Dual-Emission Fluorescent Probe for Simultaneous Visualization of Monoamine Oxidase A and Lysyl Oxidase in Alzheimer’s Disease Cell Models

Abstract

Monoamine oxidase A (MAO-A) and lysyl oxidase (LOX) are two distinct amine oxidases that play crucial roles in many neurodegenerative diseases. Despite their similar enzymatic nature, their spatial distribution in different diseases and potential synergistic effects are poorly understood, partly due to the technical difficulties in detecting them concurrently. Herein, we designed and synthesized a series of dicyanoisophorone-based fluorescent probes, MAP-X (X = 2F, 2Cl, 2Br, F, Cl, Br), among which MAP-2F exhibits dual-emission fluorescence with high selectivity, enabling real-time visualization and simultaneous imaging of MAO-A (660 nm) and LOX (530 nm) in neuronal cells. By conducting experiments on neuronal cell models of Alzheimer’s disease (AD) treated with Aβ₄₂ oligomers, aggregates, and glutamate, the probe successfully detected dynamic changes in MAO-A and LOX. Further screening of 20 AD-related natural products revealed that several of them exhibited significant inhibitory effects on MAO-A and LOX, and were associated with the production, clearance, or activity of Aβ protein. These results indicate a close relationship between the expression of MAO-A and LOX and changes in Aβ protein levels. The simultaneous detection of MAO-A and LOX in AD-model cells provides an approach for studying the pathological mechanisms of AD and developing potential therapeutic targets.

Introduction

Monoamine oxidases (MAOs) are flavin-dependent enzymes that catalyze the oxidative deamination of primary, secondary, and tertiary amines to produce the corresponding imines, which are then hydrolyzed to aldehydes. In humans, two isoenzymes have been identified, MAO-A and MAO-B, which share approximately 73% homology in sequence but are encoded by two different genes and have distinct tissue allocation systems and separate substrate specificities. , MAO-A preferentially metabolizes larger intrinsic amines, such as epinephrine, serotonin, and norepinephrine, whereas MAO-B has a higher affinity for β-phenylethylamine and benzylamine. Both isoforms are capable of oxidizing tyramine and dopamine. − Based on this, monoamine oxidase plays a key role in regulating neurotransmitter homeostasis in the nervous system, and abnormalities in its activity have been associated with the progression of a variety of neurodegenerative diseases, including AD. − Therefore, the development of fluorescence detection techniques that can specifically distinguish MAO-A from MAO-B is of great significance for dissecting their respective contributions to disease and designing isoform-selective therapeutic strategies.

Lysyl oxidase (LOX), a copper-dependent amine oxidase, represents a structurally and functionally distinct member of the amine oxidase family. − Unlike the intracellular localization of MAOs, LOX primarily operates in the extracellular matrix (ECM), where it catalyzes the oxidative deamination of lysine residues in collagen and elastin, facilitating their cross-linking and contributing to ECM structural integrity. − Abnormalities in LOX expression or activity have been associated with a variety of pathologies, including neurodegenerative diseases. − However, the current methods for real-time monitoring of LOX activity in complex biological systems remain limited, particularly in the context of AD. − Notably, despite differences in catalytic substrates and cellular localization of LOX and MAO-A/B, emerging evidence suggests that they may share commonalities in contributing to AD pathology; MAO-A/B is associated with oxidative stress and neurotransmitter dysregulation, whereas LOX may influence amyloid plaque formation and ECM remodeling, both of which are hallmarks of AD progression. Thus, the ability to monitor both enzymes simultaneously in biological systems may provide valuable mechanistic insights into their interactions and roles in neurodegenerative pathologies.

Here, we report the development of a series of near-infrared (NIR) fluorescent probes, MAP-X (X = 2F, 2Cl, 2Br, F, Cl, Br), based on dicyanoisophorone and propylamine scaffolds. We achieved selectivity by employing rational halogen substitutions to modulate the reactivity of the probes with the enzyme. Among them, MAP-2F exhibited selective and rapid detection of MAO (λ_em = 660 nm and LOD = 83.38 ng/mL) under 488 nm excitation. Remarkably, the same probe also enabled the detection of LOX at a distinct emission wavelength (λ_em = 530 nm), with a spectral separation of ∼130 nm, effectively minimizing crosstalk, and facilitated the realization of simultaneous monitoring of the two enzymes. Cellular imaging experiments confirmed the probe’s ability to simultaneously visualize endogenous MAO-A and LOX activity in live neuronal cells. In AD cellular models induced by Aβ₄₂ oligomers and glutamate, MAP-2F successfully tracked changes in enzyme activity. Moreover, screening of 20 natural products associated with AD revealed several candidates with dual-inhibitory effects on MAO-A and LOX, implicating them in the regulation of AD pathology (Figure ). Collectively, this work introduces a powerful chemical tool for the simultaneous imaging of MAO-A and LOX in neurodegenerative disease models and offers a new strategy for investigating their cooperative roles in AD pathogenesis and therapeutic development.

1.

Schematic illustration of the detection mechanism and biological application of the dual-responsive fluorescent probe MAP-2F for the simultaneous detection of LOX and MAO-A.

Experimental Section

Reagents and Instruments

The reagents and instruments employed in this study are detailed in the Supporting Information.

Synthesis of Probe MAP-X

A solution of DM-X-NBoc (1 equiv) in 5 mL of a mixed solvent of dichloromethane (DCM) and methanol (MeOH) (DCM/MeOH = 5/1) containing 4 M HCl was stirred at room temperature for 2 h. Upon completion, the reaction mixture was quenched and neutralized by the addition of saturated NaHCO₃, and then it was extracted with ethyl acetate. The organic phase was collected and dried with anhydrous magnesium sulfate. Finally, the solvent was evaporated, and the crude product was purified by silica gel column chromatography to afford the target compound MAP-X as an orange or yellow solid.

