The Role and Mechanism of the cGAS–STING/NLRP3 Signaling Axis-Regulated p38 MAPK/NF–κB Pathway in Manganese-Induced Tau Aggregation-Mediated Neurotoxicity

Abstract

Manganese (Mn) is an essential trace element, but increasing industrial and agricultural use has led to elevated levels of environmental Mn levels. Chronic overexposure to Mn can cause a Parkinson’s-like neurodegenerative disorder; however, the underlying mechanisms remain incompletely understood. This study combined in vitro and in vivo models to investigate the role of the cGAS–STING/NLRP3 axis in regulating the p38 MAPK/NF–κB pathway in the process of Mn-induced Tau aggregation leading to neurotoxicity. Our results demonstrated that MnCl₂ exposure significantly activated both the cGAS–STING/NLRP3 signaling axis and the p38 MAPK/NF–κB pathway, accompanied by increased Tau expression. Genetic ablation of cGAS, STING, or NLRP3 attenuated Mn-induced Tau upregulation, indicating that the cGAS–STING/NLRP3 signaling axis mediates Tau expression. RNA-seq analysis further revealed that the decreased Tau expression in knockout cells is associated with the MAPK/NF–κB signaling pathway. Pharmacological inhibition of p38 MAPK or NF–κB markedly downregulated cGAS–STING signaling, inflammatory cytokine release, and phosphorylated Tau (p-Tau) levels. Moreover, we observed colocalization and interaction among p-p38, p-p65, and p-Tau. Overall, these findings reveal that Mn activates the cGAS–STING/NLRP3 signaling axis regulated by the p38 MAPK/NF–κB pathway, driving pathological Tau aggregation.

1. Introduction

Manganese (Mn) is a widespread environmental pollutant, with its concentrations rising due to industrial development and intensified human activities. Human exposure to Mn occurs primarily through dermal contact, inhalation, dietary intake, and occupational exposure. Chronic or excessive Mn exposure leads to Mn²⁺ uptake into the bloodstream, where it crosses the blood–brain barrier via binding to albumin and divalent metal transporters. This results in abnormal or excessive accumulation in brain regions such as the hippocampus, globus pallidus, hypothalamus, and striatum, ultimately contributing to Mn-induced neurotoxicity, neurodegenerative disorders, and other adverse health effects.

The neurotoxicity of Mn involves multiple complex mechanisms, including neuroinflammation, oxidative stress, mitochondrial dysfunction, autophagy dysregulation, and apoptosis. Among these, neuroinflammation plays a pivotal role. Mn exposure activates microglia and impairs astrocytic mitochondria, triggering the release of pro-inflammatory cytokines and thereby promoting neuroinflammation and subsequent neuronal damage. Notably, neuroinflammation is closely linked to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). However, the precise mechanisms underlying Mn neurotoxicity remain incompletely understood.

The cGAS–STING pathway is a critical regulator of inflammatory responses. Upon nuclear or mitochondrial damageor during viral infectioncytosolic DNA fragments activate cyclic GMP–AMP synthase (cGAS), which catalyzes the synthesis of 2′3′-cGAMP. This leads to the activation of stimulator of interferon genes (STING), which in turn triggers the release of inflammatory cytokines such as the tumor necrosis factor (TNF), interleukin-1β (IL-1β), and IL-6, as well as type I interferons (IFNs), thereby initiating an inflammatory response.

Notably, Mn²⁺ is the secondary activator of cGAS, independent of double-stranded DNA (dsDNA). It enhances cGAS-mediated viral DNA sensing and cGAMP production while strengthening STING–cGAMP binding, thereby amplifying cGAS–STING signaling. , Persistent cGAS–STING activation promotes neuroinflammation, oxidative stress, endoplasmic reticulum (ER) stress, and autophagic dysfunction, all of which are implicated in neurodegenerative diseases such as AD, PD, ALS, and multiple sclerosis (MS). , Thus, the cGAS–STING pathway may be a key mediator of Mn-induced neurotoxicity.

Aberrant cGAS–STING activation triggers lysosomal rupture, upregulates pro-inflammatory gene transcription, and activates the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasomea critical driver of neurodegeneration. Mechanistically, DNA-dependent NLRP3 inflammasome activation requires cGAS–STING signaling. STING interacts with NLRP3, facilitating its ER localization and deubiquitination, thereby promoting inflammasome assembly.

Activation of the NLRP3 inflammasome is associated with the pathological processes of various inflammatory diseases. , In microglial cells, protein misfolding or the accumulation of Tau and amyloid-β (Aβ) can be recognized by the NLRP3 inflammasome, triggering inflammation and promoting the onset and progression of neurodegenerative diseases. Moreover, NLRP3 inflammasome activation is a critical factor driving Tau hyperphosphorylation. Excessive Mn exposure can cause severe lysosomal damage, leading to altered membrane permeability and the release of cathepsin B, which activates the NLRP3 inflammasome.

The mitogen-activated protein kinase (MAPK) pathway is another critical player in neuronal injury. Mn activates p38 and extracellular signal-regulated kinase (ERK) in astrocytes and microglia, increasing their phosphorylation and inducing oxidative stress, inflammation, and cellular damage. Paradoxically, MAPK/ERK signaling may attenuate Mn-induced neuroinflammation in microglia by suppressing inducible nitric oxide synthase (iNOS) expression, suggesting complex regulatory roles.

