, covered with an adhesive strip, and incubated for 60 min at 37 °C. The plate was washed five times; chromogen solution A (50 μL) and chromogen solution B were added to each well; and the mixture was gently mixed, incubated for 15 min at 37 °C, and protected from light. Finally, 50 μL of stop solution was added to each well; the absorbance was measured at 450 nm wavelength using an ELISA reader within 15 min. The experimental data were then recorded.

2.6. Statistical Analysis

The statistical analysis of the data in this study was performed by using SPSS 22.0 software. The statistical results were visualized using GraphPad Prism 8, and the fluorescence images were analyzed using ImageJ. First, normality tests were conducted for continuous variables. Data following a normal distribution were expressed as the mean ± standard deviation, and data not following a normal distribution were described using the median (interquartile range), M (P25, P75). One-way analysis of variance (ANOVA) was used for comparisons between multiple groups. For pairwise comparisons, the Student–Newman–Keuls method was employed if the homogeneity of variance was met; otherwise, the Dunnett T3 test was used. Repeated measures ANOVA was used for statistical analysis of repeated measures data. In this study, P < 0.05 was set as the threshold for statistical significance.

3. Results

3.1. Effect of Aluminum Exposure on PC12 Cell Morphology

Microscopic observation of cell morphology (Figure ) revealed that the number of cells in each maltol aluminum exposure group decreased significantly and the cells underwent obvious morphological changes. Specifically, the cell bodies became rounder, and the connections between the cells decreased. The high-dose group showed the most significant changes.

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Effect of different concentrations of aluminum on the morphology of PC12 cells after 24 h.

3.2. Effect of Aluminum Exposure on PC12 Cell Activity

Cell viability was assessed following treatment with different concentrations of maltol aluminum using the CCK-8 assay (Table and Figure ). The specific results are as follows: 0 μmol/L Al­(mal)3 (control group): cell viability was 99.997 ± 4.581, which served as the baseline for normal cell viability. In the other Al­(mal)3 treatment groups, cell viability decreased with an increasing concentration. There were significant differences between each concentration group and the previous one (P < 0.05), indicating that high concentrations of maltol aluminum significantly inhibited cell viability. This resulted in a substantial decrease in cell viability.

1. Effect of Different Concentrations of Aluminum on PC12 Cell Activity after 24 h .

groups cell viability (%) F P
0 μmol/L Al(mal)3 100.00 ± 00    
50 μmol/L Al(mal)3 93.01 ± 0.90*    
100 μmol/L Al(mal)3 90.44 ± 0.24* 169.26 <0.05
200 μmol/L Al(mal)3 85.333 ± 0.71*#
400 μmol/L Al(mal)3 81.65 ± 0.66*#&    
800 μmol/L Al(mal)3 57.43 ± 0.63*#&▲@    
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05; @: compared with 400 μmol/L Al­(mal)3, P < 0.05.

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Effects of different concentrations of aluminum on the activity of PC12 cells for 24 h. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05; a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

3.3. Effect of Aluminum Exposure on Related Protein Expression in the PKA–PGC1α–BACE1 Pathway in PC12 Cells

The expression levels of the PKA protein in PC12 cells treated with different concentrations of maltol aluminum (Table and Figure A) showed that there was no significant difference between the maltol group and the control group. The 100 μmol/L Al­(mal)3 group was significantly lower than the previous two groups, and the 200 μmol/L Al­(mal)3 group was significantly lower than the 100 μmol/L Al­(mal)3 group; the 400 μmol/L Al­(mal)3 group was significantly lower than all other groups (P < 0.05). This suggests that aluminum exposure significantly inhibits PKA protein expression, and the inhibitory effect increases with increasing dose.