MAP-2F

0.1 mmol, 28.9 mg, 75.3%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 8.04 (s, 3H), 7.55 (d, J = 9.7 Hz, 1H), 7.30 (m, 3H), 6.90 (s, 1H), 4.25 (t, J = 6.1 Hz, 2H), 2.96 (m, 2H), 2.63 (s, 2H), 2.51 (s, 2H), 2.01 (m, 2H), 1.02 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.20, 156.39, 154.95, 153.95,134.61, 132.02, 131.08, 123.55, 116.52, 113.66, 112.89, 111.71, 111.47, 77.17, 71.52, 42.18, 38.08, 35.83, 31.65, 27.58, 27.39; HRMS: calculated to be [M + H]⁺ 384.1882, found: 384.1871.

MAP-2Cl

0.1 mmol, 32.1 mg, 77.0%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 7.88 (s, 2H), 7.83 (s, 3H), 7.37 (m, 2H), 6.93 (s, 1H), 4.10 (t, J = 6.1 Hz, 2H), 3.07 (q, 2H), 2.63 (s, 2H), 2.51 (s, 2H), 2.07 (m, 2H), 1.01 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.68, 155.39, 151.12, 134.92, 134.26. 132.14, 129.30, 128.58, 124.30, 114.13, 113.34, 77.87, 71.47, 42.68, 38.56, 36.83, 32.14, 28.26, 27.90; HRMS: calculated to be [M + H]⁺ 416.1291, found: 416.1278.

MAP-2Br

0.1 mmol, 41.1 mg, 81.2%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 8.06 (s, 2H), 7.91 (s, 3H), 7.37 (m, 2H), 6.93 (s, 1H), 4.06 (t, J = 6.1 Hz, 2H), 3.06 (m, 2H), 2.63 (s, 2H), 2.49 (s, 2H), 2.11 (m, 2H), 1.01 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.71, 155.50, 152.97, 136.02, 134.32, 132.21, 132.09, 124.24, 118.63, 114.15, 113.34, 77.78, 71.23, 42.69, 38.56, 36.76, 32.12, 28.18, 27.91; HRMS: calculated to be [M + H]⁺ 506.0261, found: 506.0254.

MAP-F

0.1 mmol, 25.2 mg, 69.3%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 8.04 (s, 3H), 7.69 (dd, J = 12.9, 1.9 Hz, 1H), 7.47 (d, J = 9.2 Hz, 1H), 7.27 (m, 3H), 6.89 (s, 1H), 4.20 (t, J = 6.1 Hz, 2H), 2.95 (m, 2H), 2.62 (s, 2H), 2.52 (s, 2H), 2.06 (m, 2H), 1.02 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.29, 155.87, 152.92, 147.39, 136.36, 128.76, 125.57, 122.46, 114.83, 114.31, 114.12, 113.88, 113.08, 76.00, 65.78, 42.24, 38.12, 35.95, 31.66, 27.41, 26.65; HRMS: calculated to be [M + H]⁺ 366.1977, found: 366.1966.

MAP-Cl

0.1 mmol, 33.4 mg, 87.6%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 8.00 (s, 3H), 7.89 (d, J = 2.0 Hz, 1H), 7.64 (dd, J = 8.6, 1.9 Hz, 1H), 7.25 (m, 3H), 6.88 (s, 1H), 4.22 (t, J = 6.0 Hz, 2H), 2.98 (m, 2H), 2.62 (s, 2H), 2.52 (s, 2H), 2.07 (m, 2H), 1.02 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.79, 156.38, 154.92, 136.43, 130.46, 129.25, 129.15, 129.07, 123.02, 122.52, 114.43, 113.57, 76.54, 66.31, 42.75, 38.62, 36.57, 32.15, 27.92, 27.14; HRMS: calculated to be [M + H]⁺ 382.1681, found: 382.1671.

MAP-Br

0.1 mmol, 29.6 mg, 69.5%, ¹H NMR (400 MHz, DMSO-d ₆, 298 K) δ 8.04 (d, J = 1.9 Hz, 1H), 7.92 (s, 3H), 7.68 (dd, J = 8.6, 1.9 Hz, 1H), 7.35 (m, 2H), 7.16 (d, J = 8.7 Hz, 1H), 6.88 (s, 1H), 4.21 (t, J = 6.0 Hz, 2H), 3.00 (m, 3H), 2.62 (s, 2H), 2.51 (s, 2H), 2.06 (m, 2H), 1.02 (s, 6H); ¹³C NMR (100 MHz, DMSO-d ₆, 298 K) δ 170.78, 156.40, 155.78, 136.34, 132.22, 130.95, 129.68, 129.20, 122.99, 114.38, 114.24, 113.57, 112.24, 76.50, 66.35, 42.75, 38.62, 36.60, 32.14, 27.93, 27.14; HRMS: calculated to be [M + H]⁺ 426.1176, found: 426.1165.

Fluorescence Responses of MAP-X to Lysyl Oxidase (LOX) and Monoamine Oxidase (MAO)

First, we evaluated the time-dependent fluorescence response of MAP-X to LOX, MAP-X was incubated in borate buffer solution (BBS, 15 mM, pH 8.0, containing 20 μM Cu²⁺) with or without LOX (50 μg/mL). The probe was added to a final concentration of 10 μM, and the reaction mixture was incubated at 37 °C under gentle rotation using a tube rotator. And the fluorescence emission spectra were recorded at various time points. Simultaneously, an analogous time-course experiment was conducted to assess the response of MAP-X to monoamine oxidase (MAO). In this case, the assay was performed in phosphate-buffered saline (PBS, 10 mM, pH 7.4), with or without MAO (20 μg/mL), under otherwise identical conditions.

