Unraveling the role of abnormal AMPK and CRMP-2 phosphorylation in developmental fluoride neurotoxicity: Implications for synaptic damage and neurological disorders.

Abstract

Highlights

NaF can lead to learning and memory ability impairment in F₁ generation rats.
NaF induced developmental neurotoxicity may be related to synaptic damage.
NaF can cause abnormal phosphorylation of AMPK and CRMP-2.

Abstract

Excessive fluoride exposure can be neurotoxic, although the exact mechanism remains unknown. This study aimed to investigate the neurotoxicity of continuous sodium fluoride exposure in offspring rats, focusing on the potential effects of fluoride exposure on hippocampal synaptic function and the role of AMPK and CRMP-2 in synaptic damage. We established an SD rat model of fluoride exposure (25, 50, and 100?mg/L NaF) and found that fluoride exposure damaged the learning and memory ability of F₁ generation rats and caused ultrastructural changes in the hippocampus. Additionally, after the proteomic and phosphoproteomic analysis of rat hippocampal tissues, the Gene Ontology analysis revealed that sodium fluoride was involved in the enrichment of neuronal differentiation, synaptic signaling, and cytoskeleton-related biological processes. The Kyoto Encyclopedia of Genes and Genomes analysis showed that differential genes were enriched in synapse-related signaling pathways. Thus, we screened three differentially expressed proteins related to synaptic function for validation. The Western blotting analysis showed that AMPK and CRMP-2 were hyperphosphorylated in the hippocampus of fluoride-exposed rats. Our study found that abnormal AMPK and CRMP-2 phosphorylation leads to synaptic damage. This may be an important cause of memory impairment in fluorosis, offering new insights into the mechanism of fluoride-induced neurotoxicity.

Graphical Abstract

Keywords: Fluorosis, AMPK and CRMP-2, Proteomics, Phosphorylation, Synaptic damage

