Axonal and myelinic pathology in 5xFAD Alzheimer's mouse spinal cord

Abstract

As an extension of the brain, the spinal cord has unique properties which could allow us to gain a better understanding of CNS pathology. The brain and cord share the same cellular components, yet the latter is simpler in cytoarchitecture and connectivity. In Alzheimer's research, virtually all focus is on brain pathology, however it has been shown that transgenic Alzheimer's mouse models accumulate beta amyloid plaques in spinal cord, suggesting that the cord possesses the same molecular machinery and conditions for plaque formation. Here we report a spatial-temporal map of plaque load in 5xFAD mouse spinal cord. We found that plaques started to appear at 11 weeks, then exhibited a time dependent increase and differential distribution along the cord. More plaques were found in cervical than other spinal levels at all time points examined. Despite heavy plaque load at 6 months, the number of cervical motor neurons in 5xFAD mice is comparable to wild type littermates. On detailed microscopic examination, fine beta amyloid-containing and beta sheet-rich thread-like structures were found in the peri-axonal space of many axons. Importantly, these novel structures appear before any plaque deposits are visible in young mice spinal cord and they co-localize with axonal swellings at later stages, suggesting that these thread-like structures might represent the initial stages of plaque formation, and could play a role in axonal damage. Additionally, we were able to demonstrate increasing myelinopathy in aged 5xFAD mouse spinal cord using the lipid probe Nile Red with high resolution. Collectively, we found significant amyloid pathology in grey and white matter of the 5xFAD mouse spinal cord which indicates that this structure maybe a useful platform to study mechanisms of Alzheimer's pathology and disease progression.

Fig 1. Beta amyloid plaque deposition in the spinal cord.

(A) Representative stacked image consisting of five cervical cord cross-sections immuno-labeled with 6E10 antibody. Each section is 100μm apart and is represented by one colour. Majority of the amyloid plaques are deposited in the grey matter except Rexed laminae I and II. Some plaques are located in the white matter, with a small number distributed in the lateral and ventral white matter (arrows); most were found in the ventral part of the dorsal column (arrowheads), which corresponds to corticospinal tract in rodents. (B) Quantitative analysis of number of 6E10-positive beta amyloid plaques at the cervical (C7-C8), thoracic (T12) and lumbar (L4-L5) spinal levels at different time points. Results from 6 and 8 week old mice are omitted since no plaques were found at those time points. There was an age-dependent increase in the number of plaques in whole spinal cord at each level, however cervical spinal cord always had the most plaques regardless of age. (C) To control for the difference in cross-sectional area at different levels of the spinal cord, we also calculated the density of plaques (expressed as number of plaque/ total spinal cord area) at 27 weeks of age. Representative 6E10 labeled brain sections from 8 week (D1), 11 week (D2), 19 week (D3), and 27 week (D4) old 5xFAD mice also show progressive increase in number and size of amyloid plaques. Scale bar in A and D = 1mm.

Fig 2. Despite heavy plaque load, beta amyloid plaque did not cause significant motor neuron loss at 27 weeks of age in 5xFAD mice.

(A) Representative micrographs showing ventral horns from a 5xFAD mouse (A2) and a wild type littermate (A1). Sections were labeled with anti-choline acetyltransferase (ChAT), a motor neuron marker, and counter-stained with DAPI to label nuclei. Motor neurons in both strains appeared normal with centrally placed nuclei and no sign of atrophy or chromatolysis. (B) Counting of ChAT-positive motor neurons revealed similar numbers in both strains. (C) Results were further confirmed by TUNEL staining (arrows) which labels apoptotic nuclei. The sections were co-labeled with various cellular markers (red) and 6E10 antibody and counter-stained with DAPI. No apoptotic oligodendrocyte (C3) or motor neurons were found (C4), only a few microglia (C1) and astrocytes (C2) were positive for TUNEL. Insets of C1-3 show higher magnification of TUNEL positive nuclei in various cell types. C5 shows many TUNEL positive nuclei in dorsal horn at 7 days after spinal cord contusion injury as a positive control. MNs: motor neurons. Scale bar in A = 200μm, C = 100μm.

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).

Fig 4. Most threads are located in the peri-axonal space.