To measure the fluorescence response of MAP-2F toward LOX, MAP-2F (10 μM) was incubated in BBS buffer (pH = 8.0) containing different concentration of LOX (0, 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 55, 60, 70, 80, 90, and 100 μg/mL) at 37 °C for 2 h.

Meanwhile, to measure the fluorescence response of MAP-2F to different concentrations of MAO, MAP-2F (10 μM) were incubated in PBS buffer (pH = 7.4) containing different concentration of MAO (0, 2.4, 4.8, 7.2, 9.6, 12, 14.4, 16.8, 18, 19.2, 20.4, 21.6, 22.8, 24, 25.2, 26.4, 27.6, and 28.8 μg/mL,) at 37 °C for 2 h.

A calibration curve was constructed by plotting the fluorescence intensity against the LOX/MAO concentration, and linear regression analysis was performed over the full concentration range. The slope (k) of the linear fit was then used to calculate the limit of detection (LOD) according to the following formula:

$$\mathrm{limit}\mathrm{of}\mathrm{detection}(\mathrm{LOD})=3\sigma /k$$

The standard deviation σ of the blank signal was again determined from 20 repeated measurements of MAP-2F in BBS (for LOX, σ = 17.63) and in PBS (for MAO, σ = 8.62) without an enzyme.

The LOD of MAP-2F for LOX was then determined to be 0.92 μg/mL. The detection limit of MAP-2F for MAO was then determined to be 83.38 ng/mL.

To assess the selectivity of MAP-2F toward different analyte, MAP-2F (10 μM) was incubated with various analytes, including metal ions (Pb²⁺; Ce³⁺; Mg²⁺; Mn²⁺; Zn²⁺; Pd²⁺; Co²⁺; Cu²⁺; Al³⁺; Cr³⁺), amino acids (l-Cys; l-Ala; l-Met; l-Tyr; l-Leu; l-Phe; l-Val; l-Ile; l-Pro; l-Lys), ROS/RNS (NO₂ ^–; HS^–; ClO^–; HO•; H₂O₂; ¹O₂; TBHP), and human serum albumin (HSA), glutathione (GSH), Aβ₄₂ aggregate, LOX, and MAO. Reactions were carried out in BBS for LOX and PBS for MAO at 37 °C for 2 h. Fluorescence spectra were subsequently recorded to evaluate the probe’s response toward each analyte.

Cell Culture

Human neuroblastoma (SH-SY5Y) cells and human hepatocellular carcinoma (Hep G2) cells were obtained from the American Type Culture Collection (Manassas, VA). SH-SY5Y cells were cultured in minimum essential medium (MEM, Gibco, 24 mL) and Ham‘s F12 nutrient medium (F12, Gibco, 24 mL) supplemented with 5.6 mL of fetal bovine serum (Life Technologies, USA), nonessential amino acids (0.56 mL), and 100 U/mL penicillin/100 U/mL streptomycin. Cells were maintained and grown at 37 °C in a humidified atmosphere with a 5% CO₂ incubator. HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, PAN Biotech, Germany) supplemented with 10% fetal bovine serum (Life Technologies, USA), sodium pyruvate, nonessential amino acids, and 100 U/mL penicillin/100 U/mL streptomycin. Cells were maintained and grown at 37 °C in a 5% CO₂ incubator.

Fluorescence Imaging Studies

Living SH-SY5Y and HepG2 cells were plated in confocal imaging dishes at a density of 80,000 cells/well and allowed to adhere overnight under standard culture conditions. Following incubation, the culture media were removed, and each well was washed with MEM/F12 medium (1:1, containing 10% FBS and 1% nonessential amino acids). Subsequently, 1000 μL of fresh media containing 10 μM MAP-2F was added to each well, and the cells were incubated at 37 °C in a humidified incubator with 5% CO₂ for 2 h. After incubation, the staining medium was removed and each well was washed three times with PBS before imaging.

To evaluate the effects of different aggregations of Aβ₄₂ on the expression levels of MAO-A and LOX, SH-SY5Y cells were plated in confocal imaging dishes at a density of 80,000 cells/well and allowed to adhere for 12 h. The culture medium in each well was then replaced with 1000 μL of medium containing the various concentrations of Aβ₄₂ monomer (0, 1, 2, and 5 μM), Aβ₄₂ oligomer (0, 1, 2, and 5 μM), and Aβ₄₂ aggregate (0, 1, 2, and 5 μM), followed by incubation at 37 °C in a humidified 5% CO₂ for 12 h. After treatment, the medium was removed and each well was washed with MEM/F12 medium (1:1, containing 10% FBS and 1% nonessential amino acids). Then, cells were incubated with 1000 μL of medium containing 10 μM MAP-2F for 2 h at 37 °C. After staining, the cells were washed three times with PBS before imaging.

In a parallel time-course study, SH-SY5Y cells were treated with an Aβ₄₂ monomer, oligomer, or aggregate (each at 5 μM) for varying durations (2, 4, 6, and 8 h) under identical conditions. Following each time point, the staining and imaging procedures were performed as described above.

To investigate the impact of Glu-induced excitotoxicity on endogenous MAO-A and LOX activities, SH-SY5Y cells were plated in confocal imaging dishes at a density of 80,000 cells/well and allowed to adhere for 12 h. The culture medium in each well was then replaced with 1000 μL of fresh medium containing Glu (5 mM), and the cells were incubated at 37 °C in a humidified 5% CO₂ for different times (2, 4, 6, and 8 h). Following incubation, the medium was removed and each well was washed with MEM/F12 medium (1:1, containing 10% FBS and 1% nonessential amino acids). Cells were then treated with 1000 μL of medium containing 10 μM MAP-2F and incubated for another 2 h. After staining, cells were rinsed three times with PBS before imaging.