NF–κB is a central mediator in many immune and inflammatory responses. Mn can enhance the expression of chemokines and pro-inflammatory factors in astrocytes and microglial cells by activating NF–κB. Furthermore, the NF–κB pathway plays a role in intercellular communication during Mn-induced neurotoxicity, facilitating the transmission of inflammatory mediators between astrocytes and microglial cells. ,

The cGAS–STING/NLRP3 signaling axis and MAPK/NF–κB pathway exhibit a complex regulatory relationship. The p38 MAPK signaling pathway can activate NF–κB, regulating the expression of various inflammatory cytokines, including TNF-α and IL-1β. Studies have shown that STING activation can trigger the activation of the MAPK/NF–κB pathway. , STING promotes TBK1-dependent nuclear translocation of NF–κB, , facilitated by Ikkβ. NF–κB further amplifies STING signaling by inhibiting its lysosomal degradation. Additionally, the NF–κB pathway is a key driver of NLRP3 transcriptional regulation. Phosphorylated NF–κB (Ser536) enhances NLRP3 inflammasome formation, mediating IL-1β/IL-18 production in microglia. However, it remains unknown whether and how the cGAS–STING/NLRP3 signaling axis interacts with the p38 MAPK/NF–κB pathway to orchestrate pathological Tau aggregation following Mn exposure. This study aims to elucidate their roles and crosstalk, providing insights for targeted therapies against Mn-induced neurodegeneration.

2. Materials and Methods

2.1. Cell Lines and Cell Culture

BV2 microglial cells were obtained from Cyagen Bioscience, Inc. (Guangzhou, China) and cultured at 37 °C with 5% CO₂ in RPMI 1640 medium (VivaCell, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% penicillin–streptomycin solution (Biological Industries, Israel). Cells were passaged upon reaching 80%–90% confluence.

CRISPR/Cas9-mediated genome editing was used to generate cGAS, STING, and NLRP3 knockout cell lines. The single-guide RNA (sgRNA) plasmids targeting cGAS (5′-CGGGCCGCAGCTTTCCGCGTGGG-3′), STING (5′-GTGGATCCTTTGCCACCCAAAGG-3′), and NLRP3 (5′-TTCCTCTATGGTATGCCAGGAGG-3′) were purchased from Cyagen Biosciences, Inc. Following plasmid transfection into BV2 cells, puromycin selection was performed 24∼48 h post-transfection. After 2∼4 weeks of selection, single clones were isolated and validated by PCR and sequencing, yielding BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cell lines.

The BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cell lines were cultured and expanded in a serum-containing medium to ensure stable growth. For Mn treatment, wild-type (WT) BV2, BV2 ^STING–/– cells, and BV2 ^NLRP3–/– cells were exposed to 100 μmol/L Mn²⁺ for 6 h, while BV2 ^cGAS–/– cells were treated with 3.125 μmol/L Mn²⁺ for 6 h to simulate Mn toxicity. To inhibit the NF–κB pathway, BV2 cells were pretreated with 5 μmol/L BAY11–7082 (Selleck, USA) for 3 h. To inhibit the p38 MAPK pathway, cells were pretreated with 10 μmol/L SB203580 (Aladdin, China) for 2 h.

2.2. Animal Studies

A total of 20 C57BL/6J mice (WT) were obtained from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Animals were housed under specific pathogen-free (SPF) conditions in the Laboratory Animal Center of Lanzhou University, with controlled temperature (20 ± 2 °C), humidity (40%–60%), and 12 h light/dark cycles. Mice had ad libitum access to standard rodent chow and filtered water. After 7 days of acclimation, mice were randomly divided into two groups (n = 10 per group): WT control and WT + Mn.

Mn exposure was achieved by supplementing drinking water with MnCl₂·4H₂O (200 mg/L, calculated as Mn²⁺) for 5 weeks, as previously described. All mice were humanely euthanized under anesthesia at the experimental end point. Brains were immediately collected and processed as follows: one hemisphere was fixed in 4% neutral buffered paraformaldehyde (NBS0135, Shanghai Nonin Biological Technology) for histology, and the contralateral hemisphere was snap-frozen in liquid nitrogen and stored at −80 °C for molecular biological analysis. All mouse experiments were approved by the Institutional Review Committee of the School of Public Health, Lanzhou University (IRB23030501).

2.3. RNA Sequencing Analysis

Total RNA from BV2 cells and mouse brain tissue was extracted using TRIzol Reagent (Life Technologies, USA). RNA quality was assessed by measuring concentration (NanoDrop, Thermo Scientific, USA). Qualified RNA samples were subjected to library preparation using the following manufacturer’s protocol. Library quantification was performed by qPCR, and libraries with concentrations >2 nmol/L were selected for sequencing. Paired-end sequencing (150 bp) was conducted on the Illumina NovaSeq 6000 platform with a minimum depth of 40 million reads per sample at Biomarker Technologies. Differential expression analysis was performed using DESeq2 with thresholds of |fold change| ≥ 2 and FDR-adjusted P < 0.01. Functional enrichment analysis of differentially expressed genes (DEGs) was conducted using clusterProfiler and KOBAS to identify significantly enriched KEGG pathways (P < 0.05).

2.4. Enzyme-Linked Immunosorbent Assay

ELISA was performed to measure the levels of TNF-α and IL-6 in culture supernatants of BV2 cells treated with 100 μmol/L Mn²⁺ for 6 h, according to the manufacturer’s protocol (Elabscience, China). Supernatants were centrifuged at 1000 rpm for 10 min at 4 °C to remove debris before analysis. Briefly, biotinylated detection antibodies and HRP-conjugated streptavidin were sequentially added to form an immunocomplex. After incubation, the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added, yielding a blue product upon HRP catalysis. The reaction was terminated with an acidic stop solution, resulting in a yellow color. Absorbance was measured at 450 nm using a Bio-Rad microplate reader, and cytokine concentrations were calculated using a standard curve.