1. Introduction

Fluoride, an essential trace element for humans and animals, is found in water, air, soil, vitamin supplements, agricultural chemicals, and toothpaste (Wang and Li, 2019, Caldas and Ricomini, 2022). According to the World Health Organization, the permissible limit of fluoride in drinking water is 1.5?mg/L. Although fluoride plays a key role in preventing dental caries, excessive fluoride exposure can cause dental fluorosis, osteofluorosis, and damage to non-bone organs (Zhou and Sun et al., 2023). Fluorosis currently remains a global public health problem (Patil and Lakhkar et al., 2018). Fluoride exposure is particularly damaging to the nervous system due to the non-regenerative nature of nerve cells (Dec and Lukomska et al., 2017). Previous literature demonstrated that fluoride can accumulate in the fetal brain during the perinatal period through the placenta and the blood-brain barrier, irreversibly damaging the nervous system (Castiblanco-Rubio and Martinez-Mier, 2022). The nervous system is particularly vulnerable during development, making it more susceptible to environmental pollutants like fluoride. Therefore, fluoride exposure is more harmful to fetuses than to adults. An epidemiological study showed that maternal fluoride exposure during pregnancy is strongly associated with lower intelligence quotient (IQ) in offsprings (Green and Lanphear et al., 2019). Animal studies also found memory and cognitive dysfunction in the offsprings of rats exposed to fluoride during pregnancy and lactation (Ferreira and Aragao et al., 2021). Despite the extensive evidence showing that excessive fluoride exposure can cause neurological damage, the specific underlying mechanism of neurotoxicity remains unclear.The hippocampus is one of the regions of the mammalian brain that contains neural stem cells capable of maintaining the production of new neurons. Furthermore, it contains the CA1, CA2, and CA3 regions, as well as the dentate gyrus (DG), an important organ in the central nervous system involved in learning and memory storage (Denoth-Lippuner and Jessberger, 2021). Studies demonstrated that learning and memory impairments are associated with abnormalities in hippocampal excitatory synapses (Choopani and Kiani, 2023, Gui and Liu, 2023). The normal synaptic structure is essential to maintain the normal functioning of the nervous system (Dejanovic and Sheng et al., 2024). Previous studies showed that fluorosis can alter the hippocampal synaptic structure and affect the neuronal transmission (Chen and Ning et al., 2017). Niu et al. (Niu and Chen et al., 2018) also found that high fluoride exposure led to synaptic ultrastructural changes in mouse hippocampal neurons. These changes included blurred synaptic gaps and increased postsynaptic density. Fluoride exposure can also promote the secretion of pro-inflammatory cytokines through extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and P38/MAPK signaling pathways, causing neuroinflammation, thereby affecting synaptic neuronal plasticity (Yang and Jin et al., 2018). Chen et al. (Lingli and Hongmei et al., 2023) showed that fluoride exposure activated the ras homolog family member A (RhoA)/Rho associated protein kinase (ROCK) signaling pathway, causing cytoskeletal damage and synaptic dysfunction. Despite the evidence illustrating that fluoride exposure can cause synaptic damage, the mechanism underlying synaptic damage leading to neurotoxicity is still not fully elucidated.Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor and metabolic regulator that inhibits neuronal polarization and axonal growth while regulating synaptic remodeling in senescent neurons (Belforte and Agostinone et al., 2021). AMPK comprises 3 subunits: ?, ?, and ?. The ? subunit consists of ?1 and ?2 subunits (PRKAA1, PRKAA2), with PRKAA1-encoding subunit ?1 being the key catalytic subunit of AMPK. PRKAA1 phosphorylation plays a major role in AMPK activation (Herzig and Shaw, 2018). Yang et al. (Yang and Mohammad et al., 2022) showed that AMPK activation changes synaptic protein content and reduces the abundance and distribution of neuronal synaptic proteins. Chu et al. (Chu and Cao et al., 2019) found that AMPK activation resulted in the loss of neuronal synapses. Collapsin response mediator protein (CRMP) is highly expressed in the brain during the early postnatal period, especially in regions with extensive neuronal plasticity, such as the hippocampus and the olfactory bulb, where CRMP-2 is widely expressed and plays an important role in synaptic transmission (Stratton and Boinon et al., 2020). Zhang et al. (Zhang and Kang et al., 2016) showed that CRMP-2 is associated with synapse formation. Additionally, they demonstrated that CRMP-2-knockout mice have abnormal hippocampal dendrite formation and defective synapse formation in CA1 neurons. Furthermore, the lack of CRMP2 in CRMP-2-knockout mice led to axon guidance defects, axonal pruning in the hippocampus and visual cortex, and altered dendritic spine remodeling. Many studies confirmed that fluoride can affect the normal structure of synapses and harm the nervous system, inducing cognitive deficits (Ge and Chen et al., 2018), oxidative stress (Lai and Chen et al., 2020), and inflammation (Yang and Jin et al., 2018). It remains unclear if AMPK and CRMP-2 serve as molecular markers for fluoride-induced synaptic damage.Proteomics and phosphoproteomics have been recently widely used to study the effects of fluoride on the nervous system (Ran and Xiang, 2021, Tang and Zhang, 2023). We found abnormal phosphorylation of AMPK and CRMP-2 proteins in fluoride-induced neurotoxicity through the proteomic and phosphoproteomic analysis, which was verified by Western blotting. The aim was to identify key proteins and pathways involved in the regulation of synaptic structure and function, further providing new directions for fluoride-induced neurotoxicity and new perspectives for the treatment of fluoride-related diseases.