To assess the specificity of MAP-2F for MAO-A and LOX, SH-SY5Y cells were seeded in confocal imaging dishes at a density of 40,000 cells/well and allowed to attach for 12 h. Cells were then pretreated with selective inhibitors in 500 μL of culture medium (β-aminopropionitrile (BAPN, 200 μM) and clorgyline (CL, 20 μM)) for 2 h. Following incubation, the medium was removed, and each well was washed with DMEM containing 10% FBS. Then, cells were incubated with 500 μL of medium containing 10 μM MAP-2F for 2 h under standard conditions. After staining, cells were rinsed thoroughly with PBS before imaging.

Screening of AD-Associated Natural Products by High-Content Analysis

SH-SY5Y cells were inoculated in 12-well plates at a density of 80,000/mL and allowed to adhere for 12 h. After incubation, cells were treated with 20 different AD-associated natural products: (1) coconut oil: CoOil; (2) ginkgo biloba leaf extract: Egb; (3) ursolic acid: UA; (4) stilbene glycoside; (5) caffeic acid; (6) resveratrol; (7) astragaloside IV; (8) trans-cinnamaldehyde; (9) icariin: ICA; (10) rhamnoside; (11) ginsenoside Rg1; (12) cornelian cherry; (13) teat polyphenols; (14) crocetin; (15) curcumin; (16) artemisinin; (17) kynurenine glycosides; (18) forsythiazide B; (19) tanshinone IIA; (20) tocopherol. All of the selected natural products were specifically chosen based on their well-documented roles in Alzheimer’s disease pathology, including Aβ interactions (e.g., resveratrol for Aβ clearance, curcumin for Aβ aggregation inhibition); neuroprotective effects (e.g., tanshinone IIA for anti-inflammatory properties), etc. For compounds with defined molecular weights, a final concentration of 10 μM was used. For those without a known molecular weight (e.g., crude extracts), the working concentration was set at 10 μg/mL. Following compound treatment, cells were incubated for 12 h at 37 °C with 5% CO₂. The culture medium was then removed, and cells were washed three times with PBS. Then, cells were treated with the Aβ₄₂ oligomer (5 μM) for an additional 12 h. After this incubation, the culture medium was removed, and the cells were treated with fresh medium containing 10 μM MAP-2F and then further incubated for 2 h. Finally, cells were washed three times with PBS to remove residual probe. Cells were then harvested by trypsinization, resuspended in PBS, and transferred to U-bottom 96-well plates for fluorescence quantification. High-content analysis was performed using flow cytometry on a NovoCyte Quanteon flow cytometer (Agilent Technologies), with fluorescence signals collected in the FITC and PerCP channels to assess MAP-2F activation levels.

Results and Discussion

Optical Response and Selectivity of MAP-X Probes toward MAO-A and LOX under Physiological Conditions

The synthetic route for the MAP-X series (MAP-2F, MAP-2Cl, MAP-2Br, MAP-F, MAP-Cl, and MAP-Br) is illustrated in Scheme S1, with full structural characterization provided in Figures S1–S42 (Supporting Information). To evaluate the enzyme-responsiveness of these MAP-X probes, we systematically investigated their optical properties under simulated physiological conditions. When incubated with MAO, only MAP-2F exhibited a significant fluorescence enhancement at 660 nm (Figure a), whereas the other probes (MAP-2Cl, MAP-2Br, MAP-F, MAP-Cl, and MAP-Br) showed minimal response (Figure S43b–f). In contrast, when exposed to LOX, all MAP-X probes demonstrated fluorescence activation at 530 nm, with no signal at 660 nm (Figure S43), suggesting a wavelength-resolved distinction between MAO-A and LOX responses. To further validate the superiority of MAP-X in detecting MAO and LOX, the fluorescence changes of MAP-X were examined when incubated with MAO and LOX at different times in phosphate buffer solution (PBS, pH = 7.4) and borate buffer solution (BBS, pH = 8.5), respectively. As shown in Figure b, an obvious fluorescence enhancement (I/I₀ = 31.34) was detected in the MAP-2F and MAO systems when incubated at 37 °C within 30 min. Under the same conditions, other probes (MAP-2Cl, MAP-2Br, MAP-F, MAP-Cl, and MAP-Br) showed only weak fluorescence changes even after 90 min of incubation, highlighting the unique reactivity of MAP-2F toward MAO. In contrast, incubation with LOX in BBS buffer led to a gradual increase in fluorescence at 530 nm for all probes (Figure c), with comparable response kinetics. This suggests that the LOX-induced fluorescence enhancement may be determined by noncovalent interactions (possibly driven by the affinity of the probes to the proteins) rather than by electronic effects of chemical uncaging. These data establish MAP-2F as a superior fluorogenic probe for MAO detection, while retaining competent responsiveness toward LOX. Notably, the emission maxima of MAP-2F upon activation of MAO and LOX differ by approximately 130 nm, which results in minimal spectral overlap for dual-channel imaging, an essential feature for the simultaneous detection of intracellular and extracellular amine oxidases.

2.

Spectroscopic characterization and selectivity of MAP-2F and MAP-X series toward MAO and LOX. (a) Fluorescence emission spectra of MAP-2F before and after incubation with MAO and LOX, showing distinct emission peaks at 660 and 530 nm, respectively. (b, c) Time-dependent fluorescence responses (0–100 min) of MAP-X toward MAO (b) and LOX (c) under physiological conditions. (d) Concentration-dependent fluorescence responses of MAP-2F toward MAO (0–28.8 μg/mL). (e) Linear calibration curve of MAP-2F fluorescence intensity versus MAO concentration. (f) Selectivity profile of MAP-2F, fluorescence intensity ratios in response to various biological analytes in PBS buffer. (g) Fluorescence intensity of MAP-2F upon exposure to increasing concentrations of LOX (0–100 μg/mL). (h) Linear fluorescence response of MAP-2F as a function of the LOX concentration. (i) Fluorescence intensity ratios of MAP-2F and MAP-2F toward various analytes in BBS buffer (C _MAP‑X = 10 μM, λ _ex = 488 nm, slit = 10/10 nm, 37 °C).