2.5. Western Blotting

Total proteins were extracted using a commercial kit (Beyotime, China) and quantified by the BCA assay (Thermo Scientific, USA). Protein samples (30 μg/lane) were separated on 10% SDS-PAGE gels (100 V for 90 min) and transferred to PVDF membranes (Millipore, USA) at 300 mA for 2 h. After blocking with 5% nonfat milk (Sigma-Aldrich) in Tris-Buffered Saline Tween (TBST) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies diluted in (antibody dilution buffer): anti-cGAS (Cell Signaling Technology, USA, 31659; 1:1000), anti-STING (Abcam, Cambridge, USA, ab288157; 1:1000), anti-NLRP3 (Abcam, Cambridge, USA, ab263899; 1:1000), anti-Caspase-1 (Abcam, Cambridge, USA, ab179515; 1:1000), anti-IRF3 (Abcam, Cambridge, USA, ab68481; 1:1000), anti-p-p38 (Cell Signaling Technology, USA, 4511; 1:1000), anti-p38 (Cell Signaling Technology, USA, 8690; 1:1000), anti-p-p65 (Cell Signaling Technology, USA, 3033; 1:1000), anti-p65 (Abcam, USA, ab32536; 1:1000), anti-p-IκB-α (Cell Signaling Technology, USA, 2859; 1:1000), anti-IκB-α (Abcam, USA, ab32518; 1:1000), anti-p-Tau (Abcam, USA, ab32057; 1:1000), anti-Tau (Proteintech Group, China, 66499–1-lg; 1:1000), anti-β-actin (Proteintech Group, China, 81115–1-RR; 1:1000), and anti-GAPDH (Signalway Antibody, USA, 21612; 1:1000). After washing with TBST, membranes were incubated with HRP-conjugated goat antirabbit IgG (1:10,000; Signalway Antibody) for 1 h at room temperature. Protein bands were visualized using an ECL substrate (Pierce, USA) and quantified by densitometry using ImageJ (NIH, USA). β-Actin and GAPDH served as loading controls.

2.6. Co-immunoprecipitation

Cells were washed 2∼3 times with ice-cold PBS and lysed in buffer containing phosphatase and protease inhibitors. The lysates were collected by scraping, sonicated on ice, and centrifuged at 12,000 rpm for 15 min at 4 °C to obtain the supernatant.

Protein A/G agarose beads were washed 2∼3 times with PBS and resuspended in 100 μL of PBS using a magnetic column. The specific antibody or control IgG was added to the cell lysate supernatant and incubated at 4 °C with gentle shaking for 1 h. Then, 50 μL of the resuspended agarose beads were added and incubated overnight at 4 °C with shaking. The beads were captured using a magnetic column and washed 2∼3 times with lysis buffer. The loading buffer was added, and the samples were boiled at 100 °C for 10 min. The eluted samples were subjected to electrophoresis under the same conditions as those previously described.

2.7. Immunofluorescence

The process of immunofluorescence was carried out according to our previous study. Antibodies of p-p38 (Cell Signaling Technology, USA, 4511; 1:500), p-p65 (Cell Signaling Technology, USA, 3033; 1:500), NLRP3 (Abcam, Cambridge, USA, ab263899; 1:500), and Tau (Proteintech Group, China, 66499–1-lg; 1:500) were used to observe the expression of corresponding proteins in brain tissue.

2.8. Molecular Docking

The three-dimensional structures of the target proteins were obtained from the Protein Data Bank (PDB) (https://www.rcsb.org). Protein–ligand docking simulations were performed using GRAMM (https://gramm.compbio.ku.edu/request) to predict binding affinities and interaction modes. The resulting ligand–protein complexes were visualized with PyMOL (v2.4.1).

2.9. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 9 (San Diego, CA). Between-group comparisons were analyzed using unpaired Student’s t-tests, while multigroup comparisons employed one-way ANOVA with Tukey–Kramer posthoc testing. Data are expressed as mean ± standard deviation, with statistical significance set at P < 0.05. Immunoblot results represent at least three independent experimental replicates.

3. Results

3.1. Mn Exposure Activates the cGAS–STING/NLRP3 Signaling Axis and Induces Tau Expression

We first examined Tau levels in BV2 cells and mouse hippocampal tissue following treatment with MnCl₂. Compared with controls, Mn²⁺ exposure significantly increased Tau expression (P < 0.05) (Figure A,G). Given that abnormal Tau aggregation is a hallmark of neurodegenerative diseases such as AD and frontotemporal dementia, these results suggest that Mn may induce neurotoxicity via Tau upregulation.

1.

Mn exposure activates the cGAS–STING/NLRP3 signaling axis and upregulates Tau expression. (A–G) Representative images of the immunoblots (A) and the relative quantification of the protein levels of cGAS (B), STING (C), IRF3 (D), NLRP3 (E), Caspase-1 (F), and Tau (G) in BV2 cells by Western blotting (n = 3); (H–M) representative images of the immunoblots (H) and the relative quantification of the protein levels of cGAS (I), STING (J), IRF3 (K), NLRP3 (L), and Tau (M) in murine brain tissues by Western blotting (n = 3). *P < 0.05, **P < 0.01.

