Thiophene-Based Ligands for Specific Assignment of Distinct Aβ Pathologies in Alzheimer's Disease

Abstract

Aggregated species of amyloid-β (Aβ) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aβ deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aβ deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aβ deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aβ plaques, whereas LL-1 mainly stained cored and neuritic Aβ deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aβ plaques. The ligand-labeled Aβ deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aβ aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aβ peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aβ deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aβ species associated with different forms of AD.

Keywords: Alzheimer’s disease; amyloid-β; fluorescence; imaging mass spectrometry; ligands; protein aggregates.

Figure 1

The combination protocol based on ligands HS-276 and LL-1 shows different staining patterns when applied on brain tissue sections from sAD and fAD (PSEN1 A431E) patients. (a) Chemical structures of ligands HS-276 (top) and LL-1 (bottom). (b) Fluorescence emission spectra of HS-276 (blue solid line) and LL-1 (red dashed line) when bound to Aβ-like structures in AD. (c) Fluorescence overview images of brain tissue sections from sAD (top panel) and fAD (bottom panel) patients stained with the combination of 200 nM HS-276 (blue) and 300 nM LL-1 (red). Autofluorescence from lipofuscin granules is shown in green. Scale bar, 1 mm. (d) Fluorescence images of different Aβ-like deposits in sAD (top panel) and fAD (bottom panel) brain tissue sections stained with the combination of HS-276 (blue) and LL-1 (red). LL-1 was also labeling structures resembling DNs (arrow) and NFTs (arrowhead). Autofluorescence from lipofuscin granules is shown in orange or in white. Scale bar, 20 μm.

Figure 2

HS-276 and LL-1 label Aβ pathology in sAD and fAD (PSEN1 A431E) brain tissue sections. (a) Fluorescence overview images of tissue sections from sAD (left) and fAD (right) patients stained with 200 nM HS-276 (blue) and anti-Aβ antibody 4G8 (left panel, green), 6E10 (middle panel, green), or fibril specific antibody OC (right panel, green). Scale bar, 1 mm. (b) Fluorescence overview images of tissue sections from sAD (left) and fAD (right) patients stained with 300 nM LL-1 (red) and anti-Aβ antibody 4G8 (left panel, green), 6E10 (middle panel, green), or fibril specific antibody OC (right panel, green). Autofluorescence from lipofuscin (LF) is shown in purple. Scale bar, 1 mm.

Figure 3

Parenchymal Aβ deposit types in fAD (PSEN1 A431E) labeled with HS-276. (a) Fluorescence overview image of brain tissue section from an fAD patient stained with 200 nM HS-276 (blue) and anti-Aβ antibody 4G8 (green). HS-276 mainly labels Aβ plaques in the intermediate cortical layers, whereas 4G8 stains deposits in all layers. Autofluorescence from lipofuscin (LF) granules is shown in magenta. The white boxes define the zoomed-in regions shown in (b) and (c). Scale bar, 1 mm. (b) Zoomed-in view of the top left region highlighted in (a) showing diffuse Aβ plaques labeled with HS-276 (blue) and 4G8 (green). Autofluorescence from lipofuscin (LF) granules is shown in magenta. Scale bar, 50 μm. (c) Zoomed-in view of the bottom right region highlighted in (a) showing cored Aβ plaques (arrow) labeled with HS-276 (blue) and 4G8 (green). In the outer parts of the HS-276-positive layer of Aβ plaques, deposits stained with 4G8, but only partially with HS-276, can be seen (arrowhead). Autofluorescence from lipofuscin (LF) granules is shown in magenta. Scale bar, 50 μm.

Figure 4

Parenchymal Aβ deposit types in fAD (PSEN1 A431E) labeled with LL-1. (a) Fluorescence overview image of brain tissue section from an fAD patient stained with 300 nM LL-1 (red) and anti-Aβ antibody 4G8 (green). LL-1 labels Aβ deposits in the inner and outer cortical layers, whereas the intermediate layers only show 4G8 positivity. The white boxes define the zoomed-in regions shown in (b) and (c). Scale bar, 1 mm. (b) Zoomed-in view of the top right region highlighted in (a) showing CWPs labeled with LL-1 (red) and 4G8 (green). Autofluorescence from lipofuscin (LF) granules is shown in magenta. Scale bar, 50 μm. (c) Zoomed-in view of the bottom left region highlighted in (a) showing cored Aβ plaque in the white matter labeled with LL-1. Autofluorescence from lipofuscin (LF) granules is shown in magenta. Scale bar, 50 μm.

Figure 5

Fluorescence overview images of consecutive brain sections from an fAD (PSEN1 A431E) patient stained with the combination (left) of 200 nM HS-276 (blue) and 300 nM LL-1 (red) or with 200 or 300 nM of the conventional ligand CN-PiB (middle, white) or X-34 (right, white), respectively. CN-PiB labeled the same layer of Aβ plaques as HS-276, whereas the staining with X-34 corresponded to the staining pattern of LL-1. Scale bar, 1 mm.