To further assess the quantitative detection capabilities of MAP-2F, we performed concentration-dependent fluorescence assays. Upon exposure to increasing concentrations of MAO (0–30 μg/mL) in PBS, the emission intensity at 660 nm increased proportionally (Figure d), exhibiting an excellent linear relationship in the range of 0–0.03 μg/mL (Figure e). The limit of detection (LOD), calculated using the 3σ/slope method, was determined to be 83.38 ng/mL, suggesting ultrasensitive detection of MAO by MAP-2F in vitro. Similarly, incubation with LOX (0–100 μg/mL) led to concentration-dependent fluorescence enhancement at 530 nm (Figure g), with a strong linear relationship over the entire range (Figure h). The LOD for LOX was estimated to be 0.92 μg/mL. Importantly, no signal at 660 nm was observed in these experiments, which further confirmed the dual-analysis assay’s feasibility.

Next, to assess the selectivity of MAP-2F for MAO and LOX in biologically relevant environments, we performed a fluorescence response analysis in the presence of a range of potential interferents. These included physiologically relevant metal ions, amino acids, reactive oxygen species (ROS), reactive nitrogen species (RNS), and common proteins. For MAO detection, MAP-2F (10 μM) was incubated in PBS buffer with each analyte individually at 37 °C for 1 h. As shown in Figure f, a substantial fluorescence enhancement at 660 nm was observed only in the presence of MAO (10 μg/mL), whereas other analytes did not induce significant signal changes. A slight increase in fluorescence was recorded in the presence of LOX, yet it remained significantly lower than that induced by MAO, highlighting the preferential response of the probe. To assess the specificity for LOX, similar experiments were performed in BBS buffer to monitor the emission at 530 nm. The test results revealed that only LOX triggered a pronounced fluorescence turn-on, while MAO and other interferents produced negligible responses (Figure i). This wavelength-resolved orthogonality strongly supports the dual-analyte discrimination capability of MAP-2F. Taken together, these findings confirm that MAP-2F exhibits high selectivity for MAO and LOX through different fluorescence channels (660 and 530 nm, respectively), with minimal cross-reactivity in complex environments. This dual-responsiveness highlights MAP-2F as a powerful molecular tool for real-time, selective imaging of both intra- and extracellular amine oxidase activity in living systems.

To further evaluate the potential imaging capacities of MAP-2F for MAO and LOX in living cells, we conducted a series of cellular assays. Initially, the cytotoxicity of MAP-2F was assessed in SH-SY5Y and Hep G2 cells using the MTT assay; the results indicated no significant decrease in cell viability upon treatment with MAP-2F, suggesting good biocompatibility for both cell types (Figure S44). To examine the selectivity of MAP-2F for MAO isoforms in living cells, we utilized the different expression profiles of MAO-A and MAO-B in SH-SY5Y and HepG2 cells. , As depicted in Figure S45, negligible fluorescence was observed in Hep G2 cells following MAP-2F treatment, implying minimal interaction with intracellular MAO-B. In contrast, SH-SY5Y cells exhibited strong fluorescence upon exposure to MAP-2F. This fluorescence signal was markedly diminished by preincubation with Clorgyline (CL), a selective MAO-A inhibitor, while treatment with the MAO-B inhibitor Pargyline (PL) had almost no effect. These findings robustly confirmed the selective ability of MAP-2F to recognize and respond to MAO-A in living cells. In addition, as summarized in Table S1, MAP-2F outperforms previously reported MAO-A and LOX probes in several key aspects, including faster response kinetics, higher sensitivity, and exceptional selectivity. Notably, MAP-2F uniquely enables the dual-analytical detection of MAO-A and LOX through distinct fluorescence emission channels. This superior dual-response capability provides a critical foundation for further applications in the synergistic monitoring of the dynamic interplay and potential coexpression patterns of MAO-A and LOX in complex biological contexts.