2. Materials and methods

2.1. Animal and tissue processing

Twenty-four specific pathogen free (SPF)-grade adult sprague dawley (SD) rats were purchased from the Laboratory Animal Center of Xinjiang Medical University. The license number was SCXK (Xinjiang) 2018–0003. The Medical Ethics Committee of the First Affiliated Hospital of the Medical College of Shihezi University approved all animal experiments related to this study (approval no. A2018–011–01). All the operations were performed in strict adherence to the principles of animal experimentation. The rats were maintained at 20–25? and 50?%–60?% humidity, with a 12-h light/dark cycle.After the 7-day acclimatization, all rats were randomly divided into 4 groups (6 rats in each group) according to their body weights and caged together according to the male to female ratio of 1:2. The rats were fed in single cages after conception. Four groups of pregnant rats were exposed to sodium fluoride (NaF) using ad libitum drinking water: control (water fluoride concentration <1?mg/L), 25, 50, and 100?mg/L NaF (corresponding to 11.3, 22.6, and 45.2?mg/L fluorion, respectively) groups. These dosage was based on our previous research (Niu and Chen, 2018) and real fluoride exposure levels in the world (it has been reported that fluorion concentrations in groundwater in most fluorosis areas are higher than 0.5?mg/L and up to 48?mg/L (Mumtaz et al., 2015). Until F₁ generation weaning [i.e., 21 days after birth]. The F₁ generation was reared separately from the parental generation and continued the manner and dosage of their parental treatment until sexual maturity (i.e., 2 months of age). Then, 6?F₁ generation rats (male to female: 1:1) were randomly selected from each group and subjected to Morris water maze (MWM) experiments. After the MWM experiments, the F₁ generation rats were executed, and their hippocampal samples were collected on ice. Three rats’ hippocampi were taken from each group for electron microscopy, and the hippocampal tissues of rats in control and 100?mg/L NaF groups were selected for the proteomic and phosphoproteomic analysis. The remaining hippocampal samples were wrapped in tin foil and placed in liquid nitrogen for rapid freezing for 10?s, followed by the transfer to a ?80? refrigerator for storage.2.2. Chemical reagentsNaF was obtained from Sigma (USA). CRMP-2 antibody was provided by Proteintech (USA). p-CRMP-2 (Thr509) antibody was provided by Abcam (USA). p-CRMP-2 (Ser522) was obtained from Cusabio. PRKAA1 and p-PRKAA1 (Thr172) were obtained from Boster Biotech (China). p-PRKAA1 (Ser486) antibody was purchased from Invitrogen (USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ZB-2301) and HRP-conjugated goat anti-mouse IgG (ZB-2305) were purchased from ZSGB-BIO (China).

2.3. MWM test

The MWM instrument is an all-black circular pool with a height of 50?cm and a diameter of 180?cm, a cylindrical platform at the bottom of the pool with a height of 20?cm and a diameter of 8?cm, and a camera recording equipment above the pool. Before the experiment, water was injected into the pool, the water level was about 30?cm, and the water temperature of the pool was maintained at 22?±?1?. The pool is divided into four quadrants, and the cylindrical platform is placed in the center of the third quadrant. The platform position cannot be moved during the experiment.The MWM consists of two parts: the place navigation test (PNT) and the spatial probe test (SPT). During the first 4 days of PNT training, if the rats found the platform within 60?s, the time taken was recorded as escape latency, and then the rats were allowed to stand on the platform for 15?s. If the rat had not found the platform within 60?s, 60?s was recorded as the escape latency, and the rats were allowed to stand on the platform for 30?s. The escape latency, swimming distance, swimming speed, and swimming route of the rats were recorded during the experiment to evaluate the spatial learning and memory ability. SPT was carried out on day 5, the platform was removed, other experimental conditions remained unchanged, and the rats were allowed to enter the water from the first quadrant and swim freely in the pool for 60?s. During the experiment, the number of platform crossings, the time and distance in the target quadrant, and the swimming route were recorded to evaluate the memory retention ability of the rats.

2.4. Proteomic and phosphoproteomic analyses

2.4.1. Sample preparation

Sample preparation for proteomic and phosphoproteomic analyses was performed as previously described (Chen and Jiang et al., 2019). The hippocampal tissue was cut and grounded. Then, a lysate and phosphatase inhibitor mixture was added for cleavage, followed by extracting the protein supernatant. Each group contained 3 independent samples. Afterward, the protein concentration was measured using NanoDrop One. The total protein was incubated with dithiothreitol at 56? for 30?min and with iodoacetamide at room temperature in the dark for 15?min. Tetraethylammonium bromide was added to further dilute the sample. Then, trypsin was added and digested at 37? overnight. After digestion, the peptides were desalted with a solid phase extraction column, followed by tandem mass tag (TMT) labeling and phosphopeptide enrichment.

2.4.2. LC-MS/MS analysis

Liquid chromatography (LC) was used for gradient elution at 0.5?ml/min for 60?min. Global mass spectrometry (MS) measurement scans (mass range: 400–1600?m/z; resolution: 70,000) using Orbitrap quality analyzers. The top 25 precursor ions were selected for further analysis, and the normalized collision energy of 35?% was obtained. Protein sequences were downloaded from NCBI. Then, tandem mass spectrometry (MS/MS) data were retrieved using protein databases and known mass spectrometers. The following identification parameters were set: cleavage specificity: trypsin; maximum missed cleavage sites: 2; minimum peptide sequence: 6 amino acids. Mass tolerance was set to 10?ppm at MS level and 0.02?Da for MS/MS. The false discovery rate (FDR) for proteins and peptides was set at 1?%. Proteins or phosphoproteins between NaF-treated and control rats were identified by (1) at least one quantified unique peptide; (2) p-value <?0.05; (3) a fold-change >?1.2 or <?0.833.

