Thiophene-Based Ligands for Histological Multiplex Spectral Detection of Distinct Protein Aggregates in Alzheimer's Disease

Abstract

The aggregation and accumulation of proteins in the brain is the defining feature of many devastating neurodegenerative diseases. The development of fluorescent ligands that bind to these accumulations, or deposits, is essential for the characterization of these neuropathological lesions. We report the synthesis of donor-acceptor-donor (D-A-D) thiophene-based ligands with different emission properties. The D-A-D ligands displayed selectivity towards distinct disease-associated protein deposits in histological sections from postmortem brain tissue of individuals affected by Alzheimer's disease (AD). The ability of the ligands to selectively identify AD-associated pathological alterations, such as deposits composed of aggregates of the amyloid-β (Aβ) peptide or tau, was reduced when the chemical composition of the ligands was altered. When combining the D-A-D ligands with conventional thiophene-based ligands, superior spectral separation of distinct protein aggregates in AD tissue sections was obtained. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species, as well as offer novel strategies for developing multiplex fluorescence detection of protein aggregates in tissue sections.

Keywords: Alzheimer's disease; amyloid-β; fluorescent ligands; protein aggregates; tau.

Figure 1.. Chemical structure and photophysical properties of the novel D-A-D thiophene-based ligands for protein aggregates.

Chemical structure of D-A-D thiophene-based ligands (A) and their corresponding absorption- (B) and emission spectra (C-D). The spectra were recorded for 30 μM ligand in de-ionized water. For the blue emission spectra (C), an excitation corresponding to the absorption maximum at shorter wavelengths (blue arrows) was used, whereas an excitation corresponding to the absorption maximum at longer wavelengths (red arrows) was used for the red emission spectra (D).

Figure 2.. Fluorescence images of brain tissue sections with AD pathology stained with anti-Aβ antibody and different thiophene-based ligands.

Images showing staining of different aggregated Aβ pathologies (cored plaques, diffuse plaques and CAA lesion, highlighted by arrows) with anti-Aβ antibody 4G8 (red) and A) the tetrameric thiophene-based ligands t-HTAA, LL-8 and LL-9 (green), B) the pentameric thiophene-based ligands p-FTAA, HS-84, LL-6 and LL-7 (green) or C) the heptameric thiophene-based ligands h-FTAA and LL-1 (green). Scale bar represents 20 μm. Autofluorescent lipofuscin granules are highlighted by green arrowheads.

Figure 3.. Fluorescence images of brain tissue sections with AD pathology stained with anti-tau antibody and different thiophene-based ligands.

Images showing staining of different aggregated tau pathologies (neurofibrillary tangles, neuropil threads and dystrophic neurites, highlighted by white arrowheads) with anti-tau antibody GT-38 (red) and A) the tetrameric thiophene-based ligands t-HTAA, LL-8 and LL-9 (green), B the pentameric thiophene-based ligands p-FTAA, HS-84, LL-6 and LL-7 (green) or C) the heptameric thiophene-based ligands h-FTAA and LL-1 (green). Scale bar represents 20 μm. Autofluorescent lipofuscin granules are highlighted by green arrowheads.

Figure 4.. Fluorescence images of brain tissue sections with AD pathology stained with the pentameric D-A-D thiophene-based ligands HS-169 or LL-5.

Images showing staining of different aggregated Aβ species (cored plaques, diffuse plaques and CAA lesions) and tau pathology (neurofibrillary tangles) with A) HS-169 (green) or B) LL-5 (green). Autofluorescence from lipofuscin can be seen in blue. Single channels are shown in white to enhance visualization. Due to some spectral overlap, ligand fluorescence can occasionally also be observed in the lipofuscin channel. Scale bar represents 20 μm.

Figure 5.. Fluorescence assignment of protein aggregates in brain tissue sections with AD pathology stained with t-HTAA and HS-169.

Color-coded fluorescence images (top panel), FLIM intensity images and fluorescence decays (bottom panel) of A) CAA lesions, B) diffuse Aβ plaques, C) neuritic Aβ plaques and D) NFTs, stained with t-HTAA and HS-169. For t-HTAA, an excitation at 405 nm was used and emission was recorded between 470 nm to 545 nm, whereas HS-169 was excited at 550 nm and detection of emission between 610 nm to 745 nm was applied. Autofluorescence from lipofuscin in (B) are highlighted with white arrow heads, whereas HS-169 stained tau pathology in (C) is shown with white arrows. Scale bars represent 20 μm.

Figure 6.. Fluorescence assignment of protein aggregates in brain tissue sections with AD pathology stained with p-FTAA and LL-5.

A) Overview image of the whole tissue section showing the correlation between p-FTAA (green) and LL-5 (red) staining. B-D) Zoom in of the different regions highlighted in (A). Red square (B), blue square (C) and green square (D). E) Emission spectra from different aggregated Aβ (CAA, diffuse and neuritic plaque) and tau (NFTs and dystrophic neurites) with excitation at 490 nm. The p-FTAA emission maxima are observed at 515 nm and 545 nm, whereas LL-5 displays an emission maximum at 625 nm. F) Fluorescent decays from different aggregated Aβ pathologies (CAA, diffuse and neuritic plaque) and tau pathologies (NFTs and dystrophic neurites) with excitation at 490 nm. The distribution of decays for p-FTAA is observed between 600 ps to 1200 ps, whereas LL-5 displays distributions of decays between 2000 ps to 3500 ps. G-J) Spectral images (left panel), FLIM images (middle panel) and color-coded FLIM images (right panel) from different aggregated Aβ pathologies, CAA (G), neuritic (H) and diffuse plaque (I)) and tau pathologies, NFTs (J) with excitation at 490 nm. The color bars (right panel) represent 500 ps to 3500 ps. Scale bars represent 1000 μm (A), 200 μm (B-D), 50 μm (G) and 20 μm (H-J).

Scheme 1.. Synthesis of different D-A-D thiophene-based ligands.

Reagents and conditions: (i) PEPPSI^™-IPr, K₂CO₃, 1,4-dioxane/ toluene/ MeOH (1:1:1), 80°C, 2h; (ii) NBS, DMF/chloroform (1:1), ,−15°C to r.t., 18 h; (iii) PEPPSI^™-IPr, K₂CO₃, toluene/ MeOH (1:1), 80°C, 3h; (iv) NaOH (1M, aq.), 1,4-dioxane, 40°C; (v) PEPPSI^™-IPr, Cs₂CO₃, 1,4-dioxane, 80°C, 3h; (vi) NaOH, MeOH/CH₂Cl₂ (1:10), r.t., 24h.