A novel two-photon ratiometric fluorescent probe for imaging and sensing of BACE1 in different regions of AD mouse brain

Abstract

β-Secretase (BACE1) is the vital enzyme in the pathogenic processes of Alzheimer's disease (AD). However, the development of a powerful tool with high selectivity and sensitivity for BACE1 determination in vivo is a challenge in understanding the pathogenesis of AD. In this work, a novel two-photon ratiometric fluorescent probe (AF633mCyd) was first developed for imaging and sensing of BACE1 in live cells and deep tissues, in which the fluorescence resonance energy transfer (FRET) system was designed and synthesized by a novel two-photon donor, merocyanine derivative (mCyd), connected with an acceptor, Alexa Fluor 633 (AF633), through a peptide substrate (EVNL-DAEFRHDSGYK) with a length of less than 10 nm. The emission spectrum of mCyd possessed sufficient overlap with the absorption spectrum of AF633, resulting in the high sensitivity of the developed AF633mCyd probe. The peptide substrate which can be specifically cleaved by BACE1 was inserted between the donor and acceptor, leading to the high selectivity of the present fluorescent probe. The fluorescence emission peaks of the AF633mCyd probe were observed at 578 nm and 651 nm and the emission ratio demonstrated good linearity with the concentration of BACE1 varying from 0.1 to 40.0 nM with a detection limit down to 65.3 ± 0.1 pM. Considering the advantages of high selectivity and sensitivity, as well as long-term stability and good biocompatibility, the developed probe was successfully applied in imaging and sensing of BACE1 in different regions of AD mouse brain tissue with a depth greater than 300 μm. Using this powerful tool, it was clear that the level of BACE1 was different in various brain regions of AD mouse such as S1BF, CPu, LD, and CA1. The up-regulation of BACE1 was observed especially in the regions S1BF and CA1 in AD mouse brain. Moreover, BACE1 was also found to be closely related to AD pathogenesis caused by oxidative stress.

Scheme 1. (A) Illustration for the working principle of the designed two-photon ratiometric fluorescent probe AF633mCyd for the determination of BACE1 in neurons and mouse brain tissue slice. (B) Synthesis steps for mCyd. Reaction conditions: (a) MeCN, 78 °C, 48 h, 82%; (b) CH₃COONa, EtOH, 75 °C, 6 h, 79%.

Fig. 1. (A) UV-vis absorption spectra and one-photon fluorescence spectra of (a and b) mCyd and (c and d) AF633. (B) Photostability test of the AF633mCyd probe before (a) and after (b) addition of BACE1. (C) Two-photon fluorescence spectra of (a) mCyd, (b) AF633, (c) AF633mCyd probe, and (d) AF633mCyd probe with the addition of BACE1, excited at 820 nm. (D) Fluorescence lifetime decay curves of (a) the donor mCyd, the donor mCyd part in this probe (b) without and (c) with the addition of BACE1. Spectra and lifetime were measured in 10 mM PBS containing 0.05% DMSO, pH = 4.5.

Fig. 2. (A) Two-photon fluorescence spectra of 5.0 μM AF633mCyd probe with the addition of BACE1 at different concentrations ((a) 0.0 nM, (b) 0.1 nM, (c) 0.5 nM, (d) 2.0 nM, (e) 5.0 nM, (f) 10.0 nM, (g) 17.0 nM, (h) 25.0 nM, (i) 34.0 nM, (j) 40.0 nM, (k) 45.0 nM, and (l) 50.0 nM), excited at 820 nm. (B) The plot between F_green/F_red (F_green: 560–620 nm, F_red: 640–700 nm) of the AF633mCyd probe and BACE1 concentrations (error bars, n = 6, SD). (C) Selectivity test of 5.0 μM AF633mCyd toward proteins such as trypsin, CD, α/γ-secretase, bromelain, thrombin, IgG and pepsin (500.0 nM each). (D) Selectivity test of 5.0 μM AF633mCyd toward ROS and other anions (1.0 mM each). The titration curve and selectivity were obtained in fresh cell lysates containing 0.05% DMSO, pH = 4.5.

Fig. 3. (A) Two-photon microscope imaging of BACE1 in neurons. The green and red channels: fluorescence emission collected from 560–620 nm (F_green) and 640–700 nm (F_red), respectively. Overlay images were obtained from the F_green/F_red channel and bright image. (B) Two-photon microscope imaging of BACE1 in neurons treated with 0.0–100.0 nM Axon 1125 for 3 h. (C) The changes of F_green/F_red in neurons with increasing concentrations of Axon 1125. More than 50 neurons were evaluated for the statistical analyses (error bars, n = 50, SD). Scale bar = 25 μm.

Fig. 4. (A) Two-photon microscope imaging of BACE1 in neurons treated with 0.0–20.0 μM O₂˙^– for 12 h. (B) Western blot images of proteins (BACE1, CTFβ, and β-actin) obtained from neurons which were treated as (A). (C) F_green/F_red changes in neurons from (A) (left Y-axis); ratios of band density changes in (B) (right Y-axis). (D) Two-photon microscope imaging of BACE1 in neurons treated with 20.0 μM O₂˙^– for 0–12 h. (E) Western blot images of proteins obtained from neurons which were treated as (D). (F) F_green/F_red changes in neurons from (D) (left Y-axis); ratios of band density changes in (E) (right Y-axis). The band densities were calculated using NIH ImageJ software. More than 50 neurons were evaluated for the statistical analyses (error bars, n = 50, SD). More than 20 parallel tests were carried out in (B) and (E). Proteins were visualized with BACE1, CTFβ, and β-actin specific antibodies, respectively. Scale bar = 25 μm.

Fig. 5. Three dimensional (A) one-photon or (B) two-photon microscope images of the hippocampus region in AD mouse brain labelled with the AF633mCyd probe, excited at 552 nm and 820 nm, respectively. (C) Illustration for multiple regions of mouse brain slices (dark blue: cortex; red: striatum; green: thalamus; yellow: hippocampus). (D) Two-photon microscope images (overlay, F_green/F_red) of BACE1 using the AF633mCyd probe in S1BF, CPu, LD, and CA1 regions of AD and normal mouse brain slices. (E) F_green/F_red changes for BACE1 in S1BF, CPu, LD and CA1 regions in AD mouse brain slices compared with those in normal mouse brain. (F) Confocal microscope images of ROS using ROS commercial probe (DCFH-DA, ex/em = 480/525 nm, purchased from Yeasen Biotech Co. Ltd., China) in S1BF, CPu, LD, and CA1 regions of AD and normal mouse brain slices. Fluorescence emission collected from 500–560 nm, excited at 488 nm. (G) Fluorescence intensity changes for ROS in S1BF, CPu, LD, and CA1 regions of AD mouse brain slices compared with those of normal mouse brain. More than 20 mice were measured for the statistical analyses (error bars, n = 20, SD). Scale bar = 50 μm.