(A) Micrographs showing high magnification of sagittal spinal sections co-labeled with AB42 (red), neurofilament (blue) and myelin basic protein (A1-2; green) or CNPase (A3; green). The majority of threads are confined within the myelin cylinder. A thread coiled into a knot-like structure is visible in A1. A2 shows a long and slender thread running in parallel with an axon. Occasionally, threads are found outside myelin (arrowheads in A2 and 3). A3 shows part of the thread presumably inside the axon and part of it outside (arrowhead) and it appears to pass through an opening in the myelin (arrow and insert). (B) Cross-sectional images depicting threads outside (B1), inside (B2), and surrounding (B3) axons, as well as piercing through the myelin sheath (B4). (C) z-stack image depicting a thread (green, intersected lines) lying outside an axon (red). Scale bar in A1-A2 = 25μm; A3 = 10μm; B1-3 = 10μm in length; C = 10μm.

Fig 5. Threads appear before plaque deposition.

(A) No plaques were found in 8 week old 5xFAD mouse spinal cord, however, 6E10-positive threads can be found (boxed areas) in this sagittal cervical cord section. Only 3 threads were found over the entire length of the cervical cord section. (B) In contrast, at 19 weeks of age numerous plaques were observed in the grey matter at the cervical level in 5xFAD mouse spinal cord (B1), and many threads were observed in the white matter (boxed area in B1 enlarged in B2). Scale bar in A and B1 = 500μm; B2 = 100μm.

Fig 6. Interactions between threads and glial cells.

(A) GFAP-positive astrocytes (red) are found surrounding two 6E10-positive plaques in this micrograph, but the astrocytes do not show a similar association with 6E10-positive threads. (B) Similarly, Iba-1-positive microglia are typically found in proximity or contact with plaques in the grey matter but do not appear to interact with threads to the same extent, although 6E10-positive deposits can be found inside some microglia in the white matter (arrowheads). Co-labeling with endosome antibody LAMP-1 (red), Iba-1 (blue) and 6E10 (green) reveals these deposits confined within endosomes inside microglia, which indicates that microglia are taking up amyloid deposits in the white matter tract. Scale bar in A = 50μm; B1 = 100μm.

Fig 7. Threads may cause damage to axons.

(A) Many APP-positive axonal spheroids (red) can be seen in a representative cross-section from a 27 week old 5xFAD mouse. 6E10-positive threads (green) are often seen co-labeling with APP staining (arrow) at this age. (B) Z-stack images (0. 5μm optical sections) from the spheroid indicated by the arrow in A reveal that a thread lies on the outside of the spheroid rather than inside it. (C) Similarly, a sagittal section showed a 6E10-labeled (green) thread at the tip of an APP-positive (red) spheroid. (D) Sagittal section stained with the aminergic fibre marker, tyrosine hydroxylase (red), and 6E10 (green) shows a thread, extending from a spheroid at the end of an aminergic fibre, that forms a coil about 30μm caudal to the spheroid. Scale bar in A = 50μm; B-C = 10μm and D = 15μm.

Fig 8. Myelinopathy in 5xFAD mouse white matter.

Representative images showing Nile Red labeled spinal cord ventral white matter. 37 week old wild type mouse (A1) sample shows normal appearing myelin staining profile. Very infrequent myelin spheroid (arrow) can be seen in old wild type samples. Myelin staining appears normal in 19 week old 5xFAD mouse spinal cord (A2), but abnormalities are clearly visible at 27 weeks (A3) and are more prevalent in 37 week old 5xFAD mouse samples (A4). For example, myelin spheroid/ thickening (B1 arrowheads) and sphere formation (B2, arrowhead) are observed in a 27 week old 5xFAD mouse sample. Quantitative analysis of myelin abnormalities at 19, 27, and 37 week old 5xFAD mice and 37 week old WT mice (C). Results showed a progressive and significant increase of myelin abnormalities from 19 week to 37 week old 5xFAD. ****, p<0.001, ####, p<0.001. (D) Merging of the Nile Red (green) image with 6E10 (red) and DAPI (blue) images reveals that myelin anomalies are found in contact or close proximity to threads (arrows), but also in the absence of threads (arrowheads). Scale bar in A = 50μm, B = 20μm and D = 100μm.