2. Expression of PKA, PGC1α, and BACE1 Proteins in PC12 Cells in Different Dose Aluminum Exposure Groups (x̅± s, n = 3) .

groups PKA PGC1α BACE1
control group 1.32 ± 0.01 1.40 ± 0.05 0.23 ± 0.01
maltol group 1.35 ± 0.01 1.43 ± 0.07 0.28 ± 0.01
100 μmol/L Al(mal)3 1.13 ± 0.05*# 1.11 ± 0.07*# 0.84 ± 0.02*#
200 μmol/L Al(mal)3 0.86 ± 0.06*#& 1.00 ± 0.06*# 0.89 ± 0.01*#&
400 μmol/L Al(mal)3 0.45 ± 0.02*#&▲ 0.53 ± 0.00*#&▲ 0.96 ± 0.12*#&▲
F 128.68 11310.77 3555.61
P <0.05 <0.05 <0.05
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

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Expression of PKA, PGC1α, and BACE1 proteins in PC12 cells in different dose aluminum exposure groups. (A) PKA, (B) PGC1α, and (C) BACE1. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

Table and Figure B show the effect of different concentrations of maltol aluminum on the expression levels of the PGC1α protein in PC12 cells. PGC1α protein expression levels were slightly lower in the maltol group than those in the control group. The 100 μmol/L Al­(mal)3 group exhibited significantly lower levels than the previous two groups. The 200 μmol/L Al­(mal)3 group exhibited significantly lower PGC1α protein expression levels than the control group; however, there was no significant difference compared with the 100 μmol/L group. The PGC1α protein expression level in the 400 μmol/L Al­(mal)3 group was significantly lower than in the other aluminum-treated groups (P < 0.05). These results suggest that maltol aluminum significantly inhibits PGC1α protein expression in a dose-dependent manner.

The effect of different concentrations of maltol aluminum treatment on the expression levels of BACE1 protein in PC12 cells is depicted in Table and Figure C. Compared with the control group, there was no significant difference in the expression levels of BACE1 protein in the maltol group; the expression levels of BACE1 protein in the 100 μmol/L Al­(mal)3 group were significantly higher than those in the aforementioned two groups, being 3.65 times that of the control group (P < 0.05); the BACE1 protein expression level in the 200 μmol/L Al­(mal)3 group was significantly higher than that in the control group and the maltol group (P < 0.05), but there was no significant difference compared to the 100 μmol/L Al­(mal)3 group. The BACE1 protein expression level in the 400 μmol/L Al­(mal)3 group was significantly higher than that in the 200 μmol/L group, being 4.17 times that of the control group (P < 0.05). These results suggest that aluminum exposure significantly increases BACE1 protein expression, with the promoting effect increasing with dose.

3.4. Effects of Aluminum Exposure on the Activities of PKA and BACE1 Enzymes and the Content of Aβ in PC12 Cells

The effect of different concentrations of maltol aluminum treatment on changes in the PKA enzyme activity in PC12 cells is depicted in Table and Figure A. Compared with the control group, there was no significant difference in the PKA enzyme activity in the maltol group; the PKA enzyme activity in the 100 μmol/L Al­(mal)3 group and in the 200 μmol/L Al­(mal)3 group was significantly lower than that in the aforementioned two groups, at 80.0 and 79.3% of the control group, respectively (P < 0.05). The PKA enzyme activity in the 400 μmol/L Al­(mal)3 group was significantly lower than that in the other aluminum-treated groups, at 66.5% of the control group (P < 0.05). These results suggest that aluminum exposure significantly inhibits the PKA enzyme activity, and the inhibitory effect increases with increasing dose.

3. PKA and BACE1 Enzyme Activities of PC12 Cells in Different Dose Aluminum Exposure Groups U/L (x̅ ± s, n = 3) .

groups PKA BACE1
control group 298.585 ± 29.309 19.947 ± 0.454
maltol group 275.613 ± 46.069 22.490 ± 0.236
100 μmol/L Al(mal)3 238.868 ± 56.581*# 22.947 ± 0.212*
200 μmol/L Al(mal)3 236.777 ± 43.803*# 23.547 ± 0.225*
400 μmol/L Al(mal)3 198.648 ± 14.227*#&▲ 30.650 ± 2.318*#&▲
F 2.688 42.026
P <0.05 <0.05
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

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PKA and BACE1 enzyme activities of PC12 cells in different dose aluminum exposure groups. (A) PKA and (B) BACE1. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

Table and Figure B show the effect of different concentrations of maltol aluminum treatment on the BACE1 enzyme activity in PC12 cells. The results showed no significant difference in BACE1 activity between the maltol and control groups. However, BACE1 activity was significantly higher in the 100 μmol/L and 200 μmol/L Al­(mal)3 groups than in the control group. BACE1 activity was also significantly higher in the 400 μmol/L Al­(mal)3 group than in all other exposure groups (P < 0.05).