To explore the underlying mechanisms, we assessed the activation of the cGAS–STING/NLRP3 axis. Western blot analysis revealed that Mn²⁺ treatment elevated protein levels of cGAS, STING, IRF3, NLRP3, and Caspase-1 in BV2 cells (P < 0.05) (Figure A–G). Considering the particular vulnerability of hippocampal pyramidal neurons to Mn toxicity, we also detected upregulation of these markers (cGAS, STING, IRF3, NLRP3, and Tau) in hippocampal tissues from Mn-exposed mice (P < 0.05) (Figure H–M).

Integrating in vitro and in vivo findings, Mn²⁺ not only induced Tau pathology but also activated the cGAS–STING/NLRP3 signaling axis. These observations imply that Mn-induced Tau pathology may be mediated through the cGAS–STING/NLRP3 signaling axis.

3.2. Mn Exposure Activates the p38 MAPK/NF–κB Pathway

To investigate the mechanism through which Mn exposure activates the cGAS–STING/NLRP3 signaling axis and induces Tau expression, we performed an RNA sequencing (RNA-seq) analysis on BV2 cells and mouse hippocampal tissues following Mn treatment. The RNA-seq results showed significant upregulation of MAPK/NF–κB signaling-related genesincluding Myc, Dusp1, Dusp5, Dusp8, Atf4, Jun, Jund, Cd40, Traf5, Traf1, Ptgs2, Ddx58, Ticam1, and Cxcl2in Mn-treated BV2 cells (Figure A,C). KEGG pathway analysis further indicated that DEGs were predominantly enriched in the MAPK pathway, NF–κB pathway, NOD-like receptor pathway, Toll-like receptor pathway, and chemokine signaling pathway (Figure D). KEGG enrichment analysis of DEGs from mouse hippocampal tissues likewise identified the MAPK signaling pathway as the pathway most significantly altered upon Mn exposure (Figure E).

2.

Mn exposure activates the p38 MAPK/NF–κB pathway. (A) Volcano plot displaying DEGs in Mn-treated BV2 cells. (B) Volcano plot of DEGs in hippocampal tissues from Mn-exposed mice. (C) Heatmap of MAPK/NF–κB pathway-associated DEGs in cellular samples. (D) KEGG pathway enrichment analysis of DEGs in BV2 cells. (E) KEGG pathway annotation of DEGs in hippocampal tissues. (F–I) Representative images of the immunoblots (F) and the relative quantification of the protein levels of p-p38 (G), p38 (H), and p-p38/p38 (I) in BV2 cells by Western blotting (n = 3); (J–P) Representative images of the immunoblots (J) and the relative quantification of the protein levels of p-IκB-α (K), IκB-α (L), p-IκB-α/IκB-α (M), p-p65 (N), p65 (O), and p-p65/p65 (P) in BV2 cells by Western blotting (n = 3). Q-R ELISA quantification of TNF-α and IL-6 secretion in culture supernatants (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001.

To validate the Mn-induced activation of these pathways, we examined the expression of key signaling proteins in the p38 MAPK and NF–κB pathways in Mn²⁺-exposed BV2 cells. Compared to the control group, Mn²⁺ stimulation markedly increased the expression levels of p-p38, p-p65, and p-IκB-α (P < 0.05), as well as the ratios of p-p38/p38, p-p65/p65, and p-IκB-α/IκB-α (P < 0.05) (Figure F–P). ELISA quantification revealed a significant increase in release of NF–κB-regulated proinflammatory cytokines, specifically TNF-α and IL-6 (P < 0.01), in culture supernatants of Mn²⁺-treated BV2 microglia (Figure Q,R). Together, these results demonstrate that Mn exposure activates the p38 MAPK/NF–κB pathway by enhancing p38 phosphorylation and promoting p65 phosphorylation and IκB-α phosphorylation/degradation.

3.3. The cGAS–STING/NLRP3 Signaling Axis Mediates Tau Expression

To examine the role of the cGAS–STING/NLRP3 signaling axis in abnormal Tau expression induced by Mn exposure, we knocked out the cGAS, STING, and NLRP3 genes in BV2 cells. As reported previously, BV2 ^cGAS–/– and BV2 ^STING–/– cells were successfully constructed using CRISPR/Cas9 gene editing. In the present study, we targeted exons 3∼4 of the NLRP3 gene (located on mouse chromosome 11 and containing 11 exons) to generate BV2 ^NLRP3–/– cells (Figure A).

3.

Effects of cGAS, STING, and NLRP3 knockout on Tau expression. (A) Schematic diagram of CRISPR/Cas9-mediated NLRP3 knockout BV2 cell line construction and sequencing validation results. (B–F) Representative images of the immunoblots (B) and the relative quantification of the protein levels of cGAS (C), STING (D), NLRP3 (E), and Tau (F) in BV2, BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cells by Western blotting (n = 3). *P < 0.05, **P < 0.01.

Western blot analysis confirmed the absence of cGAS, STING, and NLRP3 protein in the respective cell lines (Figure B–E). Tau expression, both under basal conditions and after Mn²⁺ exposure, was significantly reduced in cGAS, STING, and NLRP3-deficient cells (P < 0.01) (Figure F). These results indicate that the cGAS–STING/NLRP3 signaling axis mediates Tau upregulation and further support its pivotal role in Mn-induced neurotoxicity.

Notably, cGAS knockout decreased NLRP3 expression regardless of Mn treatment (P < 0.01) (Figure B,E), suggesting that cGAS deficiency may directly or indirectly regulate NLRP3 levels and pointing to a potential interaction between cGAS and NLRP3.