2.4.3. Bioinformatic analyses

The differentially expressed proteins and phosphorylated proteins in the volcano map were visualized by GraphPad Prism. We also used Clustvis, an online web tool, to generate hierarchical clustering heat maps of differential/phosphorylated proteins using a log2 color scale to provide better details of differences between NaF-exposed and control rats. For enrichment analysis, differentially expressed or phosphorylated proteins were uploaded to Metascape. Then, Gene Ontology (GO) enrichment was performed using a standard setting.

2.5. Nissl staining

The paraffin-embedded hippocampus samples were sliced into 5?µm sections, separated by xylene, dehydrated by gradient ethanol, stained with cresol violet for 30?min, and washed with distilled water. Then soaked in 95?% alcohol for 5?min, made the slices transparent in xylene, covered with neutral gel, and finally viewed with a microscope.2.6. Transmission electron microscopy (TEM)The rat hippocampus samples were cut into 1?mm³ blocks and fixed with glutaraldehyde at 4? for 4?h. It was then dehydrated with ethanol and acetone, infiltrated with epoxy resin, and embedded. Next, the embedded blocks were cut into 50?nm sections and stained with uranyl acetate for 20?min and lead citrate for 10?min. The images were observed by TEM.2.7. Western blot analysisNanoDrop One was used to detect protein concentrations extracted from the F₁ generation hippocampus. The proteins were separated by electrophoresis of sodium dodecyl sulfate-polyacrylamide gel and imprinted on a polyvinylidene fluoride (PVDF) membrane. Afterward, the membrane was closed with 5?% skim milk for at least 1?h. Then, primary antibodies (p-CRMP-2 [Thr509], 1:2000; p-CRMP-2 [Ser522], 1:1000; CRMP-2, 1:1000; PRKAA1, 1:1000; p-PRKAA1 [Thr172], 1:1000); p-PRKAA1 [Ser486], 1:1000); and GAPDH, 1:1000) were used at 4? for 16–18?h. Subsequently, the membrane was incubated with a secondary antibody (1:20,000) at room temperature for 2?h. The protein bands were observed using enhanced chemiluminescence (ECL) reagents in a chemiluminescence apparatus. The protein band intensity was quantified using ImageJ software.2.8. Statistical analysisSPSS 26.0 software was used to analyze the data. Results were expressed as mean ±?standard deviation (SD). One-way ANOVA, followed by the Tukey’s test, was used for multiple comparisons. Statistical significance was set at P?<?0.05.

3. Results

3.1. NaF exposure impaired the learning and memory ability of F₁ generation rats

We used the MWM test to investigate the effects of NaF on learning and memory in rats. On days 3 and 4, the 100?mg/L NaF group showed significantly slower swimming speeds than the control (P?<?0.05; Fig. 1a). The daily escape latency and swimming distance showed a decreasing trend in NaF treatment and control groups. Among them, the escape latency of the 100?mg/L NaF treatment group was significantly longer than that of the control group at day 4 (P?<?0.05; Fig. 1b). The swimming distance of rats in the 100?mg/L NaF treatment group was significantly longer than that in the control group at day 1 (P?<?0.05; Fig. 1c). In the SPT experiment, the platform crossing frequency of offspring rats in 50?mg/L and 100?mg/L NaF treatment groups was significantly lower than that in the control group (P?<?0.05; Fig. 1e). The time and distance of the target quadrant in the offsprings treated with 100?mg/L NaF were significantly lower than in controls (P?<?0.05; Fig. 1f–g). Fig. 1d and h show the typical routes of PNT and SPT experiments, respectively. Fig. 1

Fig. 1. NaF exposure impaired the learning and memory ability of F₁ generation rats. (a) The mean swimming speed to the platform. (b) The mean escape latency to the platform. (c) The mean swimming distance to the platform. (d) Representative traces in the PNT. (e) The number of platform crossings. (f) Time spent in the target quadrant. (g) Distance spent in the target quadrant. (h) Representative traces in the SPT. The data are presented for six rats in each group. *P?<?0.05 versus the control group.

