Dual-ligand fluorescence microscopy enables chronological and spatial histological assignment of distinct amyloid-β deposits

Abstract

Different types of deposits comprised of amyloid-β (Aβ) peptides are one of the pathological hallmarks of Alzheimer's disease (AD) and novel methods that enable identification of a diversity of Aβ deposits during the AD continuum are essential for understanding the role of these aggregates during the pathogenesis. Herein, different combinations of five fluorescent thiophene-based ligands were used for detection of Aβ deposits in brain tissue sections from transgenic mouse models with aggregated Aβ pathology, as well as brain tissue sections from patients affected by sporadic or dominantly inherited AD. When analyzing the sections with fluorescence microscopy, distinct ligand staining patterns related to the transgenic mouse model or to the age of the mice were observed. Likewise, specific staining patterns of different Aβ deposits were revealed for sporadic versus dominantly inherited AD, as well as for distinct brain regions in sporadic AD. Thus, by using dual-staining protocols with multiple combinations of fluorescent ligands, a chronological and spatial histological designation of different Aβ deposits could be achieved. This study demonstrates the potential of our approach for resolving the role and presence of distinct Aβ aggregates during the AD continuum and pinpoints the necessity of using multiple ligands to obtain an accurate assignment of different Aβ deposits in the neuropathological evaluation of AD, as well as when evaluating therapeutic strategies targeting Aβ aggregates.

Keywords: Alzheimer’s disease; amyloid-β; fluorescence; microscopy; protein aggregation.

Figure 1

Schematic overview of the study.A, chemical structure of the five different thiophene-based ligands included in the study, as well as their suggestive binding mode to type I sporadic Alzheimer’s disease filaments (PDB:7Q4B) (19) based on previous experimental and computational studies (44, 45, 46). B, the brain tissue sections with Aβ pathology included in the study. C, graphic presentation of the dual-staining protocols, as well as the analysis of the stained tissue sections with fluorescence microscopy. The figure was created with BioRender.com. Aβ, amyloid-β; PDB, protein data bank.

Figure 2

Dual-staining of brain tissue sections from APP mouse models with ligands HS-84 and HS-259.A, chemical structures and emission spectra of HS-84 (magenta) and HS-259 (green) bound to Aβ deposits. B–G, fluorescence images of brain tissue sections from 7 months (B) or 22 months (C) old APP_ArcSwe mice, 8 months (D) or 22 months (E) APP23 mice, and 4 months (F) or 18 months old (G) APP^{NL-G-F/NL-G-F} mice. Aβ aggregates stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-84 or HS-259 are highlighted with magenta arrows (HS-84) or green arrows (HS-259), respectively. Images showing vascular Aβ deposits (VD) are indicated with VD. All other images show parenchymal Aβ deposits. Autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 20 μm. Aβ, amyloid-β; APP, amyloid-beta precursor protein.

Figure 3

Dual-staining of brain tissue sections from APP mouse models with ligands HS-335 and HS-169.A, chemical structures and emission spectra of HS-335 (magenta) and HS-169 (green) bound to Aβ deposits. B–G, fluorescence images of brain tissue sections from 7 months (B) or 22 months (C) old APP_ArcSwe mice, 8 months (D) or 22 months (E) APP23 mice, and 4 months (F) or 18 months old (G) APP^{NL-G-F/NL-G-F} mice. Aβ aggregates stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-169 are highlighted with green arrows. Images showing vascular Aβ deposits (VD) are indicated with VD. All other images show parenchymal Aβ deposits. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 20 μm. Aβ, amyloid-β; APP, amyloid-beta precursor protein.

Figure 4

Schematic presentation of different parenchymal and vascular Aβ deposits that were identified with the dual-ligand staining protocols in mouse brain tissue sections. Aβ deposits identified by all five ligands are shown in white, whereas aggregated Aβ species solely identified by HS-84 and HS-169 or HS-259 are shown in magenta (HS-84 and HS-169) or green (HS-259). Different ligand staining patterns were observed for each mouse model. In APP_ArcSwe and APP23, age-related parenchymal Aβ deposits that were solely identified by HS-84 and HS-169 (APP_ArcSwe) or HS-259 (APP23) could be observed. The figure was created with BioRender.com. Aβ, amyloid-β; APP, amyloid-beta precursor protein.

Figure 5

Dual-staining of human brain tissue sections (frontal cortex) from individuals with sAD or diAD with ligands HS-276 and HS-169.A, overview tile image of a brain tissue section from an individual with sAD. B–E, images of different Aβ aggregates, including CAA lesions (B), cored (C), and diffuse (D) Aβ plaques, as well as Aβ deposits in the white matter (E). F, overview tile image of a brain tissue section from an individual with diAD associated with the PSEN1 A431E mutation. G–J, images of different Aβ deposits, including CAA lesions (G), cored (H) and diffuse Aβ plaques (I), and cotton wool plaques (J). K, overview tile image of a brain tissue section from an individual with diAD caused by the Arctic APP E693G mutation. L–O, images of different Aβ deposits, including CAA lesions (L and M) and cotton wool-like plaques (L–O). Aβ deposits stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-276 or HS-169 are highlighted with magenta (HS-276) or green (HS-169) arrows, respectively. Tau aggregates are indicated by white arrow heads and autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 500 μm (A, F, and K) and 20 μm (B–E, G–J, and L–O). Aβ, amyloid-β; sAD: sporadic Alzheimer’s disease; APP, amyloid-beta precursor protein; CAA, cerebral amyloid angiopathy; diAD, dominantly inherited AD; PSEN1, presenilin-1.