Changes in Aβ1–42 levels in PC12 cells treated with different concentrations of maltol aluminum are depicted in Table and Figure A. The results showed that the Aβ1–42 levels in the 100 μmol/L Al­(mal)3 group were significantly higher than those in the control group and the maltol group, being 1.39 times higher than the control group; the Aβ1–42 content in the 200 μmol/L Al­(mal)3 group was significantly higher than that in the 100 μmol/L group, being 1.49 times that of the control group; and the Aβ1–42 content in the 400 μmol/L Al­(mal)3 group was significantly higher than that in the other groups, being 1.73 times that of the control group (P < 0.05). These findings suggest that aluminum exposure significantly increases Aβ1–42 levels, with the enhancing effect increasing with the dose.

4. Contents of Aβ1‑42 and Aβ1‑40 in Different Dose Aluminum Exposure Groups of PC12 Cells (pg/mL) (x̅ ± s, n = 3) .

groups Aβ1–42 Aβ1–40
control group 340.45 ± 2.22 283.79 ± 2.59
maltol group 333.07 ± 1.96 294.56 ± 4.73
100 μmol/L Al(mal)3 472.70 ± 4.24*# 379.51 ± 2.71*#
200 μmol/L Al(mal)3 505.89 ± 1.06*#& 420.76 ± 1.86*#&
400 μmol/L Al(mal)3 589.00 ± 3.03*#&▲ 463.37 ± 3.85*#&▲
F 3686.77 1495.96
P <0.05 <0.05
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

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Content of Aβ1–42, Aβ1–40 in PC12 cells in different dose aluminum exposure groups. (A) Aβ1–42 and (B) Aβ1–40. *: compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05.

Changes in Aβ1–40 levels in PC12 cells treated with different concentrations of maltol aluminum are depicted in Table and Figure B. The results showed that the Aβ1–40 levels in the 100 μmol/L Al­(mal)3 group were significantly higher than those in the control group and the maltol group; the 200 μmol/L Al­(mal)3 group was significantly higher than the control group and the 100 μmol/L group; and the 400 μmol/L Al­(mal)3 group was significantly higher than the other exposed groups (P < 0.05). Aβ1–42 and Aβ1–40 levels increased with the exposure dose, indicating a dose-dependent promotional effect of aluminum on Aβ generation.

3.5. Effect of 8-Bromo-cAMP on the Morphology of PC12 Cells

Observing PC12 cell morphology under a microscope (Figure ) revealed a significant increase in the number of cells in each 8-Bromo-cAMP exposure group as well as significant morphological changes, and intercellular connections gradually became more numerous. These changes were particularly pronounced in the aluminum-stained group.

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Morphological effect of 8-Bromo-cAMP on PC12 cells.

3.6. Effect of 8-Bromo-cAMP Intervention on Related Protein Expression in the PKA Signaling Pathway of PC12 Cells

The effect of 8-Bromo-cAMP intervention on the expression level of the PKA protein in PC12 cells was examined (Table and Figure A). The results showed no significant differences in PKA protein expression levels between the maltol group, the control +8-Bromo-cAMP group, and the maltol +8-Bromo-cAMP group compared with the control group. However, the aluminum-treated group and the 200 μM + 8-Bromo-cAMP group had significantly lower PKA protein expression levels than the control group (P < 0.05). There were no significant differences between the two groups. These results suggest that aluminum exposure significantly inhibits PKA protein expression, while 8-Bromo-cAMP intervention does not significantly affect PKA protein expression. This may be due to the activation of PKA enzyme activity, which intervenes in downstream proteins.

