A Novel Near-Infrared Fluorescence Probe THK-565 Enables In Vivo Detection of Amyloid Deposits in Alzheimer's Disease Mouse Model

Abstract

Purpose: Noninvasive imaging of protein aggregates in the brain is critical for the early diagnosis, disease monitoring, and evaluation of the effectiveness of novel therapies for Alzheimer's disease (AD). Near-infrared fluorescence (NIRF) imaging with specific probes is a promising technique for the in vivo detection of protein deposits without radiation exposure. Comprehensive screening of fluorescent compounds identified a novel compound, THK-565, for the in vivo imaging of amyloid-β (Aβ) deposits in the mouse brain. This study assessed whether THK-565 could detect amyloid-β deposits in vivo in the AD mouse model.

Procedures: The fluorescent properties of THK-565 were evaluated in the presence and absence of Aβ fibrils. APP knock-in (APP-KI) mice were used as an animal model of AD. In vivo NIRF images were acquired after the intravenous administration of THK-565 and THK-265 in mice. The binding selectivity of THK-565 to Aβ was evaluated using brain slices obtained from these mouse models.

Results: The fluorescence intensity of the THK-565 solution substantially increased by mixing with Aβ fibrils. The maximum emission wavelength of the complex of THK-565 and Aβ fibrils was 704 nm, which was within the optical window range. THK-565 selectively bound to amyloid deposits in brain sections of APP-KI mice After the intravenous administration of THK-565, the fluorescence signal in the head of APP-KI mice was significantly higher than that of wild-type mice and higher than that after administration of THK-265. Ex vivo analysis confirmed that the THK-565 signal corresponded to Aβ immunostaining in the brain sections of these mice.

Conclusions: A novel NIRF probe, THK-565, enabled the in vivo detection of Aβ deposits in the brains of the AD mouse model, suggesting that NIRF imaging with THK-565 could non-invasively assess disease-specific pathology in AD.

Keywords: Alzheimer’s disease; Amyloid; Fluorescence; Imaging.

Fig. 1

A Chemical structure of THK-565. B Fluorescence spectra of THK-565 under different pH conditions. C, D Excitation C and emission D spectra of THK-565 in PBS with and without Aβ fibril

Fig. 2

Fluorescence contour map of THK-565 solution and the THK-565/Aβ fibril complex

Fig. 3

In vitro binding of THK-565 to Aβ fibrils. A Comparison of THK-565 fluorescence intensity and that of the THK-565/Aβ fibril complex at different concentrations. *p < 0.05, compared with the fluorescence intensity of THK-565 alone. B Comparison of fluorescence intensity of THK565 alone, THK-565 with soluble Aβ, and THK-565 with Aβ fibrils. C Saturation curve of THK-565 binding to Aβ fibrils. ***p < 0.05, compared with fluorescence intensity of THK-565 with Aβ fibrils

Fig. 4

In vitro THK-565 binding to Aβ and tau deposits in the mouse models of AD. THK-565 staining of brains sections from wild-type (Wt) A, B and amyloid-β precursor protein knock-in (APP-KI) C, D

Fig. 5

Ex vivo measurement of fluorescence intensity of brain tissue after 30 min and 60 min intravenous administration of THK-565 and ICG in mice *p < 0.05, unpaired t-test (30 min p = 0.017, 60 min p = 0.025)

Fig. 6

In vivo imaging of amyloid deposits using THK-565. A Images of the brains of 11–12 month-old amyloid-β precursor protein knock-in (APP-KI) and wild-type (Wt) mice acquired after intravenous administration of THK-565 and the placement of region of interest (ROI). B Fluorescence signal intensities of the heads of 11–12 month-old APP-KI and Wt mice as a function of time after the intravenous administration of THK-565. C Average fluorescence intensity of THK-565 between 0 and 60 min after injection in APP-KI and Wt mice

Fig. 7

Comparison of in vivo fluorescence signals between compounds. A Images of the brains of 11–12 month-old amyloid-β precursor protein knock-in (APP-KI) mice acquired after intravenous administration of THK-565, THK-265, and vehicle. B Fluorescence signal intensities of the heads of 11–12 month-old APP-KI mice as a function of time after the intravenous administration of THK-565, THK-265, or vehicle. C Average fluorescence intensity of THK-565, THK-265, and vehicle between 0 and 60 min after injection in APP-KI mice.

Fig. 8

Ex vivo microscopic images. Images of brain sections from a 11–12 month-old amyloid-β precursor protein knock-in (APP-KI) mice A after intravenous administration of 0.3 mg/kg THK-565. B Aβimmunostaining in the same sections as (A). C Merged images of (A) and (B). Scale bar = 100 μm