3.2. NaF exposure caused hippocampal tissue damage in F₁ generation rats

The structure and number of Nissl bodies in hippocampal neurons were observed by Nissl staining. The Nissl bodies were neatly arranged and more numerous in the control group. After fluoride exposure, the number of Nissl bodies in the hippocampus of F₁ generation rats gradually decreased with increasing NaF dose. Additionally, the staining became shallow and blurred (Fig. 2a). The ultrastructure of the rat’s hippocampal tissue was observed by TEM. Compared with the control group, mitochondrial ridge rupture and nuclear membrane wrinkle were observed in the hippocampal tissue of F₁ generation rats in the 100?mg/L NaF group (Fig. 2b).

3.3. Proteomic and phosphoproteomic analysis of the hippocampal tissue of F₁ generation rats after NaF exposure

We analyzed the proteome and phosphorylated proteome of the hippocampal tissue of F₁ generation rats after NaF exposure using TMT quantitative mass spectrometry. The quantitative analysis showed that each of the 7157 identified proteomes had at least one unique peptide (Fig. 3a). In the quantitative phosphoproteomic analysis, 2792 phosphorylated proteins contained 9976 different amino acid sites (Fig. 3b). The proportion of pSer, pThr, and pTyr was 85.32?%, 14.41?%, and 0.27?%, respectively (Fig. 3c). Differentially expressed and phosphorylated proteins were selected by calculating the fold changes in the hippocampal tissues of control and NaF F₁ generation rat groups, with ratios >?1.2 or <?0.833 defined as upregulation or downregulation, respectively (Fig. 3d).

3.4. GO enrichment analysis of proteins and phosphorylated proteins in the hippocampal tissues of F₁ generation rats after NaF exposure

We performed combined proteomic and phosphoproteomic analyses of the rat’s hippocampal tissue to further determine the effects of fluoride on the tissue. Proteomic detection showed 10 upregulated and 7 downregulated differentially expressed proteins in the rat’s hippocampus. The phosphoproteomic detection showed 96 upregulated and 110 downregulated differentially expressed proteins in the rat’s hippocampus. These results were visualized in volcano plots and clustered heatmaps (Fig. 4a–d). Additionally, the GO enrichment analysis revealed that most differentially expressed genes were involved in the neuronal structure and function, with some of them playing a role in maintaining the synaptic structure and function (Fig. 4e).

3.5. Abnormal AMPK and CRMP-2 phosphorylation plays an important role in fluoride-induced synaptic damage

Based on the proteomic and phosphoproteomics analyses, we screened 3 differentially expressed proteins associated with synaptic function in the phosphoproteomics results (Fig. 5a). Western blot analysis validated the phosphoproteomic findings, revealing increased p-PRKAA1 (S486) levels in NaF-treated groups. Furthermore, the hippocampal p-PRKAA1 (T172) protein level was significantly increased in the 100?mg/L NaF-treated group (P?<?0.05; Fig. 5b–c). Compared with the control group, p-CRMP-2 (S522) levels in the hippocampus of rats treated with 50 and 100?mg/L NaF were notably increased. As well, p-CRMP-2 (T509) levels in the hippocampus of rats treated with 100?mg/L NaF were significantly increased (P?<?0.05; Fig. 5d–e).

4. Discussion

The present study suggests that developmental fluoride neurotoxicity might be mediated by synaptic damage. Specifically, NaF could cause synaptic tissue damage in the hippocampal tissues of F₁ generation rats by upregulating AMPK and CRMP-2 phosphorylated proteins, leading to memory and learning deficits, as well as neurological damage. Moreover, the enrichment of differential and phosphorylated proteins revealed the overexpression of proteins associated with synaptic structure and function, cytoskeletal structure, calcium transport, and protein deubiquitination. Importantly, an analysis revealed that AMPK and CRMP-2 phosphorylation motifs were significantly enriched in phosphopeptides. Hence, AMPK and CRMP-2 proteins are involved in fluoride-induced developmental neurotoxicity by affecting synaptic function, causing neurological damage and learning and memory impairments.