5. Expression of PKA, PGC1α, and BACE1 Proteins in PC12 Cells Intervened by 8-Bromo-cAMP (x̅ ± s, n = 3) .

groups PKA PGC1α BACE1
control 0.62 ± 0.02 1.15 ± 0.19 3.24 ± 0.07
maltol 0.70 ± 0.08 0.84 ± 0.03* 1.87 ± 0.04*
200 μM 0.09 ± 0.01*# 0.62 ± 0.02*# 1.20 ± 0.01*#
Maltol+8-Bromo-cAMP 0.68 ± 0.01 2.82 ± 0.08*#& 3.94 ± 0.01*#&
Control+8-Bromo-cAMP 0.65 ± 0.03 1.96 ± 0.04*#&▲ 3.71 ± 0.03*#&▲
200 μM+8-Bromo-cAMP 0.08 ± 0.00*#▲a 1.08 ± 0.03*#&▲a 2.12 ± 0.02*#&▲a
F 867.85 1111.51 3273.21
P >0.05 <0.05 <0.05
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05, a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

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Effect of 8-Bromo-cAMP intervention on the protein expression of PKA, PGC1α, and BACE1 in PC12 cells. (A) PKA, (B) PGC1α, and (C) BACE1. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05; a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

The effect of 8-Bromo-cAMP intervention on the expression level of the PGC1α protein in PC12 cells was examined (Table and Figure B). The results showed that the expression levels of PGC1α protein were significantly lower in the maltol and maltol-aluminum groups than in the control group. The expression levels were significantly higher in the control +8-Bromo-cAMP group than in the control group, in the maltol +8-Bromo-cAMP group than in the maltol group, and in the 200 μM + 8-Bromo-cAMP group than in the maltol-aluminum group (P < 0.05). Aluminum exposure inhibited PGC1α protein expression, while 8-Bromo-cAMP intervention increased it, suggesting that 8-Bromo-cAMP may upregulate PGC1α expression by activating PKA enzyme activity.

The effect of 8-Bromo-cAMP intervention on the expression level of BACE1 protein in PC12 cells was examined (Table and Figure C). The maltol group had significantly lower BACE1 protein expression levels than the control group, while the maltol-aluminum group had significantly higher expression levels than the control group. The control +8-Bromo-cAMP group had significantly lower protein expression levels than did the control group, as did the maltol +8-Bromo-cAMP group, which had significantly lower expression levels than did the maltol group. Finally, the 200 μM + 8-Bromo-cAMP group had significantly lower protein expression levels than the maltol-aluminum group (P < 0.05). These results suggest that 8-Bromo-cAMP intervention significantly reduces the BACE1 protein expression by activating the PKA enzyme activity.

3.7. Effect of 8-Bromo-cAMP Intervention on the Activities of Enzymes Related to the PKA Signaling Pathway and the Content of Aβ in PC12 Cells

The effect of 8-Bromo-cAMP on PKA enzyme activity in PC12 cells was examined (Table and Figure A). The results revealed that PKA activity was significantly lower in the maltol and aluminum chloride groups than in the control group. Conversely, the PKA activity was significantly higher in the control +8-Bromo-cAMP, maltol +8-Bromo-cAMP, and 200 μM + 8-Bromo-cAMP groups than in the control group. Notably, the maltol +8-Bromo-cAMP group exhibited significantly higher PKA activity than the maltol group, and the 200 μM + 8-Bromo-cAMP group demonstrated significantly higher activity than the maltol-aluminum group (P < 0.05). These results suggest that 8-Bromo-cAMP may partially reverse the inhibitory effect of aluminum chloride by activating the PKA enzyme activity.

