Induction of Amyloid-β42 Production by Fipronil and Other Pyrazole Insecticides

Abstract

Generation of amyloid-β peptides (Aβs) by proteolytic cleavage of the amyloid-β protein precursor (AβPP), especially increased production of Aβ42/Aβ43 over Aβ40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aβ production originates from specific mutations of AβPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aβs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aβ42/Aβ43 over Aβ40 and shorter Aβs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aβ42/Aβ43 production. Nine pyrazole insecticides were found to induce a β- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aβ42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aβs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AβPP by highly purified γ-secretase toward Aβ42/Aβ43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aβ42/Aβ43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aβ42/Aβ43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

Keywords: Alzheimer’s disease; Aβ38; Aβ40; Aβ42; Aβ42/Aβ40 ratio; Aβ43; aftins; alzheimerogen; amyloid-β; amyloid-β protein precursor; fipronil; human chemical exposome; pesticides; phenylpyrazoles; prevention; pyrazoles; triazines; γ-secretase.

Fig. 1.

Some pyrazoles trigger the production of extracellular amyloid Aβ₄₂. Effect of 18 pyrazoles on extracellular amyloid Aβ₄₂ production by N2a-APP695 and CHO-7PA2-APPsw cells. Cells were treated with 100 μM of each compound for 18 h and cell supernatants were collected for extracellular Aβ₄₂ levels measurement by an ELISA assay. Aftin-5 was used as a positive control and the corresponding volume of vehicle (DMSO) was used as a negative control. Levels are expressed as fold change, ± SE, of Aβ₄₂ levels over the Aβ₄₂ level of control, vehicle-treated cells. Average of five experiments performed in triplicate. Horizontal dotted lines indicate levels for 1- and 3- fold increases in Aβ₄₂ concentration.

Fig. 2.

Molecular structure of the nine active pyrazoles.

Fig. 3.

Molecular structure of fipronil and some of its metabolites and derivatives.

Fig. 4.

Extracellular Aβ₄₂ production induced by pyrazoles is inhibited by β-secretase inhibitor IV, γ-secretase inhibitors DAPT and BMS 299897, and γ-secretase modulator ‘Torrey Pines’. N2a-APP695 cells were exposed to 10 μM β-secretase inhibitor IV, 2 μM BMS 299897, 2 μM DAPT or 10 μM ‘Torrey Pines’ compound. 1.5 h later cells were exposed to 50 μM (2,5,8,14,16,18) or 25 μM (3, 4, 6) pyrazoles or 50 μM aftin-5. Extracellular Aβ₄₂ levels were measured after 18 h and are expressed as fold change, ± SE, of Aβ₄₂ level in pyrazole-treated cells over the Aβ₄₂ level of control, vehicle-treated cells. Representative of two independent experiments performed in triplicates. Errors bars represent SE of all six values.

Fig. 5.

Absolute quantification of Aβ₃₈, Aβ₄₀, and Aβ₄₂ using LC-MS/MS. Levels of the three Aβs were determined by mass spectrometry in supernatants of N2a-APP695 cells following 18 h treatment with DMSO, 100 μM of pyrazoles 18,20, or 21. Amyloid levels are expressed as percentage of levels in vehicle-treated cells (average ± SE of quadriplicate values; absolute values in control cell supernatants are indicated) and Aβ₄₂/Aβ₄₀ ratios are indicated in parentheses. Horizontal dotted line indicates basal Aβ levels versus the values in DMSO-treated cells.

Fig. 6.

Pattern of Aβs produced by N2a-APP695 cells exposed to pyrazoles 18, 20, or 21. Cells were treated for 18 h with DMSO or 20 μM of each pyrazole. Cell supernatants were collected and analyzed as described. Quantification of all Aβs in N2a-APP695 cells supernatants are presented as percentage of total amyloids. Note the decrease in peptides Aβ_1–19, Aβ_1–37, Aβ_1–38, the increase in Aβ_1–40 and the appearance of Aβ_1–42 and Aβ_1–43 in the supernatants of pyrazole-treated cells (these two peptides are undetectable in the supernatant of DMSO-treated cells).

Fig. 7.

Pyrazoles trigger enhanced production of Aβ₄₂ versus Aβ₄₀ in neurons differentiated from human induced pluripotent stem cells. Neurons were derived from iPSCs obtained from healthy donor (APP WT, white bars) or from an AD patient with APP K724N mutation (grey bars). They were exposed for 24 h to DMSO (control), 100 μM aftin-5 or the nine pyrazoles. Cell supernatants were collected for extracellular Aβ₄₀ and Aβ₄₂ levels measurement by an ELISA assay. Levels are expressed as Aβ₄₂/Aβ₄₀ ratios ± SE of triplicate values. Horizontal dotted line indicates level for basal Aβ₄₂/Aβ₄₀ ratios.