Previous studies demonstrated the developmental neurotoxicity of fluoride (Zhang and Tang et al., 2023). By constructing rat NaF exposure models, we found that exposure to NaF during pregnancy caused memory and learning impairments in F₁-generation rats. The current findings are consistent with research by Chen et al. (Chen and Niu et al., 2018), which demonstrated that fluoride exposure during the critical neurodevelopmental period impairs the learning and memory abilities of rat offsprings. Thus, overexposure to NaF during pregnancy increases escape latency and swimming distance and decreases time and distance spent in the target quadrant. Interestingly, these results are consistent with epidemiological surveys showing that the children of women exposed to high fluoride during pregnancy have lower IQs and poorer memory and cognitive abilities (Bashash and Thomas, 2017, Dewey and England-Mason, 2023). Our findings showed that excessive NaF exposure resulted in memory and learning deficits in rat offsprings. Hence, the rat model of developmental fluoride neurotoxicity was successfully constructed, suggesting that fluoride impairs neurodevelopment.

The hippocampus is a part of the brain important for maintaining learning, memory, and emotional regulation, representing an essential target for fluoride action (Bittencourt and Dionizio et al., 2023). Our study found that NaF exposure impaired the structure and quantity, as well as the ultrastructure, of Nissl bodies in the hippocampus of F₁ generation rats. Ran et al. (Ran and Xiang et al., 2023) demonstrated chronic exposure to 100?mg/L fluoridated tap water resulted in damage to the CA3 region in the hippocampus of SD rats, with nuclear membrane crumpling, chromatin aggregation, and alteration of neuron morphology and characteristics. Xu et al. (Xu and Hu et al., 2023) showed that with increasing NaF exposure during pregnancy, the number of Nissl bodies in offspring rats decreased, the staining became shallow and blurred, and the ultrastructure of the hippocampal tissue was damaged. Li et al. (Li and Lu et al., 2022) also found that NaF exposure caused damage to mouse hippocampal neurons using Nissl staining, while the TEM results showed that the nuclei of the hippocampal neurons in the 100?mg/L NaF group were atrophied and irregularly shaped. Furthermore, synaptic damage was observed in the rats’s hippocampal neurons. These results are consistent with our findings that prenatal fluoride exposure could decrease the number of Nissl bodies, making the Nissl bodies staining shallow and blurred in offspring rats’ neurons. Therefore, fluoride can cause neuronal damage.

From the proteomic and phosphoproteomic analyses, we found that differential proteins were mainly concentrated in the pathways of neuronal differentiation, synaptic signaling, and cytoskeleton (Fig. S1). Jiang et al. (Jiang and Li et al., 2019) found that treatment with 50?mg/L and 100?mg/L NaF inhibited glycogen synthase kinase-3 beta (GSK-3?)/?-catenin signaling in a dose-dependent manner and impaired neurogenesis and synaptic plasticity. Furthermore, Ge et al. (Ge and Chen et al., 2018) found that exposure of mice to NaF (50 and 100?mg/L) during pregnancy impaired the cytoskeleton in the cerebral cortex of the offspring mice, which exhibited impaired cognitive function with disrupted synapse-associated protein expression and diminished neuronal function. Thus, NaF exposure impairs synaptic function and signaling, causing neuronal damage. Moreover, we found that most differential genes were enriched in the cyclic adenosine monophosphate (cAMP) signaling pathway, oxytocin signaling pathway, and MAPK signaling pathway (Fig. S2). The cAMP signaling pathway is a cyclic nucleotide signaling pathway continuously triggered in astrocytes (Zhou and Ikegaya et al., 2019). An increase in the cAMP in astrocytes leads to the release of lactate, which is important for maintaining synaptic plasticity and memory (Zhou and Okamoto et al., 2021). Niu et al. (Niu and Chen et al., 2018) found that 60?mg/L NaF reduced cAMP response element binding protein (CREB) expression in the hippocampus of mice and damaged synaptic ultrastructure in the hippocampus. These results suggest the importance of the cAMP signaling pathway in maintaining the synaptic structure and function. MAPK plays an important role in the formation of synapses (Wakatsuki and Takahashi et al., 2021). Gopalan et al. (Gopalan and Venkatramanan, 2023) revealed that the disruption of the brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling pathway mediated by fluoride through the MAPK/ERK1/2 signaling pathway leads to synapse dysfunction in human neuroblastoma SH-SY5Y cells, ultimately causing developmental neurotoxicity. Hence, fluoride exposure mostly affects the synaptic structure and function, which is consistent with the results of our analyses.