6. 8-Bromo-cAMP Intervention on the Enzyme Activities of PKA and BACE1 in PC12 Cells (x̅ ± s, n = 3) .

groups PKA BACE1
control group 304.06 ± 7.52 19.96 ± 1.26
maltol group 277.10 ± 2.65* 20.06 ± 0.38
200 μM group 228.94 ± 7.99*# 26.64 ± 0.85*#15.93 ± 0.14*#&
C+8-Bromo-cAMP 383.25 ± 2.54*#&
M+8-Bromo-cAMP 355.66 ± 6.16*#&▲ 15.35 ± 1.18*#&
200 μM+8-Bromo-cAMP 263.27 ± 9.77*#&▲a 20.22 ± 0.50&▲a
F 228.19 71.85
P <0.05 <0.05
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*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05, a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

8.

8

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8-Bromo-cAMP intervention on PKA and BACE1 enzyme activities in PC12 cells. (A) PKA and (B) BACE1. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05, a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

The effect of 8-Bromo-cAMP on the BACE1 enzyme activity in PC12 cells was examined (Table and Figure B). The results showed that the BACE1 enzyme activity was significantly increased in the maltol-aluminum group, while the control group +8-Bromo-cAMP group was significantly lower than the control group; the maltol +8-Bromo-cAMP group was significantly lower than the maltol group; and the 200 μM + 8-Bromo-cAMP group was significantly lower than the maltol aluminum group (P < 0.05). These results suggest that 8-Bromo-cAMP may inhibit BACE1 expression by activating the PKA signaling pathway.

The effect of 8-Bromo-cAMP on the levels of Aβ1–42 and Aβ1–40 in PC12 cells was examined (Table and Figure A,B). The results revealed that the levels of Aβ1–42 and Aβ1–40 were significantly lower in the control +8-Bromo-cAMP group than in the control group. Additionally, the levels were significantly lower in the maltol +8-Bromo-cAMP group than in the maltol group and in the 200 μM + 8-Bromo-cAMP group than in the 200 μM group (P < 0.05). These results suggest that 8-Bromo-cAMP may inhibit BACE1 activity by activating the PKA signaling pathway, thereby reducing Aβ levels.

7. 8-Bromo-cAMP Intervention on the Content of Aβ1‑42, Aβ1‑40 in PC12 Cells (x̅± s, n = 3) .

groups Aβ1–42 Aβ1–40
control group 340.79 ± 1.90 278.99 ± 3.61
maltol group 324.64 ± 5.57* 278.60 ± 8.09*
200 μM group 518.72 ± 16.88*# 323.02 ± 51.71*#
C+8-Bromo-cAMP 232.67 ± 5.19*#& 247.25 ± 10.72*#&
M+8-Bromo-cAMP 222.39 ± 3.18#&▲ 234.71 ± 6.44#&▲
200 μM+8-Bromo-cAMP 412.92 ± 105.72*#&▲a 274.93 ± 51.74*#&▲a
F 250.07 3.03
P <0.05 <0.05
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a

*: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05, a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

9.

9

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8-Bromo-cAMP intervention on the content of Aβ1–42, Aβ1–40 in PC12 cells. (A) Aβ1–42 and (B) Aβ1–40. *: Compared with 0 μmol/L Al­(mal)3, P < 0.05; #: compared with 50 μmol/L Al­(mal)3, P < 0.05; &: compared with 100 μmol/L Al­(mal)3, P < 0.05; ▲: compared with 200 μmol/L Al­(mal)3, P < 0.05; a: compared with 400 μmol/L Al­(mal)3, P < 0.05.

4. Discussion

Aluminum is a widely distributed metallic pollutant in the environment, and its potential neurotoxic effects are a popular research topic. Numerous studies have demonstrated a close association between the accumulation of aluminum in the nervous system and the onset of neurodegenerative diseases. Epidemiological studies have found that chronic aluminum exposure increases the risk of cognitive impairment and AD, a finding supported by biological research evidence. Aβ plays a key role in the pathobiology of AD, and increased aluminum levels can lead to the accumulation of Aβ plaques, which are primarily composed of two forms: Aβ1–40 and Aβ1–42, with Aβ1–42 being more neurotoxic. , Aβ generation critically depends on β-secretase (BACE1) and γ-secretase activities. BACE1, the Aβ-producing rate-limiting enzyme, directly elevates Aβ levels upon hyperactivity. Research on the mechanisms underlying Aβ deposition in the brain has focused primarily on the effects of BACE1 protein and its gene, but the mechanisms by which aluminum induces changes in BACE1 remain unclear. , This study found that aluminum may promote the BACE1 protein expression and enzyme activity by inhibiting PKA activity and reducing PGC1α expression, thereby increasing Aβ production.