Fig. 8.

Mass spectrometric analysis of the Aβs generated in cell-free γ-secretase assays, in the presence of DMSO or fipronils 18, 20, or 21. A) The Aβs generated by highly purified γ-secretase in the presence of 100 μM of fipronil (18), fipronil sulfone (20) or fipronil desulfinyl (21) or DMSO (vehicle) were pooled from triplicates of the activity assays, immunoprecipitated overnight with the anti-Aβ antibody 4G8 and protein G and analyzed by MALDI-TOF in a reflectron mode. The Aβs generated from the recombinant APP-C99 contain an N-terminal methionine, which results in a mass shift of +149m/z when compared to endogenous Aβ peptides. B) Note the increased Aβ₄₂/Aβ₄₀ ratio for fipronils 18, 20, and 21, and the increased Aβ₄₃/Aβ₄₀ ratio for fipronil sulfone (20).

Fig. 9.

Pyrazoles alter the cleavage pattern of Alcadeinα, leading to increased p3-Alcα38 production. A) Immunoprecipitation/mass spectrometry spectra of p3-Alcα peptides produced by HEK cells expressing full length Alcadeinα exposed to various pyrazoles or DMSO (control). Cells were treated for 48 h with 100 μM of each reagent and p3-Alc peptides were analyzed by MALDI-TOF/MS. A) Representative profile for each product showing the the p3-Alcα2N+34, p3-Alcα2N+35, p3-Alcα2N+36, p3-Alcα2N+37 and p3-Alcα2N+38 peaks. B) Relative quantification of p3-Alcα peptides produced by cells exposed to DMSO (control), fipronil (18), fipronil sulfone (20) or fipronil desulfinyl (21). Levels of each peptide are presented as ratios of p3-Alcα2N+38 versus p3-Alcα2N+35 (average ± SE of triplicate values).

Fig. 10.

Proteomics and phosphoproteomics analysis of N2a-APP695 cells exposed to fipronil sulfone (20) and dipropetryn. A) N2a-APP695 cells were exposed for 18 h to 20 μM fipronil sulfone (20), 100 μM dipropetryn or DMSO. This led to increased extracellular Aβ42 expression (3.58 ± 0.05 and 7.87 ± 0.81 fold change for fipronil sulfone (20) and dipropetryn, respectively, compared to DMSO-treated cells). Up/downregulated proteins in fipronil sulfone- (1634) and dipropetryn- (1638) treated cells versus DMSO-treated cells were identified and compared. Among the 261 common proteins, 178 proteins (listed in Supplementary Table 1) were either upregulated by both treatments or downregulated by both treatments. B) DAVID analysis of the 178 proteins common to fipronil sulfone (20) and dipropetryn treatments. Data are selected with p-value < 0.01 and FDR < 0.05. FDR, False Discovery Rate.

Fig. 11.

Short-term (72 hrs) time-course of fipronil (18) and fipronil sulfone (20) bioaccumulation in brain (A) and epididymal adipose tissue (B) following a single oral administration of fipronil (18) to mice. Fipronil (18) (10 mg/kg) was administrated by oral gavage at time 0. Animals were sacrificed at various times and plasma, epididymal adipose tissue and brain were collected. Fipronil (18) and fipronil sulfone (20) levels were quantified by LC-MS/MS. Concentrations are expressed as μg/brain or (μg/epididymal adipose tissue.

Fig. 12.

Long-term (56 days) time-course of fipronil sulfone (20) production and accumulation following single (A) or repeated (B) oral administrations of fipronil (18) to mice. A) Fipronil (18) (10 mg/kg) was administrated by oral gavage on day 0. Animals were sacrificed at various times and plasma, epididymal adipose tissue and brain were collected. Fipronil sulfone (20) levels were quantified by LC-MS/MS. B) Fipronil (18) (10 mg/kg) was administrated by oral gavage 5 days/week for 3 weeks (times of administration are indicated by black dots). Animals were sacrificed at various times and plasma, epididymal adipose tissue and brain were collected. Fipronil sulfone (20) levels were quantified by LC-MS/MS. Concentrations are expressed as μg/brain, μg/epididymal adipose tissue or μg/mL plasma.