Threads may cause damage to axons.

<p>(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.</p

Primary antibodies used in the study.

<p>Primary antibodies used in the study.</p

Threads appear before plaque deposition.

<p>(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.</p

Threads may cause damage to axons.

<p>(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.</p

Most threads are located in the peri-axonal space.

<p>(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.</p

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

<p>(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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188218#pone.0188218.ref019" target="_blank">19</a>]. 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).</p

Interactions between threads and glial cells.

<p>(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.</p

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

<p>(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.</p

(A) Representative examples of raw mEPSCs recorded in mature (12–16 DIV) WT and PrP-null hippocampal neurons in culture

In PrP-null neurons, NMDA-mediated mEPSCs were observed to be larger and showed prolonged decay times. (B) Event histograms for mEPSC amplitude (top) and decay time (bottom). Note that mEPSCs in PrP-null neurons exhibit a shift toward larger amplitude events and increased decay time constants. (C) Cumulative probability plots for mEPSC amplitude and decay times showing a shift in each summed distribution toward larger events with longer decay times (P < 0.05; Kolmogorov-Smirnov test). (D) Mean values for mEPSC waveform parameters showing increased EPSC amplitudes and prolonged decay times. Here, decay time refers to the time required for an e-fold reduction in peak current amplitude. Data are represented as mean ± SEM (error bars), with statistical significance denoted as *, P < 0.05 and **, P < 0.001. Numbers in parentheses indicate the number of cells.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p

(A) Western blot analysis of the NR2D subunit protein expression in neonatal and adult hippocampal tissue obtained from the WT and PrP-null mouse

α-Actin expression was used as a loading control. (B) NMDAR subunit surface expression as visualized by immunolabel reactivity with an antibody targeted against an extracellular (N terminus) epitope of NR2D. A punctate pattern of receptor distribution is visualized along dendritic processes. The depth of field is ∼1 μm. (C) Surface expression of NR2D relative to total cellular NR2D protein content as quantified using an ELISA assay in permeabilized (P) and nonpermeabilized (NP) cells. The number of neuronal culture samples is indicated in parentheses. Error bars represent SEM. (D) Coimmunoprecipitation of PrP and NR2D using both permutations of tag and probe showing that PrP and NR2D are in a complex. In the top panel, the blot was probed with a PrP antibody, and in the bottom panel, membrane was probed with NR2D antibody. The lane labeled control reflects beads without antibody. The experiment is a representative example of four different repetitions for both neonatal and adult mouse hippocampal tissue. (E) Western blot demonstrating the lack of coimmunoprecipitation between NR2B and PrP, whereas NR2B can be detected in brain homogenate (input). (F) Costaining of WT mouse hippocampal neurons for PrP (red) and NR2D (blue). The cells were not permeabilized, thus allowing for the selective staining of cell surface protein. The white line in the top left panel indicates the position of the linescan shown in the bottom left panel. The rectangle in the merged image (top right) corresponds to the magnified images shown at the bottom right. The arrowheads highlight examples of clear colocalization between NR2D and PrP. Bars: (B, top left) 7.5 μm; (B, top right and F, top) 10 μm; (B, bottom) 1 μm; (F, bottom) 2 μm.<p><b>Copyright information:</b></p><p>Taken from "Prion protein attenuates excitotoxicity by inhibiting NMDA receptors"</p><p></p><p>The Journal of Cell Biology 2008;181(3):551-565.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364707.</p><p></p