PC12 cells are a commonly used neural cell model and are widely applied in the study of neurotoxic mechanisms. , Simulating aluminum exposure in PC12 cells allows for a closer examination of the effects of aluminum on cell morphology, activity, and signaling pathways. This not only helps to validate phenomena observed in animal experiments but also further reveals the molecular mechanisms of aluminum exposure at the cellular level. In PC12 cell experiments, the effects of aluminum exposure on the cell activity and morphology were first evaluated. CCK-8 assay results showed that aluminum exposure significantly inhibited PC12 cell activity in a dose-dependent manner. Microscopic observation revealed that aluminum exposure significantly reduced cell numbers and altered the cell morphology. Specifically, cells exhibited a tendency toward rounded bodies and reduced intercellular connections. These changes were particularly pronounced in the high-dose group. These results suggest that aluminum exposure is toxic to PC12 cells, affecting their survival and morphology.

To further investigate the mechanisms underlying the effects of aluminum exposure on PC12 cells, this study examined the expression of signaling pathway proteins and enzyme activities associated with Aβ production and synaptic function. PKA plays a crucial role in intracellular signal transduction within the central nervous system, mediating processes such as neuronal differentiation, plasticity, and long-term learning. Inhibition of PKA has been shown to impair long-term memory. This study shows that aluminum exposure significantly reduces the protein expression level of PKA in PC12 cells, while PKA enzyme activity is also significantly reduced, indicating that aluminum exposure affects normal cell function by inhibiting the PKA signaling pathway, and the inhibition of PKA is one of the important mechanisms of aluminum neurotoxicity. Aluminum exposure also significantly reduced the protein expression levels of PGC1α. Changes in the level of PGC1α protein expression may be regulated by PKA and may be associated with mitochondrial dysfunction and abnormal energy metabolism. Cheng’s research results indicate that impaired mitochondrial function may lead to an insufficient cellular energy supply, thereby affecting cellular survival and function. Additionally, the downregulation of PGC1α may further weaken the cellular antioxidant capacity and increase oxidative stress damage. Results from BACE1 protein expression and enzyme activity assays showed that aluminum exposure significantly increases BACE1 protein expression and activity levels, indicating that aluminum exposure promotes Aβ generation by enhancing BACE1 expression and activity and affecting cellular function. Aluminum exposure significantly increased Aβ1–40 and Aβ1–42 levels in PC12 cells, which is consistent with increased BACE1 activity and confirms the impact of aluminum exposure on Aβ generation. In summary, we verified the effects of aluminum exposure on the cell activity, morphology, and signaling pathways through PC12 cell experiments. These results suggest that aluminum exposure may influence Aβ production and cell function by regulating the PKA–PGC1α–BACE1 signaling pathway.