3.4. Regulatory Role of the cGAS–STING/NLRP3 Signaling Axis on the p38 MAPK/NF–κB Pathway

To investigate the mechanism through which the cGAS–STING/NLRP3 signaling axis mediates Tau expression, we performed RNA-seq analysis on BV2 cells following the knockout of cGAS, STING, or NLRP3. Principal component analysis (PCA) showed a clear separation between WT cells and knockout cells after Mn²⁺ treatment (Figure A), indicating that gene knockout altered the cellular transcriptional profile.

4.

Transcriptomic profiling of Mn²⁺-treated BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cells by RNA-seq analysis. (A) PCA plot showing global gene expression patterns. (B–D) Volcano plots of DEGs in WT vs STING ^–/– (B), WT vs cGAS ^–/– (C), and WT vs NLRP3^–/– (D); (E) heatmap of DEGs associated with the MAPK/NF–κB signaling pathway. (F–H) KEGG pathway enrichment analysis of DEGs, WT vs STING ^–/– (F), WT vs cGAS ^–/– (G), and WT vs NLRP3^–/– (H).

Compared to Mn²⁺-treated WT cells, BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cell lines exhibited 1,961, 1,304, and 1557 upregulated DEGs, and 2225, 1559, and 1885 downregulated DEGs (|log2FC| > 1, P < 0.05), respectively (Figure B–D). The number of down-regulated genes exceeded that of up-regulated genes in each case. Heatmap analysis revealed that knockout of cGAS, STING, or NLRP3 downregulated key genes in both the MAPK pathway (including Atf4, Dusp1, Dusp5, Jun, and Jund) and the NF–κB pathway (including Cd40, Traf1, Ptgs2, Ddx58, and Ticam1) (Figure E). This downregulation was particularly pronounced in BV2 ^STING–/– cells (Figure E), suggesting that STING may play a central role in Mn²⁺-mediated activation of the MAPK/NF–κB pathway.

KEGG pathway enrichment analysis indicated that DEGs in the knockout cells were primarily enriched in the MAPK pathway (Figure F–H). Notably, BV2 ^STING–/– cells showed a specific enrichment of 33 DEGs (2.38%) in the NF–κB pathway (Figure F), implying STING-dependent regulation of NF–κB activity by Mn²⁺. DEGs were also associated with neurodegenerative diseases, including AD and PD (Figure F–H). Together, these transcriptional changes suggest that the cGAS–STING/NLRP3 axis may contribute to Mn-induced neurodegeneration by regulating the p38 MAPK/NF–κB pathway.

To further define this regulatory relationship, we examined key molecules of the p38 MAPK and NF–κB pathways in the knockout lines. Under basal conditions, cGAS, STING, or NLRP3 knockout did not significantly alter the expression of p-p38 or total p38 (Figure A–D). After Mn²⁺ treatment, p-p38 levels increased in all groups (P < 0.05) (Figure A–D). However, compared with Mn²⁺-treated WT cells, the p-p38/p38 ratio was significantly lower in each knockout group (P < 0.05) (Figure A–D), indicating that loss of cGAS, STING, or NLRP3 partially suppresses Mn-induced activation of the p38 MAPK pathway.

5.

Effects of the cGAS–STING/NLRP3 axis blockade on the p38 MAPK/NF–κB pathway. (A–D) Representative images of the immunoblots (A) and the relative quantification of the protein levels of p-p38 (B), p38 (C), and p-p38/p38 (D) in BV2, BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cells by Western blotting (n = 3). (E–K) Representative images of the immunoblots (E) and the relative quantification of the protein levels of p-IκB-α (F), IκB-α (G), p-IκB-α/IκB-α (H), p-p65 (I), p65 (J), and p-p65/p65 (K) in BV2, BV2 ^cGAS–/–, BV2 ^STING–/–, and BV2 ^NLRP3–/– cells by Western blotting (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

In the NF–κB pathway, STING knockout reduced the p-p65/p65 ratio under basal conditions, yet Mn²⁺ treatment still increased p-IκB-α and p-p65 levels (P < 0.001) and elevated the p-IκB-α/IκBα ratio (P < 0.001) (Figure E–K). This suggests that STING knockout inhibits p65 phosphorylation, but Mn²⁺ can partially reactivate NF–κB signaling.

For the cGAS knockout, basal levels of p-IκB-α and p-p65 were elevated (Figure E–K). However, Mn²⁺ treatment did not significantly alter the p-IκB-α/IκB-α and p-p65/p65 ratios (Figure E–K), indicating a complex Mn-independent effect of cGAS on NF–κB regulation.

NLRP3 knockout did not significantly affect the p-IκB-α/IκBα and p-p65/p65 ratios under basal conditions, but the p-IκB-α/IκBα ratio increased after Mn²⁺ treatment (Figure E–K), suggesting a minimal role for NLRP3 in modulating the NF–κB pathway during Mn exposure.

The above results show that blocking the cGAS–STING/NLRP3 signaling axis partially inhibited Mn-induced activation of the p38 MAPK pathway, and STING knockout additionally attenuated NF–κB pathway activation. These findings underscore the important regulatory role of the cGAS–STING/NLRP3 signaling axis in modulating the p38 MAPK/NF–κB pathway.

3.5. Inhibition of the p38 MAPK/NF–κB Pathway Ameliorated Mn-Induced Tau Pathology

To validate the critical role of the p38 MAPK/NF–κB pathway in Mn-induced Tau pathology, we performed intervention experiments using SB203580, a specific p38 MAPK inhibitor, and BAY 11–7082, a specific NF–κB pathway inhibitor.