Elucidation of the Fluorescence Response Mechanism of MAP-2F to MAO and LOX

The distinct response mechanisms of MAP-2F toward MAO and LOX were investigated using ¹⁹F nuclear magnetic resonance (¹⁹F NMR) spectroscopy, which takes advantage of the unique 3,5-difluoro substitution of probe MAP-2F, and ¹⁹F NMR analysis characterized by high sensitivity and minimal biological background. As shown in Figure a, the ¹⁹F NMR signal of MAP-2F exhibited a chemical shift at −127.24 ppm. Upon incubation with MAO, a new signal appeared at −134.28 ppm, which was closely aligned to the chemical shift of the free fluorophore DM-2F (−134.34 ppm), indicating cleavage at the response site and subsequent release of the fluorophore. This cleavage event initiates an intramolecular charge transfer (ICT) process, thereby accounting for the observed fluorescence enhancement. Fluorescence emission measurements further supported these findings; the emission profile of MAP-2F upon reaction with MAO was consistent with that of DM-2F (Figure b), further confirming a fluorogenic reaction driven by enzymatic cleavage and fluorophore release. This mechanism is consistent with previous literature studies on MAO substrates, wherein oxidative deamination of the amine moiety generates an aldehyde intermediate that triggers β-elimination and fluorophore release. − In contrast, the ¹⁹F NMR spectra of MAP-2F treated with LOX displayed a modest downfield shift of MAP-2F at −126.96 ppm without the appearance of new peaks (Figure d), suggesting a noncleavage mechanism. This observation indicates that the interaction between MAP-2F and LOX induces a restriction of intramolecular motion rather than a chemical transformation. To validate this hypothesis, we further examined the fluorescence emission in solvents of varying viscosity. The fluorescence intensity at 530 nm increased gradually with medium viscosity (Figure e), further suggesting that restricted molecular rotation of MAP-2F, potentially due to interaction with LOX, would obstruct the twisted intramolecular charge transfer (TICT) process and enhance fluorescence emission. Moreover, molecular docking analysis further confirmed these mechanistic differences. MAP-2F exhibited significantly stronger binding affinity toward LOX compared to MAO-A (Figures c, f, S46; Table S2), supporting the model wherein the LOX interaction stabilizes the molecular conformation of MAP-2F, restricts torsional freedom, and suppresses the TICT, thereby enhancing the fluorescence emission at 530 nm. In contrast, the MAO-A interaction facilitates the chemical transformation and release of DM-2F, producing a red-shifted emission centered at 660 nm. Taken all together, these results highlight that MAP-2F can distinguish between MAO-A and LOX, through a distinct response mechanism, a cleavage-induced ICT activation for MAO-A, and a conformationally restricted TICT suppression for LOX (Figure g). These mechanistic differences result in spectrally resolved fluorescence outputs, thereby minimizing spectral overlap for dual-channel imaging. This dual-response capability not only improves detection accuracy but also provides a rational reference for designing multianalyte fluorescent probes targeting structurally or functionally similar biomolecules in complex biological systems.

3.

Mechanistic studies of the MAP-2F interactions with MAO and LOX. (a, d) The ¹⁹F NMR spectra of DM-2F, MAP-2F, and MAP-2F after response to MAO (a) and LOX (d); (b) fluorescence emission spectra of DM-2F, MAP-2F, and MAP-2F after response to MAO. (c, f) Molecular docking simulations showed two-dimensional interaction maps between MAP-2F and MAO (c) or LOX (f), indicating distinct binding orientations and key amino acid interactions. (e) Fluorescence spectra of MAP-2F in various viscosity system (water/glycerol, v/v, f _glycerol from 0 to 90%, λ_ex = 488 nm, slit = 10/10 nm). (g) Proposed enzymatic activation mechanisms of MAP-2F with MAO-A (left) and LOX (left), respectively.

Dual-Channel Cellular Imaging of MAO-A and LOX under Aβ₄₂ Exposure Stresses

Based on the extracellular fluorescence results indicating the dual-channel response of MAP-2F to MAO-A (660 nm) and LOX (530 nm), we further assessed the feasibility of using MAP-2F for simultaneous imaging of both enzymes in live cells. Confocal fluorescence imaging in SH-SY5Y cells, using excitation at 488 nm, revealed two distinct emission signals corresponding to green and red channels (Figure ), which were consistent with the LOX- and MAO-A-mediated responses, respectively. Notably, pretreatment with clorgyline, a MAO-A inhibitor, significantly suppressed the red fluorescence signal in the red channel, confirming the specificity of MAP-2F for MAO-A in this spectral window. In contrast, β-aminopropionitrile (BAPN), a well-established LOX inhibitor, exhibited only a minor inhibitory effect on green channel fluorescence. This result aligns with our proposed mechanism that MAP-2F interacts with LOX through noncatalytic nucleophilic binding rather than enzymatic oxidation and thus may not be completely inhibited by BAPN. Intriguingly, dual-channel imaging also revealed distinct subcellular localization patterns of the red and green signals, suggesting spatial segregation of MAO-A and LOX activity in the cellular environment. These findings collectively highlight that MAP-2F serves as an effective probe for dual-channel imaging of MAO-A and LOX activity dynamics in living neuronal cells, offering a valuable tool for elucidating the interactions between these two oxidative enzymes in complex biological systems.

4.

Fluorescence imaging of MAO-A and LOX activity in SH-SY5Y cells using MAP-2F. (a) Confocal fluorescence images of SH-SY5Y cells incubated with MAP-2F (10 μM), clorgyline (CL, 20 μM; MAO-A inhibitor), and β-aminopropionitrile (BAPN, 200 μM; LOX inhibitor). Dual-channel imaging reveals enzyme-specific signals: green channel (λ_em = 525 nm) for LOX and red channel (λ_em = 617 nm) for MAO-A. λ_ex = 488 nm, scale bars = 10 μm. (b, c) Quantification of mean fluorescence intensities from panel (a) in both emission channels. Data are presented as the mean ± standard deviation of three replicates. Multiple comparisons were performed by one-way ANOVA: *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Given the link between abnormal metabolism of MAO-A and LOX in neurodegeneration diseases, particularly in AD, and the central role of amyloid-β (Aβ₄₂) in the pathogenesis of AD, ,− ,− ,− , we further explored whether MAP-2F could be used to monitor the impact of varying degrees of Aβ₄₂ peptide on the activity dynamics of MAO-A and LOX in live neuronal cells. SH-SY5Y cells, previously validated as a suitable cell model for dual imaging with MAP-2F, were exposed to different concentrations of Aβ₄₂ monomers, oligomers, and aggregates for 12 h, followed by incubation with MAP-2F for 2 h. As shown in Figure , a dose-dependent increase in fluorescence was observed in SH-SY5Y cells treated with Aβ₄₂ oligomers and Aβ₄₂ aggregates, whereas minimal fluorescence changes were observed in Aβ₄₂ monomer-treated cells. These observations revealed that Aβ₄₂ oligomers and aggregates significantly upregulate the intracellular levels and activities of MAO-A and LOX in live neuronal cells. To further investigate whether these effects on enzyme activity of MAO-A and LOX were time-dependent, SH-SY5Y cells were treated with Aβ₄₂ monomers, oligomers, or Aβ₄₂ aggregates for different times and then subjected to MAP-2F imaging (Figure S47). We found that the fluorescence signals of SH-SY5Y cells in the red and green channels increased significantly over time in the presence of Aβ₄₂ oligomers and aggregates, further confirming the time- and dose-dependent upregulation of MAO-A and LOX under Aβ₄₂ exposure stresses. Collectively, these findings reveal a clear correlation between Aβ₄₂ oligomer-aggregate exposure stresses and the aberrant upregulation of MAO-A and LOX in live neuronal cells. The ability of MAP-2F to simultaneously visualize these enzymes in real-time and at distinct wavelengths makes it a powerful tool for dissecting the molecular mechanisms underlying AD pathology and potentially for evaluating therapeutic strategies targeting oxidative stress and enzyme dysregulation in neurodegenerative disorders.