Figure 6

Comparing the combination staining protocol in fAD (PSEN1 A431E) with binding of LCOs and examining the effect on the staining result when increasing the concentration of HS-276 or LL-1. (a) Chemical structure of the LCO ligand h-FTAA. (b) Fluorescence images of fAD brain tissue section stained with h-FTAA (green) and anti-Aβ antibody 4G8 (red). An overview of the staining result is depicted in the top image, whereas the bottom image shows the binding pattern in more detail. H-FTAA showed complete colocalization with the antibody, confirming that the LCO ligand was binding to all Aβ deposits in the sample and not just specific types of plaques as HS-276 and LL-1. Scale bars, 1 mm (top panel) and 50 μm (bottom panel). (c,d) Fluorescence images of brain tissue section from fAD patient stained with 2 μM HS-276 (green) and anti-Aβ antibody 4G8 (red) (c) or 1 μM LL-1 (green) and anti-Aβ antibody 4G8 (red) (d). Overviews of the staining result are depicted in the top panels, whereas the bottom panels show the binding patterns in more detail. Even at the higher concentrations, both ligands were still mainly staining the same type of Aβ plaque as at the lower concentrations used in the combination protocol. Scale bars, 1 mm (top panel) and 50 μm (bottom panel).

Figure 7

Distribution of tau pathology in fAD (PSEN1 A431E) brain tissue sample. (a) Fluorescence overview images of fAD brain sections stained with 200 nM HS-276 (top panel, blue) or 300 nM LL-1 (bottom panel, red) together with anti-tau antibody GT-38 (green). There is a marked increase in tau deposits accumulating just below the layer with HS-276-positive Aβ plaques. The pathology was extending into the upper part of the inner LL-1 labeled layer. Scale bar, 500 μm. (b) Fluorescence images showing the staining results in panel (a) in more detail. In many CWPs localized in the upper part of the LL-1 layer, antibody staining of DNs (arrow) could be seen. LL-1, but not HS-276, showed costaining with the tau antibody also in NFTs (small arrow) and neuropil threads (arrowhead). Scale bar, 50 μm. (c) Fluorescence images showing the staining results in panel (b) in a higher magnification. In several of the CWPs, DNs positive for LL-1 (red) and the tau antibody (green) can be seen (top panel, arrow). The LL-1 ligand is also labeling immunopositive neuropil threads (top panel, arrowhead) and NFTs (top/bottom panel, small arrow). Scale bar, 20 μm. (d) Fluorescence images of sAD brain tissue section stained with 300 nM LL-1 (red) and anti-tau antibody GT-38 (green). LL-1 is labeling immunopositive DNs (top panel, arrow), neuropil threads (bottom panel, arrowhead), and NFTs (bottom panel, small arrow). Scale bar, 20 μm.

Figure 8

Correlative chemical imaging identifies HS-276 and LL-1 staining-associated Aβ deposition patterns. (a) Bright-field and (b–d) fluorescence microscopy ((c) and (d) are magnifications of (b)) images of fAD (PSEN1 A431E) frontal cortex brain tissue stained with HS-276 (blue) and LL-1 (red). Autofluorescent lipofuscin (LF) can be seen in orange. (e–i) MALDI MSI of Aβ peptides performed on brain section closely adjacent to the section labeled with HS-276 and LL-1. (e) Segmentation map using bisecting k-means cluster analysis (CA) identifies plaque-associated signatures following the plaque staining distribution patterns identified with HS-276 and LL-1. (f,g) Loading spectra and cluster tree of three spatial amyloid patterns retrieved by CA (1: blue, 2/1: yellow, and 2/2: green (e)). Inspecting the cluster-associated variable spectra shows the primary content within each cluster. Specifically, plaques within cluster 1 are dominated by Aβx-40 peptides as further highlighted in the corresponding single ion map (h-I). Cluster 2 comprises a different pattern with two subtypes: 2/1 (e–g, yellow) and 2/2 (e–g, green). Plaques within cluster 2/2 show a significantly higher content of Aβ3pE-42 (h-II) and Aβ11pE-42 (h-III) and follow distribution of LL-1. In contrast, Aβ4-42 and Aβ1-42, while higher in cluster 2/2 (green), are also abundant in cluster 2/1 (yellow) as indicated by the single ion maps (h-IV and h-V) following HS-276 staining. (i) Overlay of Aβ3pE-42 and Aβ1-42 showing the complementary pattern outlined by CA (e), which is in line with LL-1/HS-276 staining (d). Scale bars, 1 mm (b–d) and 500 μm (h).