Combined proteomic and phosphoproteomic analyses revealed the hyperphosphorylation of synapse-associated proteins in the hippocampus of rats exposed to fluoride during pregnancy. This was evidenced by a significant increase in rat hippocampal p-PRKAA1 (S486), p-PRKAA1 (T172), p-CRMP-2 (S522), and p-CRMP-2 (T509) levels, which aligns with the results of Yang et al. (Yang et al., 2022). They found that PRKAA1 (T172) hyperphosphorylation decreased synaptic plasticity and inhibited neurite growth in SH-SY5Y cells. Lee et al. (Lee and Kondapalli et al., 2022) found that overactivation of Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-AMPK could induce mitochondrial fission and mitochondrial autophagy, leading to synaptic loss. Belforte et al. (Belforte and Agostinone et al., 2021) found that AMPK hyperactivation can inhibit mechanistic target of rapamycin complex 1 (mTORC1) function, promoting dendrite retraction, synaptic loss, and neuronal dysfunction. Attenuation of AMPK activity restores mTORC1 function and rescues dendrite and synaptic contacts. Cole et al. (Cole and Knebel et al., 2004) found that CRMP-2 hyperphosphorylation plays a role in axonal damage caused by GSK-3, which is related to the occurrence of Alzheimer’s disease. Petratos et al. (Petratos and Li et al., 2008) also showed that threonine phosphorylation of CRMP-2 increased in the transgenic mouse model of Alzheimer’s disease. Furthermore, this increase was related to the decreased ability of CRMP-2 to bind tubulin. Phosphorylated CRMP-2 interferes with tubulin assembly in neurites and affects synaptic transmission. Therefore, an increase in AMPK and CRMP-2 phosphorylated leads to synaptic damage. Interestingly, our combined omics analysis found that fluoride triggered PRKAA1 (S486), CRMP-2 (S522), and CRMP-2 (T509) hyperphosphorylation in the rat’s hippocampal tissue, with consistent Western blotting results. However, Li et al. (Li and Lu et al., 2022) found that exposure to fluoride during pregnancy can lead to decreased expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYN) in the hippocampus of offspring, resulting in synaptic dysfunction and thus cognitive impairment. Therefore, abnormal AMPK and CRMP-2 phosphorylation may be new molecular markers of synaptic damage.

Conclusion

In conclusion, our study identified a new factor of fluoride-induced developmental neurotoxicity, namely, the upregulation of AMPK and CRMP-2 phosphorylation, which leads to structural and functional abnormalities of synapses in F₁ SD rats. The abnormalities further cause NaF-induced nerve damage. These results clarify the role of abnormal AMPK and CRMP-2 phosphorylation in fluoride-induced neurological injury and may guide the development of drugs targeting AMPK and CRMP-2 phosphorylation status for the prevention or treatment of fluoride-induced neurological injury. This also provides new ideas for understanding and treating fluoride-associated neurodegenerative diseases.

Statement

This work has received approval for research ethics from Animal Experimental Ethical Inspection Committee of First Affiliated Hospital, Shihezi University School of Medicine and a proof/certificate of approval is available upon request.

CRediT authorship contribution statement

Niu Qiang: Writing – review & editing, Resources, Funding acquisition, Conceptualization. Zhang Jingjing: Writing – original draft, Methodology, Formal analysis. Xu Panpan: Writing – review & editing, Methodology. Zhang Yue: Writing – review & editing. Li Tingting: Visualization, Investigation. Ding Xueman: Visualization, Investigation. Liu Li: Visualization, Investigation. Yao Ping: Writing – review & editing, Funding acquisition.

Declaration of Competing InterestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.AcknowledgmentsThis study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 82360671 and 82060580), the Shihezi University International Science and Technology Cooperation Promotion Programme Project (No. GJHZ202308), the Open Research Fund of Laboratory of Xinjiang Endemic Diseases Shihezi University, Ministry of Education (NO. KF2021–3), Shihezi University independently funded and supported university-level scientific research projects (No. ZZZC2023027), as well as the Bingtuan Program of Science and Technology Innovation (Grant No. 2021CB046).Appendix A. Supplementary material

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Supplementary material

Data availabilityThe data that has been used is confidential.

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Unraveling the role of abnormal AMPK and CRMP-2 phosphorylation in developmental fluoride neurotoxicity: Implications for synaptic damage and neurological disorders.

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