5.

Fluorescence imaging of MAO-A and LOX activity in SH-SY5Y cells exposed to different Aβ₄₂ species. (a–c) Confocal fluorescence images of SH-SY5Y cells treated with increasing concentrations of (a) Aβ₄₂ monomers, (b) Aβ₄₂ oligomers, and (c) Aβ₄₂ aggregate for 12 h, followed by incubation with MAP-2F (10 μM) for 2 h (λ_ex = 488 nm; Green channel corresponds to LOX activity: 525 nm; red channel corresponds to MAO-A activity: 617 nm). (d–f) Quantification of mean fluorescence intensities from panels (a–c), respectively, revealing dose-dependent modulation of enzyme activity by different Aβ₄₂ species. Data are shown as the mean ± standard deviation of three replicates. Statistical significance was assessed using one-way ANOVA with multiple comparisons: ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Glutamate (Glu), an essential excitatory neurotransmitter in the brain, is known to exert neurotoxic effects and has been widely used in cellular models of AD in vitro. Based on studies that have shown an increase in LOX levels in neuronal cells after treatment with Glu, a subsequent evaluation of the changes in MAO-A and LOX levels in SH-SY5Y neuronal cells post-Glu treatment was undertaken, as shown in Figure S48. The imaging findings indicated that the fluorescence intensity of the green channel, representing the LOX activity, increased in a time-dependent manner. In contrast, the fluorescence intensity of the red channel, representing MAO-A, increased relatively only slightly. These results demonstrate that Glu primarily upregulates LOX rather than MAO-A, thus emphasizing the critical role of LOX in Glu-induced neurotoxicity and its potential contribution to AD pathophysiology. To validate these observations, we further performed a series of complementary experiments, including ELISA, Western blotting, and immunofluorescence assays (Figure S49). Consistently, the outcomes of these experiments all confirmed that the expression levels of both MAO-A and LOX were elevated in SH-SY5Y cells following treatment with AD-related factors such as Aβ₄₂ oligomers, Aβ₄₂ aggregates, and Glu. These results collectively support the notion that AD-related stimulus stresses promote differential yet simultaneous up-regulation of MAO-A and LOX in neuronal cells and further demonstrate the utility of MAP-2F in monitoring these concurrent changes in live AD-model cells. Furthermore, it has been established that overactivation of MAO-A contributes to the generation of Aβ monomers by facilitating β-and γ-secretase-mediated cleavage of amyloid precursor protein (APP), which ultimately leads to the formation of Aβ plaques. , Additionally, LOX has also been implicated in the cross-linking of extracellular matrix metabolism and aggregation of Aβ₄₂ protein, thereby contributing to the pathology of AD. These established roles prompted us to investigate a potential feedback mechanism wherein LOX facilitates the aggregation of Aβ₄₂ monomers, potentially triggering further upregulation of MAO-A and LOX. To test this hypothesis, SH-SY5Y cells were exposed to Aβ₄₂ oligomers or Glu for 4 h, followed by treatment with Aβ₄₂ monomers for an additional 12 h. Subsequently, cells were treated with MAP-2F and subjected to fluorescence imaging (Figure S50a–f). The results demonstrated that the cotreatment resulted in a significant increase in fluorescence intensity in the green and red channels compared to the control groups with Glu or Aβ₄₂ oligomers alone. These observations suggest that LOX, upregulated by Aβ₄₂ oligomers or Glu, may facilitate the aggregation of intracellular Aβ₄₂ monomers, which in turn stimulate MAO-A and LOX expression. Moreover, Western blot analysis confirmed this finding, showing elevated levels of MAO-A and LOX proteins in the cotreated cells compared to controls (Figure S50g–i). This observation further confirmed that the upregulation of MAO-A and LOX may be associated with the aggregation of Aβ₄₂ monomers, thus supporting a self-reinforcing cyclical relationship between Aβ₄₂ aggregation and the overexpression of MAO-A and LOX in neuronal cells.

