Fig 3. Beta amyloid-positive threads in 5xFAD mouse spinal cord.

(A) Cervical spinal cross-section stained with AB42 (red) and myelin basic protein SMI99 (green) as shown in insert. For clarity of plaque and thread labeling only the AB42 staining is shown in the large cross-sectional image. The image was processed in grey scale and colour-inverted. Many black puncta were seen in the white matter, largely localized to the descending tracts outlined and colour-coded in the image as follows: corticospinal tract (red); rubrospinal tract (green); caudal and rostral reticulospinal tracts (blue); and medial and lateral vestibulospinal tracts (yellow). The outlines are based on the work of Watson and Harrison [19]. On closer examination, the puncta have unique structure such as thread or ring like as shown in insert (rectangle in cross-section). (B) Sagittal spinal section co-labeled with 6E10 (green) and AB42 (red) antibodies revealed thread structure and confirmed that they consisted of beta amyloid peptide (boxed area in B1 is enlarged in B2-3). (C-E) We used three conformationally-sensitive amyloid probes to confirm our findings. The threads are positive for pFTAA, ThT and K114 (arrows), indicating they possess beta sheet secondary structure. K114-positive threads (arrows in E2) are found in plaque laden (arrowhead) corticospinal tract (E1) and are positive for AB42 antibody (E3-4). No beta amyloid-positive threads are found in any wild type samples (E5). (F) K114 spectral emission red-shifts when bound to amyloid fibrils at high pH. (G) When truecolour images of K114-labeled plaques (G1) are converted to spectral pseudo-colour images (G2), it is clear that the emission spectrum of K114 varies considerably in different regions of a single plaque. (H) Truecolour images and the corresponding pseudo-colour (heat map) images (inserts) show spectral heterogeneity of K114 bound to amyloid threads as well. Arrowheads in H1 point to a broken thread; arrowheads in H2 point to the edge of the thread being more blue-shifted than the core. Scale bar in A = 1mm; B1, H1-2 = 50μm; B2-3 = 20μm; C-D, E5 = 100μm; E2 = 25μm; and G1-2 = 15μm. Abbreviations: background (BG); corticospinal tract (CST); ependymal cell layer (EC); grey commissure (GC).