Compared with the Mn²⁺-treated group, the SB203580 + Mn group showed significantly lower expression of p-p38 and a reduced p-p38/p38 ratio (P < 0.05) (Figure A–G), confirming that SB203580 effectively inhibited Mn²⁺-induced activation of the p38 MAPK pathway. Similarly, the BAY 11–7082+Mn group exhibited decreased levels of p-IκB-α and p-p65, as well as lower p-IκB-α/IκB-α and p-p65/p65 ratios relative to the Mn²⁺-treated group (P < 0.05) (Figure H–N), demonstrating that BAY 11–7082 suppresses IκB-α phosphorylation and subsequent NF–κB pathway activation. Following blockade of the p38 MAPK/NF–κB pathway, the increased p-Tau expression induced by Mn²⁺ treatment was significantly inhibited (P < 0.05) (Figure A,E,H,O). These results indicate that Mn-induced Tau pathology depends on the p38 MAPK/NF–κB pathway.

6.

Inhibition of the p38 MAPK/NF–κB pathway attenuates Mn-induced Tau expression. (A–G) Representative images of the immunoblots (A) and the relative quantification of the protein levels of p-p38 (B), p38 (C), p-p38/p38 (D), p-Tau (E), STING (F), and cGAS (G) in BV2 cells by Western blotting (n = 3); (H–R) representative images of the immunoblots (H) and the relative quantification of the protein levels of p-IκB-α (I), IκB-α (J), p-IκB-α/IκB-α (K), p-p65 (L), p65 (M), p-p65/p65 (N), p-Tau (O), cGAS (P), STING (Q), and IRF3 (R) in BV2 cells by Western blotting (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

To explore the interplay between the p38 MAPK/NF–κB and cGAS–STING pathways in Mn-induced Tau upregulation, we examined the cGAS–STING pathway components after pharmacological inhibition. Treatment with SB203580 significantly suppressed Mn-induced STING upregulation (P < 0.05) (Figure H,Q). Likewise, BAY 11–7082 attenuated Mn²⁺-activated expression of cGAS, STING, and IRF3 (P < 0.05) (Figure H,P–R). Together, these findings demonstrate that inhibition of the p38 MAPK/NF–κB pathway suppresses Mn-triggered activation of the cGAS–STING pathway, suggesting that the p38 MAPK/NF–κB pathway contributes to Mn²⁺-induced Tau dysregulation by modulating the cGAS–STING pathway.

3.6. Interactions among NLRP3, p38 MAPK, NF–κB, and Tau

Immunofluorescence analysis revealed a marked increase in the expression of p-p38, p-p65, NLRP3, and Tau in hippocampal tissues of Mn-exposed mice, as indicated by stronger fluorescence signals compared with the controls (Figure A). These observations are consistent with the immunoblotting data. Moreover, p-p38, p-p65, and Tau showed clear colocalization in the hippocampal region (Figure A). Molecular docking analysis predicted a direct interaction between NF–κB p65 and Tau, with a calculated binding free energy of −16.7 kcal/mol (Figure B). Co-immunoprecipitation (Co-IP) assays further demonstrated that both p38 and p-p65 proteins were enriched in p-Tau immunoprecipitates (Figure C), indicating direct or indirect interactions between p-Tau and p38 as well as between p-Tau and p-p65. In contrast, no interaction was detected between p-Tau and NLRP3. Together, these results establish a protein interaction network linking p38 MAPK, NF–κB, and Tau, which likely plays a critical role in mediating Mn-induced neurotoxicity.

7.

Interaction analysis among p38 MAPK, NF–κB, NLRP3, and p-Tau protein. (A) Immunofluorescence staining showing spatial distribution of p-p38, p-p65, NLRP3, and Tau proteins in mouse brain tissue sections. (B) The docking model shows the interaction between p65 and Tau, with the binding free energy reaching −16.7 kcal/mol. (C) Co-IP assays demonstrating protein–protein interactions between p-Tau and p38 and p-Tau and p-p65 in BV2 cells. Immunoblots are representative of three independent experiments.

4. Discussion

Environmental Mn exposure levels have risen progressively with industrial development. Chronic or excessive Mn exposure can lead to abnormal accumulation, which has been closely linked to the pathogenesis and progression of neurodegenerative diseases such as AD and PD. Tau, a microtubule-associated protein, plays a key role in stabilizing neuronal structure and facilitating intracellular transport. Under pathological conditions, Tau undergoes abnormal post-translational modifications including hyperphosphorylation and acetylation, leading to its detachment from microtubules and formation of neurofibrillary tangles (NFTs). Mn neurotoxicity involves neuronal damage, neuroinflammation, and Tau aggregation, linked to microglial overactivation. As key immune cells in the brain, microglia modulate neurodegeneration by balancing neuroprotective and neurotoxic responses, and pathological Tau has also been detected in glial cells. The BV2 cell line, an immortalized murine microglial cell line, exhibits high similarity to primary microglia in morphology, function, and molecular expression. Therefore, we used BV2 cells to establish an in vitro Mn cytotoxicity model.

Excessive activation of the cGAS–STING pathway can trigger severe inflammatory responses and contribute to neuroinflammation. Mn²⁺ has been shown to enhance the sensitivity of cGAS and its downstream adaptor STING, and it can directly activate the cGAS–STING pathway independent of dsDNA. Recent studies demonstrate that Mn induces mitochondrial dysfunction by downregulating mitofusin 2 (MFN2) and vacuolar protein sorting 35 (VPS35), thereby activating the NLRP3 inflammasome. Moreover, leucine-rich repeat kinase 2 (LRRK2) and KH-type splicing regulatory protein (KHSRP) have been identified as critical mediators in Mn-induced NLRP3 inflammasome activation. , Our in vitro and in vivo data demonstrate that Mn exposure upregulates the expression of cGAS, STING, IRF3, and NLRP3 in BV2 cells and mouse brain tissues, indicating activation of the cGAS–STING/NLRP3 signaling axis. These findings align with previous reports showing that Mn²⁺ potently activates the cGAS–STING-IFN-I pathway and that Mn exposure increases NLRP3 gene expression in the substantia nigra pars compacta while activating the NLRP3 inflammasome pathway in the hippocampus.