To explore the molecular mechanisms underlying the effects of aluminum exposure and identify potential intervention strategies, we treated PC12 cells with 8-Bromo-cAMP, a known PKA agonist. Activating the PKA signaling pathway allowed us to verify whether it could reverse the alterations induced by aluminum exposure and mitigate the negative effects on cellular function. The microscopic observations revealed an improved cell morphology after 8-Bromo-cAMP intervention, including reduced rounding of the cell body and increased intercellular connections. These results suggest that 8-Bromo-cAMP can effectively counteract the toxic effects of aluminum exposure on PC12 cells. This study measured the expression levels of PKA, PGC1-α, and BACE1 proteins, as well as the activity of related enzymes. 8-Bromo-cAMP intervention significantly increased the PKA enzyme activity in PC12 cells; however, there was no significant change in the PKA protein expression. These results suggest that 8-Bromo-cAMP can activate the PKA enzyme activity to regulate downstream proteins rather than directly regulate the PKA protein expression. The intervention significantly increased PGC1α protein expression levels, suggesting that 8-Bromo-cAMP may upregulate the PGC1α expression by activating the PKA enzyme activity. This could help improve mitochondrial function and energy metabolism by alleviating an aluminum-induced mitochondrial dysfunction. The intervention also reduced BACE1 protein expression levels and significantly decreased the BACE1 enzymatic activity. These results suggest that 8-Bromo-cAMP can reduce Aβ generation by inhibiting BACE1 activity and mitigate the negative effects of aluminum on cellular function. The results of Aβ content detection showed that 8-Bromo-cAMP intervention significantly reduced the levels of Aβ1–40 and Aβ1–42 in PC12 cells, consistent with the reduction in BACE1 activity, confirming the inhibitory effect of 8-Bromo-cAMP on Aβ production. 8-Bromo-cAMP enhances the PGC1α expression by activating the PKA enzyme activity, thereby inhibiting the BACE1 expression and activity and reducing Aβ production. These results suggest that 8-Bromo-cAMP can effectively target key steps in the Aβ production and mitigate the negative effects of aluminum exposure on cellular function.

In summary, this study investigated the effects of aluminum on neurological function and Aβ production as well as its potential mechanisms through cellular experiments. Using the PC12 cell model, the study confirmed that aluminum exposure negatively affects cellular activity, morphology, and signaling pathways. The study also revealed the molecular mechanism by which aluminum exposure influences the Aβ production by regulating the PKA–PGC1α–BACE1 signaling pathway. Furthermore, the study reversed the signaling pathway alterations caused by aluminum exposure, significantly reduced Aβ production, and improved cellular morphology and function by activating the PKA signaling pathway using 8-Bromo-cAMP. Research on the mechanisms of Aβ deposition in the brain has primarily focused on aluminum’s effects on BACE1. However, these studies have concentrated on the effects on the BACE1 protein and the gene itself. The mechanisms underlying aluminum-induced changes in BACE1 remain unclear. The findings of this study provide new insights into the mechanisms underlying aluminum-induced neurodegenerative diseases and offer important theoretical foundations for the development of preventive and therapeutic strategies that target aluminum toxicity. However, although this study has provided experimental evidence at the cellular level, it lacks supporting data from population studies. Future research should investigate the specific mechanisms of the PKA–PGC1α–BACE1 signaling pathway in aluminum toxicity and explore additional effective intervention targets to provide new strategies and tools for preventing and treating neurodegenerative diseases.

5. Conclusions

This study examined aluminum-induced cognitive impairment and the underlying mechanisms via cellular experiments. Results demonstrate that aluminum exposure influences the Aβ production by modulating the PKA–PGC1α–BACE1 signaling pathway, elevating Aβ production, and resulting in cognitive impairment. Furthermore, 8-bromo-cAMP, a PKA agonist, was found to significantly enhance the PKA activity, promote PGC1α expression, and inhibit BACE1 activity. Consequently, the level of Aβ production was reduced. These findings provide new insights into the neurotoxic mechanisms of aluminum exposure and offer a theoretical basis for developing intervention strategies.

All the data supporting the conclusions of this study have been fully presented in the main text of the paper. Readers can directly access them without the need for additional applications.

#.

R.W., and F.K. contributed equally to this work and share first authorship. Conceptualization, F.K. and B.P.; data curation, R.W.; formal analysis, R.W., F.K., Z.W., and W.H.; funding acquisition, B.P.; investigation, Z.W. and Z.P.; supervision, B.P.; validation, W.H., Z.P., J.S., and X.L.; visualization, F.K.; writingoriginal draft, R.W.; writingreview and editing, R.W., F.K., Z.W., J.S., X.L., and B.P. All authors have read and agreed to the published version of the manuscript.

This study was supported by the Fundamental Research Program of Shanxi Province (grant no 202303021211255) and the Scientific Research Projects of Six Special Actions (grant no Z2025002).

The authors declare no competing financial interest.

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