High-Throughput Screening of MAO-A/LOX Regulators in the AD Cellular Model with MAP-2F

Finally, to further evaluate the efficacy of MAP-2F as a screening tool for potential MAO-A and LOX modulators in AD, we sought to combine MAP-2F staining with flow cytometry to develop a fluorescence-based high-throughput screening platform (Figure a). Living SH-SY5Y cells were first pretreated with various natural products possessing potential anti-AD properties for 12 h, followed by treatment with Aβ₄₂ oligomers for 24 h, and subsequent MAP-2F staining. Flow cytometric analysis of the FITC (LOX) and PerCP (MAO-A) channels revealed distinct modulation patterns. Specifically, compounds such as ursolic acid, resveratrol, triptolide, corni fructus, forsythoside B, and stigmasterol significantly reduced LOX-associated fluorescence signal in the FITC channel (Figure b,c), suggesting effective suppression of LOX upregulation. Meanwhile, Icariin (ICA), curcumin, geniposidic acid, forsythoside B, tanshinone IIA, and stigmasterol markedly reduced the fluorescence of PerCP, suggesting that they inhibited the up-regulation of MAO-A (Figure d, e). Notably, forsythoside B and stigmasterol were the only compounds that effectively downregulated both MAO-A and LOX. Conversely, ursolic acid, Resveratrol, and Triptolide inhibited LOX activity while enhancing MAO-A fluorescence, a phenomenon previously observed following BAPN treatment, possibly indicating compensatory crosstalk between these two enzymes. Further literature review revealed that the mechanisms of action of these compounds are closely linked to Aβ pathology. For example, ursolic acid, curcumin, stigmasterol, forsythoside B, and cornuside attenuated Aβ-induced cytotoxicity; resveratrol enhanced Aβ clearance; and ICA, tanshinone IIA, geniposidic acid, and triptolide inhibited Aβ production. − These findings further confirmed the correlation between intracellular Aβ load and abnormal MAO-A/LOX expression, reinforcing their role as dual biomarkers and therapeutic targets for AD. Collectively, the integration of MAP-2F with flow cytometry offers a robust, sensitive platform for high-throughput screening of modulators targeting MAO-A and LOX. The unique spectral response of MAP-2F allows for simultaneous, interference-free detection of both enzymes, enabling rapid evaluation of potential modulators in complex cellular models of AD.

6.

Evaluation of natural products on intracellular MAO-A and LOX activity in Aβ₄₂ oligomer-induced SH-SY5Y cells using MAP-2F and flow cytometry. (a) Flow cytometric analysis of SH-SY5Y cells pretreated with Aβ₄₂ oligomers followed by exposure to various natural compounds, with MAP-2F used to simultaneously detect changes in LOX and MAO-A activity. (b, c) FITC channel: used to analyze the change in LOX levels. (d, e) Per CP channel: used to analyze the changes in MAO-A levels. Tested natural products are as follows: (1) coconut oil: CoOil; (2) ginkgo biloba: Egb; (3) ursolic acid; 4. 2,3,5,4′-tetrahydroxy stilbene-2-Ο-β-d-glucoside; (5) caffeic acid; (6) resveratrol; (7) astragaloside IV; (8) trans-cinnamaldehyde; (9) icariin: ICA; (10) triptolide; (11) ginsenoside Rg1; (12) corni fructus; (13) tea polyphenols; (14) crocin; (15) curcumin; (16) artemisinin; (17) geniposidic acid; (18) forsythoside B; (19) tanshinone IIA; (20) stigmasterol.

Conclusions

In summary, we have rationally designed and synthesized a series of dicyanoisophorone-based fluorescent probes, MAP-X (X = 2F, 2Cl, 2Br, F, Cl, Br), among which MAP-2F, with a 3,5-difluoro substitution, exhibited excellent performance in the selective detection of MAO-A and LOX through different fluorescence emission channels and independent recognition mechanisms. Notably, MAP-2F enabled the simultaneous, spatially resolved imaging of endogenous MAO-A and LOX within living neuronal cells, enabling dual-target visualization without overlapping fluorescent signals for the first time. Utilizing this capability, we applied MAP-2F to dynamically monitor the enzyme activity status under AD-related pathological stimuli, including Aβ₄₂ oligomer, Aβ₄₂ aggregate, and Glu treatment, revealing a potential feed-forward mechanism whereby upregulation of LOX promotes the polymerization of Aβ₄₂ monomers, which in turn exacerbates the expression of MAO-A and LOX. These findings suggest that a pathologic amplification cycle contributes to the development of AD. To further explore the potential for its application, we integrated MAP-2F into a high-throughput screening platform for evaluating the MAO-A and LOX regulatory activities across a panel of 20 natural products with reported anti-AD properties. Several Aβ-interacting natural products (ICA, curcumin, geniposidic acid, forsythoside B, tanshinone IIA, stigmasterol, ursolic acid, resveratrol, triptolide, and corni fructus) were identified as effective MAO-A/LOX inhibitors, which is consistent with their known interactions with Aβ pathology. Interestingly, we found dual-inhibitory activities of forsythoside B and stigmasterol, further confirming the dual readout capacity of MAP-2F. Overall, this work established MAP-2F as a powerful molecular tool for real-time, multiplexed imaging of MAO-A and LOX activity in neurodegenerative disease models, enabling simultaneous screening of pharmacologically active modulators. While the probe demonstrates exceptional potential for cellular imagingparticularly through its NIR emission (660 nm) and low cytotoxicityits translation to in vivo applications requires further optimization to address challenges in BBB penetration and metabolic stability. Future studies will (i) engineer derivatives with enhanced brain delivery (e.g., lipid-soluble modifications or BBB-targeting moieties) and (ii) validate their performance in AD animal models. Beyond direct biological applications, this study provides a conceptual and methodological framework for designing next-generation fluorescent probes capable of concurrent monitoring of disease-associated enzyme networks. Critically, it offers mechanistic insights into the feed-forward relationship between MAO-A/LOX dysregulation and Aβ-mediated neurotoxicity, opening new avenues for therapeutic target discovery and intervention strategies in Alzheimer’s disease.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00102.

• Additional experimental details, materials, methods, and additional figures (PDF)

Y.Q., J.Z., and Y.C. conceived the project; Y.C. performed the measurements; Y.C. conducted the experiments; Y.Q., J.Z., and Y.C. wrote the paper; J.Z., L.Z., W.W., and X.W. supervised the project. All authors discussed the results and commented on the manuscript.

The authors declare no competing financial interest.