Aberrant Tau aggregation serves as a hallmark of neuroinflammation and neurodegenerative diseases. Our study shows that Mn exposure upregulates Tau expression in both in vitro and in vivo, suggesting that the cGAS–STING/NLRP3 signaling axis activated by Mn promotes Tau dysregulation and aggregation, thereby contributing to neurotoxicity. Notably, Tau upregulation was significantly attenuated in cGAS, STING, and NLRP3 knockout cells, supporting the view that the cGAS–STING/NLRP3 signaling axis mediates Tau dysregulation in Mn-induced neurodegeneration. Of particular interest is that cGAS knockout substantially reduced NLRP3 expression, indicating that cGAS directly or indirectly modulates NLRP3 levels. This further validates the existence of a functional cGAS–STING/NLRP3 signaling axis. Previous studies report that STING binds specifically to NLRP3, facilitating its ER localization and deubiquitination, thereby enhancing inflammasome activation. ,, Our results corroborate the established role of cGAS–STING as an upstream regulator of NLRP3, highlighting cGAS as a critical modulator of neuroinflammation and neurodegeneration via NLRP3-dependent mechanisms. However, in the present study, immunofluorescence analysis did not show colocalization of NLRP3 and Tau in the mouse hippocampus, and Co-IP in BV2 cells revealed no direct interaction, suggesting that NLRP3 may regulate Tau expression indirectly through alternative pathways. It is important to note that while this study delineates a key molecular mechanism linking Mn exposure to Tau pathology, the functional cognitive consequences of this pathway remain to be established.

The p38 MAPK pathway is a key regulator of inflammatory mediator production in microglia and plays a pivotal role in Mn-induced neurotoxicity. Previous studies have demonstrated that Mn exposure induces phosphorylation of p38 and ERK in human microglia, triggering oxidative stress and cytotoxicity. Furthermore, Mn activates the p38 MAPK pathway, initiating protein kinase A signaling cascades that ultimately lead to neuronal apoptosis. Subchronic Mn exposure has also been shown to increase phosphorylation of ERK and p38 in the rat thalamus and hippocampus. In this study, RNA-seq analysis also revealed significant upregulation of MAPK pathway-related genes in Mn-exposed BV2 cells and mouse hippocampal tissues. Importantly, immunofluorescence studies identified the colocalization of p-p38 and Tau in the hippocampus of Mn-treated mice. These collective findings establish a pivotal role for the p38 MAPK pathway in mediating Mn-induced Tau dysregulation. To further investigate the role of p38 MAPK in Mn-induced Tau pathology, we used SB203580, a selective p38α/β inhibitor that blocks downstream signaling. SB203580 pretreatment markedly attenuated Mn-induced p-Tau expression, and Co-IP confirmed an interaction between p38 and p-Tau. These results demonstrate that Mn drives Tau dysregulation via p38 MAPK activation, identifying this pathway as a potential therapeutic target for Mn neurotoxicity.

The NF–κB pathway is vital for regulating neuroinflammatory responses and is critically involved in Mn-induced neurotoxicity. Mn activates NF–κB through multiple mechanisms, including upregulation of the immunoproteasome catalytic subunit PSMB8 to promote p65 phosphorylation, IKK2 phosphorylation, p65 nuclear translocation, and suppression of excitatory amino acid transporter 2 (EAAT2). Through NF–κB-dependent mechanisms, Mn activates microglia and subsequently enhances inflammatory responses in astrocytes while also promoting NOS2 expression. Our study demonstrates that Mn treatment upregulates the levels of p-p65 and p-IκB-α and elevates the p-p65/p65 and p-IκB-α/IκB-α ratios. Concurrently, we observed an increased secretion of the NF–κB-dependent cytokines TNF-α and IL-6. Furthermore, we found colocalization and interaction between p-Tau and p-p65. These findings suggest that Mn exposure not only activates the NF–κB pathway to induce Tau expression but also promotes downstream inflammatory factor production. Previous studies show that Mn activates NF–κB in astrocytes, increasing proinflammatory cytokines, whereas IKK2 depletion or Bay 11–7082 (an IκB-α phosphorylation inhibitor) suppresses these effects. Consistent with Kirkley et al., we found that Bay 11–7082 treatment in BV2 cells significantly reduced Mn-induced p-Tau expression. Collectively, these findings underscore the central regulatory role of the NF–κB pathway in Mn-induced neurotoxicity and demonstrate that its inhibition can mitigate abnormal Tau expression.