Figure 6

Dual-staining of human brain tissue sections (frontal cortex) from individuals with sAD or diAD with ligands HS-84 and HS-259.A, overview tile image of a brain tissue section from an individual with sAD. B–E, images of different aggregated Aβ pathologies, including CAA lesions (B), cored (C), diffuse (D), and Aβ deposits in the white matter (E). F, overview tile image of a brain tissue section from an individual with diAD associated with the PSEN1 A431E mutation. G–J, images of different Aβ deposits, including CAA lesions (G), cored (H), and diffuse Aβ plaques (I), and cotton wool plaques (J). K, overview tile image of a brain tissue section from an individual with diAD caused by the Arctic APP E693G mutation. L–O, images of different Aβ deposits, including CAA lesions (L and M) and cotton wool-like plaques (L–O). Aβ deposits stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-84 or HS-259 are highlighted with magenta (HS-84) or green (HS-259) arrows, respectively. Tau aggregates are indicated by white arrow heads and autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 500 μm (A, F, and K) and 20 μm (B–E, G–J and L–O). Aβ, amyloid-β; sAD: sporadic Alzheimer’s disease; CAA, cerebral amyloid angiopathy; APP, amyloid-beta precursor protein; diAD, dominantly inherited AD; PSEN1, presenilin-1.

Figure 7

Dual-staining of human brain tissue sections (frontal cortex) from individuals with sAD or diAD with ligands HS-84 and HS-169.A, overview tile image of a brain tissue section from an individual with sAD. B–E, images of different Aβ deposits, including CAA lesions (B), cored (C), diffuse (D), and Aβ aggregates in the white matter (E). F, overview tile image of a brain tissue section from an individual with diAD associated with the PSEN1 A431E mutation. G–J, images of different Aβ deposits, including CAA lesions (G), cored (H), and diffuse Aβ plaques (I) and cotton wool plaques (J). K, overview tile image of a brain tissue section from an individual with diAD caused by the Arctic APP E693G mutation. L–O, images of different Aβ deposits, including CAA lesions (L and M) and cotton wool-like plaques (L–O). Aβ deposits stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-84 or HS-169 are highlighted with magenta (HS-84) or green (HS-169) arrows, respectively. Tau aggregates are indicated by white arrow heads and autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 500 μm (A, F, and K) and 20 μm (B–E, G–J and L–O). Aβ, amyloid-β; sAD: sporadic Alzheimer’s disease; diAD, dominantly inherited AD; CAA, cerebral amyloid angiopathy; APP, amyloid-beta precursor protein; PSEN1, presenilin-1.

Figure 8

Dual-staining of human brain tissue sections (frontal cortex) from individuals with sAD or diAD with ligands HS-276 and HS-259.A, overview tile image of a brain tissue section from an individual with sAD. B–E, images of different Aβ deposits, including CAA lesions (B), cored (C), diffuse (D), and Aβ aggregates in the white matter (E). F, overview tile image of a brain tissue section from an individual with diAD associated with the PSEN1 A431E mutation. G–J, images of different Aβ deposits, including CAA lesions (G), cored (H), and diffuse Aβ plaques (I), and cotton wool plaques (J). K, overview tile image of a brain tissue section from an individual with diAD caused by the Arctic APP E693G mutation. L–O, images of different Aβ deposits, including CAA lesions (L and M) and cotton wool-like plaques (L–O). Aβ deposits stained by both ligands are indicated by white arrows, whereas Aβ aggregates only stained by HS-276 or HS-259 are highlighted with magenta (HS-276) or green (HS-259) arrows, respectively. Tau aggregates are indicated by white arrow heads and autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 500 μm (A, F, and K) and 20 μm (B–E, G–J, and L–O). Aβ, amyloid-β; sAD: sporadic Alzheimer’s disease; diAD, dominantly inherited AD; CAA, cerebral amyloid angiopathy; APP, amyloid-beta precursor protein; PSEN1, presenilin-1.

Figure 9

Images of human brain tissue sections, collected from frontal or visual cortex, from an individual with sAD, stained with a dual-staining protocol comprised of ligands HS-276 and HS-259.A, overview tile image of brain tissue sections from frontal (left) or visual (right) cortex. B and C, images of different Aβ deposits in the gray (B) or white (C) matter region of visual cortex. Aβ deposits only stained by HS-276 or HS-259 are highlighted with magenta (HS-276) or green (HS-259) arrows, respectively. Tau aggregates are indicated by white arrow heads and autofluorescence from granular lipofuscin is indicated by blue arrow heads. The staining was performed using 200 nM of the respective ligand in phosphate buffered saline (PBS, pH 7.4). Scale bars represent 500 μm (A) and 20 μm (B and C). Aβ, amyloid-β; sAD: sporadic Alzheimer’s disease.

Figure 10

Schematic presentation of different parenchymal Ab deposits that were identified with each dual-ligand staining protocols in human brain tissue sections. Parenchymal Aβ deposits, such as cored plaques (CP), diffused plaques (DP), cotton wool plaques (CWP), and cotton wool like plaques (CWLP) identified by both ligands are shown in white, whereas Aβ deposits solely identified by one of the ligands are shown in magenta (HS-276, HS-84, or HS-335) or green (HS-169 and HS-259). Distinct ligand staining patterns were observed between different types of AD, as well as between different brain regions, frontal cortex (fc), and visual cortex (vc) in sporadic AD (sAD). The figure was created with BioRender.com. Aβ, amyloid-β; AD, Alzheimer’s disease.