Differentiating Aβ40 and Aβ42 in amyloid plaques with a small molecule fluorescence probe†

Differentiating amyloid beta (Aβ) subspecies Aβ40 and Aβ42 has long been considered an impossible mission with small-molecule probes. In this report, based on recently published structures of Aβ fibrils, we designed iminocoumarin–thiazole (ICT) fluorescence probes to differentiate Aβ40 and Aβ42, among which Aβ42 has much higher neurotoxicity. We demonstrated that ICTAD-1 robustly responds to Aβ fibrils, evidenced by turn-on fluorescence intensity and red-shifting of emission peaks. Remarkably, ICTAD-1 showed different spectra towards Aβ40 and Aβ42 fibrils. In vitro results demonstrated that ICTAD-1 could be used to differentiate Aβ40/42 in solutions. Moreover, our data revealed that ICTAD-1 could be used to separate Aβ40/42 components in plaques of AD mouse brain slides. In addition, two-photon imaging suggested that ICTAD-1 was able to cross the BBB and label plaques in vivo. Interestingly, we observed that ICTAD-1 was specific toward plaques, but not cerebral amyloid angiopathy (CAA) on brain blood vessels. Given Aβ40 and Aβ42 species have significant differences of neurotoxicity, we believe that ICTAD-1 can be used as an important tool for basic studies and has the potential to provide a better diagnosis in the future.


Materials and instruments
All chemicals were purchased through commercial vendors and used without further purification. Absorption spectra were recorded on SpectraMax (Molecular Device, San Jose, CA). A fluorescence spectrophotometer F-7100 (Hitachi High-Tech Corporation, Japan) was employed fluorescent spectra recording. 13 C NMR and 1 H NMR spectra were collected on a JOEL 500-MHz Spectrometer (JEOL USA, Inc.), and the values of δ are in ppm respect to TMS. LC-MS was carried out on an Agilent 1200 Series apparatus with an LC/MSD trap (Agilent Technologies) and Daly conversion dynode detector with UV detection at 254 nm. Tissue imaging was performed on Nikon Eclipse 50i (Nikon Corporation). Two-photon imaging in vivo was conducted on an Olympus BX-51 microscope (Olympus Life Science). Transgenic female 5xFAD mice and age-matched wildtype female mice were purchased from Jackson Laboratory. All animal experiments were performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. An IVIS Spectrum animal imaging system (Perkin Elmer) was used for in vitro plate imaging. Selectivity of ICTAD-1 towards Aβ fibrils over metal ions, BSA and HSA. Fluorescent spectra of ICTAD-1 (10 μM) with different analytes in PBS (10 mM, pH 7.4, 1% DMF) were recorded, and concentrations of metal ions, amino acids, and thiols were 1 mM. PBS solutions of BSA and HSA were also tested with ICTAD-1 (10 μM) (Ex = 430 nm, slit 5/5, 400 V for PMT).

Experimental Procedures
Plate imaging of ICTAD-1. ICTAD-1 (250 nM) was incubated with different concentrations of Aβ40 or Aβ42 (2.5 μM, 10 eq) in PBS (10 mM, pH 7.4, 1% DMF). The resulting solution was added to wells on a 96-well plate, and the final volume of each well was 200 μL. Triplicate samples were used for each condition. Plate images were recorded on the IVIS System. Moreover, the plate imaging of ICTAD-1 with Aβ40 fibrils and Aβ42 fibrils (2.5 μM, 10 eq) in the presence of BSA (2.5 μM, 10 eq) or mouse brain homogenate (BH, 0.1 mg/mL) was also performed similarly to the above procedure.
In vitro histological staining. A fresh brain tissue from a 24-month old APP/PS1 mouse was fixed in 4% formaldehyde for 24 hours and transferred into 30% sucrose at 4 °C until the tissue sunk. Then the tissue was embedded in OCT with gradual cooling over dry ice. The OCT embedded tissue block was sectioned into 25-μm slice with a cryostat. The tissue was washed in diluting buffer (0.4% Triton X-100, 1% goat serum, S5 2% BSA in TBS) for 3x10min, then blocked in 20% goat serum for 30min at room temperature. Then the slice was incubated with primary antibody 6E10 overnight at 4°C. After washed with diluting buffer for 3x10min, the slice was incubated with secondary antibody for 2 hours at room temperature. Then the slice was washed with TBS for 3x10min. 25 uM of ICTAD-1 in 20% ethanol/PBS was prepared as the staining solution. The brain slice was incubated with freshly prepared staining solution for 15 min at room temperature and then washed with 50% ethanol for 2x1min, followed by washing with double distilled water twice. Then the slice was covered with FluoroShield mounting medium (Abcam) and sealed with nail polish. Florescence images were obtained using the Nikon Eclipse 50i microscope with blue and red-light excitation channel.
Confocal spectral unmixing imaging. The above brain slice was placed under a Nikon confocal microscope (A1R HD25). Excitation laser of 468 nm was used, and emission was collected from 505 nm to 690 nm with 2.5 nm steps. A region of interest (ROI) was selected from the core and peripheral area of a plaque respectively, and a spectrum from each ROI was extracted and stored into the system as the spectral library.
Spectral unmixing was performed with the spectra stored in the library. The unmixing spectra was achieved by averaging the data collected from three individual plaques (core and periphery respectively).
In vivo two-photon imaging. These animal experiments were performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. A 15month-old 5xFAD female mouse was anesthetized with 2% isoflurane, and a cranial imaging window was surgically prepared as described [2] . Before ICTAD-1 injection, two-photon images of capillary were acquired using the 800-nm laser (Prairie Ultima) with 500 to 550 nm emission by injection of FITC-Dextran. A bolus i.v.