The neurotoxic effects of Mn exposure involve the complex regulation of multiple signaling pathways. Among these, the cGAS–STING, p38 MAPK, NF–κB, and NLRP3 pathways are critically involved in inflammatory responses and may collectively contribute to Mn-induced neuroinflammation and neuronal damage through potential crosstalk. Previous studies have demonstrated that STING activation in both humans and mice triggers MAPK pathway activation. Similarly, our transcriptomic sequencing revealed that the genetic knockout of cGAS, STING, or NLRP3 improved Tau pathology in a manner primarily associated with the p38 MAPK pathway. To further investigate their regulatory relationship, we treated BV2 cells with the p38 MAPK inhibitor SB203580 in combination with MnCl₂ and assessed the expression of cGAS–STING pathway-related proteins. Our results showed no significant change in cGAS levels, whereas STING expression was significantly reduced, suggesting that the p38 MAPK pathway may exert feedback regulation on the cGAS–STING pathway by modulating STING expression. We propose a bidirectional regulatory mechanism for Mn-induced neurotoxicity: Mn²⁺ activates the p38 MAPK pathway via the cGAS–STING/NLRP3 signaling axis, and the p38 MAPK pathway, in turn, regulates STING expression, thereby modulating the cGAS–STING/NLRP3 axis. This reciprocal interaction likely plays a pivotal role in Mn-induced neuroinflammation and neuronal injury.

Dysregulated activation of the cGAS–STING and NF–κB pathways has been closely associated with the pathogenesis and progression of various neurodegenerative disorders, including AD, PD, and poliomyelitis. Studies have demonstrated that upon STING activation, TBK1 undergoes dimerization and translocates to the Golgi apparatus or ER intermediate compartment, subsequently initiating NF–κB and IRF3 activation, which ultimately leads to interferon and proinflammatory cytokine production. Molecules such as UL138, Bcl10, Ikkβ, and inhibitor of kappa B kinase epsilon (IKKε) play crucial roles in activating the cGAS–STING–NF–κB signaling axis. Our investigation revealed that the NF–κB pathway was also among the pathways enriched for DEGs following STING knockout. BV2 ^STING–/– cells exhibited a significantly decreased p-p65/p65 ratio after Mn²⁺ treatment, suggesting that Mn²⁺ may regulate NF–κB pathway activation through an STING-dependent mechanism. However, elevated p-IκB-α and p-p65 expression was observed in BV2 ^STING–/– cells both before and after Mn exposure, indicating that Mn²⁺ can still partially activate the NF–κB pathway, independent of STING. These findings collectively suggest that Mn exposure mediates NF–κB activation in part through the cGAS–STING pathway. In contrast, NLRP3 knockout had no significant effect on p-IκB-α/IκBα or p-p65/p65 ratios under basal conditions, indicating that NLRP3 plays a negligible role in regulating the NF–κB pathway. Zhang et al. reported that NF–κB pathway activation impedes STING trafficking from the Golgi to lysosomes while inhibiting STING degradation, thereby prolonging and amplifying STING signaling. To elucidate the interplay between cGAS–STING and NF–κB pathways in Mn-induced neurotoxicity, we treated BV2 cells with the NF–κB inhibitor BAY 11–7082 combined with MnCl₂. This intervention significantly reduced protein expression of cGAS, STING, and IRF3, demonstrating that NF–κB pathway inhibition downregulates cGAS–STING signaling components and confirming the existence of NF–κB-mediated feedback regulation on the cGAS–STING pathway. Thus, during Mn-induced aberrant Tau expression, while STING modulates NF–κB pathway activity, the NF–κB pathway conversely regulates cGAS–STING signaling, revealing a complex bidirectional interaction mechanism between these two pathways. Although our findings reveal that multiple Mn-activated pathways converge on a core Tau pathological mechanism, the potential distinctions between this Mn-driven pathology and canonical Alzheimer’s disease pathology represent an important avenue for future investigation.

5. Conclusions

Mn exposure activates both the cGAS–STING/NLRP3 signaling axis and the p38 MAPK/NF–κB pathway, leading to pathological Tau aggregation (Figure ). Within this process, p-p38, p-p65, and p-Tau interact directly or indirectly. Blocking the cGAS–STING/NLRP3 signaling axis attenuates Mn-induced activation of the p38 MAPK/NF–κB pathway, thereby reducing the level of abnormal Tau aggregation. Conversely, the pharmacological blockade of the p38 MAPK/NF–κB pathway also suppresses cGAS–STING signaling, further diminishing Tau pathology. These findings reveal a reciprocal regulatory network that drives Mn-induced neurotoxicity and highlight potential therapeutic targets for intervening in Mn-associated neurodegenerative processes.

8.

Schematic diagram demonstrates that Mn exposure activates both the cGAS–STING/NLRP3 axis and the p38 MAPK/NF–κB pathway, leading to the induction of Tau expression.

†.

S.Z. and Q.Z. contributed equally to this work. Shiyin Zhong: methodology, writingoriginal draft preparation. Qiongli Zhou, Jirui Yang, Zhimin Zhang, Xueting Chai, Jinghao Luo, Xin Zhang, Jingjing Liu, Yangzong Yixi, Lei Shi, Xuhong Chang: methodology, investigation, formal analysis, data curation. Hui Wang: conceptualization, writingreview and editing. All authors have read and agreed to the published version of the manuscript.

This work was supported by the Natural Science Foundation of Gansu Province (25JRRA653; 21JR11RA100); Gansu Province’s Longyuan Young Talents; Lanzhou Youth Science and Technology Talent Innovation Project (2023-QN-60); Science and Technology Support Special Project of Lanzhou Chengguan District (2025-zc-21); National Natural Science Foundation of China (31802256); Disease Prevention and Control Research Project of Gansu Province (GSJKKY2025-01); Startup Fund for the Construction of the Double First-Class Project, Lanzhou University, China (561121203); and Project of Education and Science Innovation Fund of Gansu Province (2022B-029).

Ethical Approval Statement: The animal study was reviewed and approved by the Institutional Review Committee of the School of Public Health, Lanzhou University (IRB23030501). All experiments complied with the WMA Statement on animal use in biomedical research for experimental design and analysis in pharmacology care.

The authors declare no competing financial interest.